MXPA97002152A - Cloning of a membrane aminopeptidase, dependent of insulin, from deglu vesicles - Google Patents

Abstract

A peptidase that divides an insulin has been purified from vesicles containing GLUT-4 and has been cloned. The peptidase has a measured mass of approximately 165 kD, but is 110 kD in its deglycosylated state. A mass of 117 239 Daltons was predicted based on the predicted amino acid sequence of the cDNA in the figure (SEQ ID Nos: 15 and 16). It includes peptides that have the amino acid sequences Phe-Ala-Ala-Thr-Gln-Phe-Glu-Pro-Leu-Ala-Ala (sec id NO: 1) and Ile-Leu-Gln-Asn-Gln-Ile-Gln -Gln-Gln-Thr-Arg-Thr-As p-Glu-Gly-Xaa-Pro-Xaa-Met (SEQ ID NO: 2) and binds to the antibodies raised against the peptide identified as (SEQ ID NO: 1) ). Modulators and a method for the treatment of insulin resistance syndrome, which includes diabetes, are also claimed through the administration of such modulation.

Description

CLONING OF A MEMBRANE AMINOPEPTIDASE, nKPKNDTRMTiü i? OF INSULIN, FROM GLUT-4 VESICLES • FIELD OF THE INVENTION This request is related to the biochemistry of diabetes, and more particularly, to an inopeptidase which is involved in the regulation of insulin.

BACKGROUND OF THE INVENTION Adipocytes and myocytes contain vesicles that contain intracellular GLUT4, which fuse with the plasma membrane after stimulation with insulin. The resulting increase in GLUT4 in the plasma membrane is responsible for a 10 to 20-fold increase in glucose transport in the state stimulated by insulin. It is believed that the movement of vesicles containing GLUT4 from the cytoplasm to fuse with the plasma membrane, a process called translocation, is abnormal both in insulin resistance and in non-insulin-dependent diabetes mellitus (NIDDM) (B. Rahn, J Clin Invest., 89, 1367-1374, (1992)). The normal molecular mechanism of insulin-stimulated translocation of vesicles containing REF: 24408 GLUT4 towards the plasma membrane and the concomitant increase in glucose uptake by the affected cells is still largely unknown. The proposed mechanisms include both entrapment and fusion of vesicles containing GLUT4 with the plasma membrane. The vesicles containing GLUT4 are only a minor fraction of the population of total intracellular vesicles within the adipocytes (James et al, J. Biol. Chem.,: 2_62, 11817-11824, (1987)). These GLUT4 vesicles can be isolated from the low density microsomal fraction using immunoaffinity methods with antibodies to the cytoplasically oriented C-terminal GLUT4 (James et al, J. Biol. Chem., 262, 11817-11824, (1987); Thoidis, et al, J. Biol. Chem., 268, 11691-11696, (1993)). The vesicles enriched with GLUT4 have been shown to contain several proteins similar to those of the synaptic vesicles including the membrane protein associated with the vesicles (VAMP) (Cain et al, J. Biol. Chem., 267, 11681-11684, (1992)), membranal protein associated with the secretory carrier (SCAMP) (Lauri et al, J. Biol. Chem., 268, 19110-19117, (1993)), and certain ras analog proteins originally identified in the brain (proteins Rab) (Coron et al, J. Biol. Chem., 2_68, 19491-19497, (1993)). The pharmacological therapy so far for NIDDM is not aimed at insulin resistance which it is the key to the pathophysiological abnormality of the disease. The existing therapies are 1) diet and exercise; 2); sulfonylureas, which stimulate insulin secretion; 3) α-glucosidase inhibitors, which inhibit the enzymatic digestion of complex carbohydrates and therefore decrease the postprandial absorption of glucose; 4) metformin, whose mechanism probably includes the improvement of hepatic sensitivity to insulin, and 5) insulin injections. A therapeutic agent that targets insulin resistance and improves insulin sensitivity may have significant advantages over the therapies listed above. Such a therapeutic agent is the object of the present invention. An object of this invention is to increase insulin sensitivity by modulating the aminopeptidase associated with GLU 4 vesicles.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a novel aminopeptidase, here designated GTVap, which is a component of the vesicles containing GLUT4 and is involved in the insulin signaling pathway. It is also related to the use of therapeutic modulators of this protein to treat diabetes. Additional aspects of the invention will be discussed later. The deregulation of peptide signaling molecules is important to maintain normal homeostasis. More particularly, the deregulation or removal of active insulin is important to prevent hyperinsulinemia with the resulting hypoglycemia in non-diabetic individuals. The current dogma is that all circulating insulin degrades after binding to the insulin receptor, its internalization, and its eventual intracellular proteolysis by an enzyme that degrades previously characterized insulin (IDE). The data presented here indicates that there is an additional mechanism for insulin degradation, namely that the GTVap of the cell surface of the adipocytes removes the N-terminal amino acids from the insulin molecule, altering its physiological activity. Although this may be a normal mechanism of insulin processing, the increased levels of enzyme present in obese individuals or in conditions of increased enzymatic activity that lead to conditions in which plasma insulin has been partially inactivated. This explains the association of obesity with insulin resistance and NIDDM.

Another indication that GTVap is important in the insulin signaling pathway is the fact that the GTVap is found primarily in insulin-sensitive tissues, which also contain the GLUT4 protein. It is known that obesity is associated with a variety of clinical manifestations, as well as an increase in body fat with a concomitant increase in GTVap. The increase in body fat and GTVap may result in inactivation or inappropriate activation of other important circulating polypeptides in addition to insulin. Consequently, the pharmacological and genetic manipulation of GTVap constitutes a novel therapeutic objective for the relief of insulin resistance, NIDDM, and obesity. The present invention relates to a protein which is a component of the vesicles containing GLUT4 in the natural state. This protein has aminopeptidase activity, a molecular weight of about 165 D, 155kD, or 120kD depending on the degree of glycosylation, and about HOkD in its deglycosylated form. This includes the amino acid sequences Phe-Ala-Ala-Thr-Gln-Phe-Glu-Pro-Leu-Ala-Ala (SEQ ID NO: 1) and Ile-Leu-Gln-Asn-Gln-Tle-Gln- Gln-Gln-Thr-Arg-Thr-Asp-Glu-Gly-Xaa-Pro-Xaa-Met (SEQ ID NO: 2), and reacts with the antibodies produced against the identified peptide, (SEQ ID NO: 1). This is encoded by the cDNA encoding the full length sequence of the GTVap (SEQ ID NOs: 15 and 16) and its amino acid sequence is essentially identical to that estimated for the GTVap (SEQ ID NOs: 15 and 16). The present invention also relates to the muteins of this protein. The present invention also relates to the cDNA encoding the full-length sequence of the GTVap and the estimated protein sequence (SEQ ID NOs: 15 and 16). The invention further relates to a method for treating insulin resistance syndromes, which comprises administering to a subject exhibiting an insulin resistance syndrome, an effective amount of a GTVap modulator in a pharmaceutically acceptable carrier. The other aspects of the invention will be discussed in the detailed description below.

DESCRIPTION OF THE DRAWINGS The invention will be more fully understood from the consideration of the following description and the glossary of terms, taken in conjunction with the drawings, in which: Figure 1 is a Western blot showing immunoaffinity purification of GLUT4 vesicles; Figure 2 is a staining of purified GLUT4 vesicle proteins by immunoaffinity; Figure 3 shows the CLAP profiles of tryptic digestion of GLUT4 vesicle proteins of 165kD (above the fat level) and 155kD (below the fat level); Figure 4 is a Western blot showing the enrichment of the 165kD and 155kD proteins in the GLUT4 vesicles; Figure 5 is a Western blot of the 165kD and 155kD proteins (LDM) of the low density microsomes after treatment with N-Glicosidase F; Figure 6 is a graph showing the quantitative enrichment of aminopeptidase activity in GLUT4 vesicles; Figure 7 is a Western blot of the 165kD, 155kD and 120kD proteins in rat adipocyte membrane fractions; Figure 8 is the activity of the GTVap enzyme in fractions of rat adipocyte membranes; Figure 9 is a graph showing the optimal pH of the GTVap; Figure 10 is a graph showing the temperature stability of the GTVap; Figure 11 is a graph of the effect of several ions on the activity of GTVap: Co (solid cadres); Zn (open boxes); Mg (solid diamonds); Mn (open diamonds); Ca (solid triangles); Figure 12 is a graph of the ionic increase in stability of the GTVap: control (open diamonds); Mg 2 mM (solid triangles); Mn 0.02 mM (open triangles); Ca 2 mM (solid circles); Zn 1 μM (solid frames); Co 1 μM (open boxes); 0.2 mM DTT (solid diamonds); Figure 13 is a graph showing the effects of different protease inhibitors on the activity of GTVap: phenanthroline (solid cadres); DTT (open boxes); LLPAC (solid diamonds); dipyridyl (open diamonds); Figure 14 is a graph of the effects of different aminopeptidase inhibitors on the activity of GTVap: bestatin (solid cadres); actinonin (open boxes); leutión (solid diamonds); to astatine (open diamonds); Figure 15a is a graph of the activity of the GTVap showing a chromatographic separation by anion exchange of the purified GTVap with WGA; Figure 15b is a Western blot of GTVap-120, 155 and 165 following an anion exchange chromatographic separation of the purified GTVap with WGA; Figure 16 is a graph of the activity of the GTVap following an anion exchange enzymatic activity profile of the purified GTVap with WGA and IDAC: through the flow of IDAC (solid diamonds), IDAC binding (open circles); Figure 17 is a Western blot of the GTVap of the plasma membrane fraction before and after insulin stimulation; Figure 18 is a graph of activity of the GTVap in the plasma membrane before and after insulin stimulation; Figure 19a, 19b, and 19c are graphs of mass analysis of insulin after digestion with GTVap, at times 0 hr., 3.5 hr., And 2 hr., Respectively; Figure 20 shows the cDNA sequences of the GTVap and the sequences of the proteins estimated from the cDNAs (SEQ ID Nos. 15 and 16). Figure 21 shows the consensus sequence of retention and classification of GTVap and GLUT4 (SEQ ID NO: 20), the consensus sequence of inverse retention and classification (SEQ ID NO: 21), and the respective segments of the GTVap (in orientation from N to C; (SEQ ID NOs: 17 and 18)) and GLUT4 (in orientation from C to N; (SEQ ID NOs: 19)) from which they were derived. The asterisk indicates that the sequence is written in the non-conventional C to N direction. Xaa means any amino acid; Xaa means Pro, Glu, or Asp; and Xaa means Arg or Lys.

DETAILED DESCRIPTION OF THE INVENTION LDM vesicles containing GLUT4 from rat adipocytes have been purified by an immunoaffinity procedure and their protein composition has been compared with LDM vesicles that lack GLUT4. It has been hypothesized that GLUT4 vesicle proteins are involved in insulin-induced translocation of vesicles, which includes movement from their resident intracellular compartment, and attack with and infusion with the plasma membrane, and the reabsorption or endocytosis of the plasma membrane after the termination of the insulin signal. The present purification strategy resulted in the identification of several unique proteins with molecular weights in the vicinity of 160kD.

Two of the identified proteins that are unique to the GLUT4-containing vesicles have molecular weights of approximately 165kD and 155kD, respectively. Initial attempts at N-terminal sequencing indicated that the N-terminus of each was blocked. The digestion of both 165kD and 155kD proteins followed by the CLAP preparation of the micropore of the peptide fragments indicated that these proteins are closely related, since the CLAP-UV profiles of the digestions are very similar. A peptide sequence had a homology of 100 ?; with a previously published 15 amino acid peptide, claimed to be a fragment of a plasma pregnancy-related inopeptidase. None of the remaining peptide sequences had any homology with the placental aminopeptidase or any other known. Experiments with a variety of synthetic and natural aminopeptidase substrates have shown that 165kD proteins, 155kD and 120kD within the adipocyte are aminopeptidases and are glycosylated since they bind to the wheat germ lecithin affinity resins. The 165kD and 155kD materials can be deglycosylated using N-glycosidase F and result in an HOkD protein. The material of 120kD is related to the aminopeptidases of 165kD and 155kD since the antibodies that react with the 165kD and 155kD forms they also react with 120kD aminopeptidase. In the following text, each of the aminopeptidases of the vesicles containing GLUT4 will be referred to as Glucose Transport Vesicle Aminopeptidase (GTVap), with a molecular weight in kD appended to the designation. Although a 165kD GTVap peptide sequence had 100% homology with a known pregnancy-related aminopeptidase of placental origin, the two aminopeptidases have significantly different properties and are presumably members of a larger aminopeptidase family. Immunoreactivity and enzymatic activity of GTVap have also been found in rat muscle, human fat, 3T3-L1 adipocyte cell lineage. High levels of immunoreactivity in the spleen can also be found with lower amounts in other tissues. With respect to the isolated and purified aminopeptidase which is a component of the vesicles containing GLUT4 in the natural state, the activity of the aminopeptidase, the molecular weight, certain amino acid sequences, and its reactivity with the antibodies raised against the identified peptide, [SEQ ID NO: 1] have been summarized above. This protein is useful for identification of modulators that may have utility in the treatment of diabetes. The protein is also characterized by having optimal activity at neutral pH; its relative activity towards substrates of synthetic amino acid p-nitroanilide is: leucine > > proline, alanine > valine, glycine; its relative activity towards synthetic amino acid naphthylamide substrates is leucine > power plant > arginine > methionine > Alanine > phenylalanine; its activity is modulated by the divalent ions of Co, Zn, Mg, Mn, and Ca; the minimum inactivation temperature is between 40 ° C and 50 ° C; its activity is stabilized by Ca ions; its activity is reduced by phenanthroline, dipyridyl, leutiol, amastatin, actinonin, and bestatin; and has at least three glycosylated forms having molecular weights of approximately 165kD, 155kD, and 120kD, respectively. These characteristics are developed in the experimental section later. In a second aspect, the invention relates to a method for purifying GTVap and separating glycosylated species therefrom, comprising the following steps: (a) extracting the GTVap from at least one source; (b) contacting the resulting GTVap extract with lectin affinity resin to absorb the glycosylated GTVap; (c) eluting the glycosylated GTVap from the lectin affinity resin; (d) contacting the resin eluate lectin affinity with a chelation chromatography resin, which includes a metal ion which interacts simultaneously with the resin and with the GTVap; (e) collecting unbound or unbound material from the chelation chromatography resin; (f) eluting the bound material from the chelation chromatography resin; (g) separately contacting the bound and eluted GTVap containing fractions of the chelation chromatography resin with anion exchange resin; (h) eluting the bound GTVap species from the anion exchange resin; and (i) detecting the GTVap species separated from the anion exchange resin. In the claimed method of GTVap purification and separation of the glycosylated species thereof, the step of extracting the GTVap from at least one source of biological origin is preferably carried out using a mixture of at least one metal ion stabilizer and a Detergent. As shown in the experimental section below, the activity of the enzyme decreases with time and in the absence of stabilizing agents. It has now been found that the calcium ion preferably at a concentration of 2 mM, serves as one of a potentially larger number of metal ions that serve to stabilize the activity of the protein. A variety of different detergents, non-ionic detergents can be used such as the Triton X-100 are preferred. The step of contacting the GTVap extract with lecithin affinity resin to absorb the glycosylated GTVap is carried out using any of a number of commercially available lecithins known to those skilled in the art for their ability to absorb glycoproteins. Those include Concanavalin A, wheat germ, Helix, common lentil lectins and Li ulus. Wheat germ lectin was used in this invention, others could have been used. Elution of the glycosylated GTVap from the lectin affinity resin was performed using any of a variety of methods to break the lectin-carbohydrate linkage. Examples include the use of a competent carbohydrate ligand, and varying the pH conditions. The chelation chromatography step was conducted by contacting the eluate of the lectin affinity resin with a chelation chromatography resin comprising a metal ion which interacts simultaneously with the resin and with the GTVap. There are numerous suitable commercially available chelation chromatography resins. Those resins are typically treated with metal ions which can interact with the protein or glycoprotein that is being purified, and with the resin. In the present invention it is preferred to use zinc as the metal ion, since it can interact with the GTVap and the iminodiacetate resin used. The elution The attached material of the chelation chromatography resin is typically carried out by adding a chelating agent such as EDTA or changing the pH. Preferably, conditions are used that do not alter the intrinsic activity of the enzyme. The step of separately contacting the bound and eluted GTVap containing fractions of the chelation chromatography resin with anion exchange resin can be carried out using a variety of different supports containing functionally active ligands known to those skilled in the art. . The process of this invention preferably uses a resin such as Resource Q which has a high resolution capacity and excellent flow rates when run using rapid liquid chromatographic procedures. The elution of the bound GTVap species from the anion exchange resin is preferably achieved by the use of an augmenting amino gradient, or alternatively, by changing the pH of the buffer. The step of detecting the GTVap species separately from the anion exchange resin is carried out by measuring the enzymatic activity. The activity of the GTVap can be detected using any number of substrates, including those derived from p-nitroanilide or ß-naphthylamide amino acid as well as native protein substrates such as insulin. Other substrates could also be used. This procedure is useful for the purification of the enzyme, which in turn also allows its further characterization, as well as the preparation of the antibodies for the enzyme, and the identification of the modulators of the activity of the enzyme. In a third aspect, the invention relates to a method for identifying the modulators of GTVap activity, comprising the following steps: (a) provides the GTVap or the material containing GTVap having a testable amount of enzymatic activity; (b) incubating the GTVap or the material containing the GTVap with a test substance to be tested for its ability to modulate the activity of the GTVap; (c) adding a GTVap substrate; (d) verify the activity of the GTVap as a function of time; Y (e) determining the modulating effect of the test substance on the GTVap. With respect to the method for identifying modulators of GTVap activity, the step of providing the GTVap or the material containing the GTVap having a workable amount of enzymatic activity involves the use of biological tissues including but not limited to adipose tissue, tissues of skeletal muscle, cardiac muscle, spleen tissue, cell lineages derived from those materials, or recombinant sources made using methods known to those skilled in the art. Such tissues could typically be of human or rodent origin, although GTVap can also be derived from other species. Preferably, the human source of the enzyme is used. A recombinant agent could be, for example, the insertion of the gene encoding GTVap into a prokaryotic or eukaryotic cell line capable of transferring the inserted gene, resulting in the expression of an enzymatically active GTVap molecule. Such procedures are understood by those skilled in the art. The step of incubating the GTVap or the material containing the GTVap with a test substance to be tested for its ability to modulate the activity of the GTVap, the possible test substances could include the analogues of the aminopeptidase inhibitors, and Synthetic and natural test substances without prior known activity. The step of adding a GTVap substrate could typically involve the use of a synthetic aminopeptidase substrate such as leucine p-nitroanilide, or a synthetic or natural polypeptide substrate in which the N-terminal amino acids could be removed by the particular enzyme. Two examples of suitable polypeptides are insulin and synthetic insulin. The step of verifying the activity of the GTVap as a function of time is generally carried out by verifying the disappearance of the substrate or the appearance of the product of the enzymatic activity on the substrate. This is typically accomplished by spectroscopic or chromatographic means, although a wide variety of other techniques could be used for that purpose, depending on the individual case. The step of determining the modulating effect of the test substance on the GTVap is carried out by comparing the rate of substrate cleavage of the GTVap in the presence and absence of the test substance. In a fourth aspect the invention relates to an antibody specific for GTVap, produced using substantially pure GTVap or a fragment thereof. The antibody specific for GTVap can be produced as follows. Polyclonal antiserum to GTVap is produced by injecting intact GTVap or fragments thereof into any of a variety of host animals, such as rabbits, mice, goats and sheep. The antibodies produced in such host animals will be polyclonal in nature, in which case the specific antibodies can be partially purified by methods known to those skilled in the art. In addition, monoclonal antibodies can be produced by fusion of immunocompetent spleen cells with myeloma cells to produce a hybrid cell line that produces essentially monoclonal immunoglobulin. Under the current conditions of the art, such animals include mice and rats. The antibodies could find use in the detection and quantification of GTVap in biological materials. From In addition, such antibodies could be used for the purification of GTVap using the immunoaffinity methods known to those skilled in the art. In a fifth aspect, the present invention relates to a method for preparing proteins and peptides truncated at the amino terminus comprising the step of incubating a protein or peptide with GTVap. Regarding the method for preparing proteins and truncated peptides, the process is carried out by incubating the subject protein under conditions that increase the activity of the GTVap, that is, under conditions of neutral pH and in the presence of ions that increase or stabilize the activity of the GTVap. The truncated product is isolated from the reaction by any suitable separation procedure. The material that is truncated is insulin or any polypeptide with an amino terminal capable of being cleaved by GTVap. The method of preparation of truncated peptides and proteins was used to derive the novel pharmacologically active polypeptides. In a sixth aspect the invention relates to a method for determining GTVap in biological material, comprising the following steps: (a) preparing a specimen of biological material to optimally expose the immunoreactive epitopes; (b) incubating this specimen or an extract thereof with antibody specific for GTVap; (c) remove the unbound antibodies of the specimen; and (d) quantify the antibodies bound to the GTVap in the specimen. With respect to the method to determine GTVap in biological material, the step of preparing a specimen of biological material to optimize the exposure of the immuno-active epitopes is carried out by washing with buffer or treating under more chaotropic conditions, which will expose the epitopes that are buried or masked in the native conformation of the protein. Such chaotropes could include solutions containing acetone or alcohol, SDS, and other denaturants with similar properties. These preparative methods allow detection of GTVap either native or denatured. The step of incubating the specimen or an extract thereof with the antibody specific for GTVap is carried out in a normal manner, and preferably employs the GTVapl antibody of the present invention. The antibody specific for GTVap can be directly labeled, with fluorophores, biotin, or radioisotopes, or an enzyme such as alkaline phosphatase or horseradish peroxidase. The quantification of the antibodies bound to the GTVap in the specimen is carried out by identifying the aforementioned marks. Alternatively, the bound antibody can be detected using secondary antibodies which are themselves marked and that will bind specifically to the primary antibody, which in turn binds to the GTVap. In a seventh aspect the invention relates to an oligonucleotide probe specific for a nucleic acid sequence encoding a segment of GTVap. Examples of oligonucleotide probes for an Nucolic acid sequence encoding a segment of the GTVap are the following materials, which are shown below in conjunction with the amino acid sequences identified in the GTVap, referenced in the Table. I: Peptide: Phe-Ala-Ala-Thr-Gln-Phe-Glu-Pro-Leu-Ala-Ala (SEQ ID NO: 1) Which corresponds to the oligonucleotide "Sense": 5'TTY GCN GCN ACN CAR TTY GAR CCN YTN GCN GCN 3 '(SEQ ID NO: 3) Which corresponds to the "Antisense" oligonucleotide: 5 'NGC NGC NAR NGG YTC RAA YTG NGT NGC NGC RAA 3' (SEQ ID NO: 4) Peptide: Ile-Leu-Gln-Asn-Gln-Ile-Gln-Gln-Gln-Thr-Arg-Thr-Asp-Glu-Gly (SEQ ID NO: 5) Which corresponds to the oligonucleotide "Sense": 'ATH YTN CAR AAY CAR ATH CAR CAR CAR ACN MGN ACM GAY GGN 3' (SEQ ID NO: 6) Which corresponds to the oligonucleotide "Antisense": 5 'NCC YTC RTC NGT NCK NGT YTG YTG YTG DAT YTG RTT YTG NAR DAT 3 '(SEQ ID NO: 7) In the above sense and antisense oligonucleotides the designation of a single standard letter was used for the individual bases and the letter N designates the degenerate positions. N is A, T, C, G, or I (inosine); R is A or G; And it is C or T; M is A or C; K is G or T; S is C or G; W is A or T; H is A, C, or T; B is C, G, or T; V is A, C, or G; and D is A, G, or T. Fragments of the above oligonucleotides and additional nucelotide replacements known to those skilled in the art constitute additional examples. These oligonucleotides can be used to determine the nucleic acids encoding GTVap in biological sources. In an eighth aspect, the invention relates to a method for determining a nucleic acid sequence encoding a segment of the GTVap in a biological specimen, comprising the following steps: (a) preparing a biological specimen for the analysis of the material of nucleic acid; (b) incubate the nucleic acid material of this biological specimen with an oligonucleotide or acid probe nucleic acid specific for a nucleic acid sequence encoding a segment of the GTVap; (c) removing the oligonucleotide or unbound nucleic acid probe from the nucleic acid material; and (d) determining the oligonucleotide probe bound to a nucleic acid sequence encoding a segment of the GTVap. With respect to the method for determining a nucleic acid sequence encoding a segment of the GTVap in a biological specimen, the step of preparing a biological specimen for the analysis of the nucleic acid material is carried out by lysis in detergent or in an alkaline medium, or through the use of chaotropic agents. This is followed by the purification of the DNA or RNA using affinity chromatography (this is useful for the mRNA, and uses oligo-dT to purify the mRNA), or by centrifugation in gradients in CsCl (useful for both DNA and RNA), or by the destruction of contaminating material using proteases (to destroy the protein) and specific nucleases (to destroy undesirable DNA or RNA). The material can be applied directly to a membrane, separated by gel electrophoresis, or further analyzed by restriction digestion before gel electrophoresis. The final material is immobilized on a membrane capable of binding to the nucleic acid. The steps of incubating the nucleic acid material of this biological specimen with a probe oligonucleotide specific for a nucleic acid sequence encoding a segment of GTVap, removing the unbound oligonucleotide probe from the nucleic acid material, and determining the oligonucleotide probe bound to a nucleic acid sequence encoding a segment of GTVap are brought to performed in the manner known to those skilled in the art. In a ninth aspect, the invention relates to a nucleic acid sequence encoding GTVap. More particularly, the invention relates to a purified nucleic acid consisting essentially of a nucleic acid encoding a GTVap having the amino acid sequence described in the sequences ID NOs 15 and 16, or a nucleic acid capable of hybridizing to the complement it under strict conditions and coding for a GTVap. Those are shown in Figure 20. It should be noted that some DNA clones include a series of AGC nucleotides which are absent in other clones, resulting in a GLN in certain of the resulting estimated proteins; this is shown in the boxes in Figure 20, and in the sequences identified as SEQ ID NO: 15 (which contain the insert) and SEQ ID NO: 16 (which does not contain the insert), which are the same execpto for this difference. This nucleic acid sequence can be used to identify or purify the sequence of nucleic acid coding for GTVap from biological sources using methods known to those skilled in the art. Oligonucleotides derived from the region of this nucleic acid sequence or capable of hybridizing to this sequence or its complement can be used to identify mutations that are present in this sequence in biological sources. The nucleic acid can also be inserted into a vector possessing a heterologous promoter and the recombinant protein expressed by inserting it into a cell. The recombinant protein may be the full-length GTVap or a mutein thereof. The preferred expression system is a homogeneous cell population, possessing a cloned nucleic acid encoding a GTVap or a mutein thereof having the amino acid sequence described in Figure 20 (SEQ ID NOs 15 and 16) or a nucleic acid capable of hybridizing to the complement thereof under stringent conditions and coding for a GTVap. Preferred cells are mammalian cells stably expressing the GTVap protein, although prokaryotic and other eukaryotic cells can also be used. A method for producing the GTVap comprises the step of culturing cells having a cloned nucleic acid encoding a GTVap as defined above in a medium to form a population of cells expressing GTVap, and purifying the GTVap from the cells of the medium. culture. The GTVap can be purified using the method described above or other methods known to those skilled in the art. Recombinant GTVap can be used as a source of enzymatically active aminopeptidase for research or therapeutic purposes. In a tenth aspect, the invention relates to a polypeptide which codes for the retention sequence of a protein, which is selected from the group consisting of a first polypeptide, which comprises the following residual amino acid sequence: -XLaa-Xaa- (Xxaa) 3- (Xaa) 2-3-Tyr-Glu- (Xaa)? 3-Ser- (Xaa) 0-? ~ X2aa- (Xaa) 0_4-Leu-Leu- SEQ ID NO: 20), and a second polypeptide, the inverse of the first, comprising the following sequence of residual amino acids: -Leu-Leu- (Xaa) 0-4-X2aa- (Xaa) 0 -? - Ser- (Xaa)? _3-Glu-Tyr - (Xaa) 2-j- (X1aa) 3-Xaa-X1aa- (SEQ ID NO: 21 *); wherein Xaa represents any residual amino acid, Xaa represents Pro, Glu, or Asp, and X2aa represents Arg or Lys, and muteins thereof. Figure 21 shows those retention and classification sequences of GTVap and GLUT4. Particular examples of such polypeptides are those in which the retention sequence is His Pro Leu Glu Pro Asp Glu Val Glu Tyr Glu Pro Arg Gly Ser Arg Leu Leu (SEQ ID NO 7); His Glu Met Asp Glu Asp Glu Glu Asp Tyr Glu Ser Be Ala Lys Leu Leu (SEQ ID NO 18); o Leu Leu Glu Gln Glu Val Lys Pro Ser Thr Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Glu (SEQ ID NO 19). The retention sequence represented by sequence ID No. 20 is found in the GTVap as the sequences ID NOs 17 and 18. The one represented by SEQ ID NO 21 can be found in GLUT4 as the sequence ID NO. 19. Those polypeptides (SEQ ID Nos 17-21) can be synthesized by chemical means known to those skilled in the art or expressed by recombinant means, and could serve as useful reagents for the identification of intracellular adhesion modulators. A method for identifying an insulin-sensitive retention sequence of a protein different from the GTVap whose classification is regulated by insulin comprising determining the amino acid sequence of the protein, comparing its sequence with the amino acid sequence of the SEQ polypeptide ID NO. 20 6 21, and identify an amino acid sequence of the protein that contains or is homologous to the amino acid sequence of the polypeptide (SED ID Nos. 20 or 21). Once such retention sequences have been identified in other proteins, these proteins are implicated as materials which are classified as GLUT4 or GTVap, and could thus be regulated by insulin. The invention also relates to a method for identifying an intracellular sorting protein or an active fragment thereof that binds to a sequence of insulin-sensitive retention of a second protein. This method comprises 1) incubating the intracellular sorting protein with the retention sequence under favorable conditions for binding between the sorting protein and the retention sequence and 2) identifying the specifically linked sorting protein. Such a method could involve immobilization of the retention sequence, followed by the addition of a cellular extract containing the intracellular sorting protein. Non-specifically bound proteins could be removed, then the intracellular sorting protein could be eluted and analyzed. An alternative method could involve the expression of the retention sequence as a bait in the "two hybrids" system and the selection of a library to identify a DNA encoding a protein that interacts specifically with the sequence. Such a protein could be the intracellular sorting protein. This material, in combination with the retention sequence, will allow the identification of the compounds that break their interaction. Accordingly, a method for identifying a compound capable of altering the intracellular classification of at least one of GLUT4 and GTVap comprises: a) incubating an intracellular sorting protein, or active fragment thereof with the polypeptide retention sequence of sequences ID NOos 20 21 low favorable conditions for binding between the sorting protein and the retention sequence in the presence and absence of a test compound; b) quantify the specifically bound complexes of the sorting protein and the retention sequence; and c) comparing the degree of complex formation in the presence and absence of the test compound. Such compounds could be useful for treating insulin resistance syndromes. The method for treating insulin resistance syndromes is related to the treatment of conditions such as diabetes, obesity, impaired glucose tolerance, hyperinsulinemia, and other conditions associated with hyperinsulinemia such as, but not limited to, the Syndrome X, hyperlipidemia, and hypertension. Doses and administration times could be according to standard protocols, with the main therapeutic objective of decreasing blood glucose levels, plasma glucose levels, control markers such as glycated proteins such as hemoglobin A? C, and insulin levels, such as, for example, (depending on the specific modulator) orally, transcutaneously or intranasally, or in an injectable form, subcutaneously, intramuscularly, or intravenously, or by other suitable means, and could have a mimetic activity of insulin or insulin-reinforcing activity. The method claimed may be used alone or in combination with other therapies for the treatment of insulin resistance and related syndromes. The additional details will be readily apparent without undue experimentation to those skilled in the art.

Materials and methods The following list of terms, sources, instruments, etc. is provided. to help the reader understand and reproduce the experimental work that will be presented below.

Abbreviations and descriptions: Cell membrane fractions: PM (plasma membrane), HDM (high density microsomes), and LDM (low density microsomes) are cell membrane fractions defined by their intrinsic density and sedimentation behavior during centrifugation and by their enrichment. particular enzymes that typify those membranes.

GTVap means aminopeptidase of Glucose Transport Vesicles. GTVapl refers to the rabbit monoclonal antibody produced from the peptide Phe-Ala-Ala-Thr-Gln-5 Phe-Glu-Pro-Leu-Ala-Ala- [Cys] [SEQ ID NO: 8] which reacts with the different forms of GTVap. KRBH buffer is 120 mM NaCl, 4 mM KH2P04, 1 mM CaCl2, 10 mM NaHCO3 and 30 mM HEPES, and has a pH of 7.4. RACE is Rapid Amplification of the Ends of 0 cDNA TBS (Tris saline buffer) is 10 mM Tris, 150 mM NaCl, 0.01% Thimerosal, pH 8.0 TBS-TW is TBS with 0.05% Tween 20. TES buffer (Tris-EDTA-sucrose) is 20 mM Tris, 5 mM EDTA, 250 mM sucrose, pH 7.5. SDS-PAGE is electrophoresis on sodium dodecyl sulfate - polyacrylamide gel. 0 Reagents: Actinonin was obtained from Sigma Chemical Co., St, Louis, MO. Amastatin was obtained from Sigma Chemical Co., St, \ i > Louis, MO. ? -aminohexyl Sepharose 4B was obtained from Sigma Chemical Co. , St. Louis, MO. The AmpliTaq DNA polymerase was obtained from Perkin Elmer, Norwalk, CT. Benzamidine was obtained from Sigma Chemical Co., St.

Louis, MO. Bestatin was obtained from Sigma Chemical Co., St. Louis, MO. BSA (Fraction V sero bovine albumin) was obtained from Sigma Chemical Co. St. Louis, MNO. Collagenase was obtained from Worthington, Freehold, NJ. Comassie blue G-250 and SDS (sodium dodecyl sulfate) were obtained from BioRad, Hercules, CA. Cyalasin B was obtained from Sigma Chemical Co., St. Louis, MO. DFP (diisopropyl fluorophosphate) was obtained from Aldrich, Milwaukee, Wl. EDTA is ethylenediaminetetraacetic acid and was obtained from Sigma Chemical Co., St. Louis, MO. Expanded thermostable DNA polymerase was obtained from Boehringer Mannheim, Indianapolis, IN. HEPES is N- (2-hydroxyethyl) piperazin-N '- (2-ethanesulfonic acid) and was obtained from Sigma Chemical Co., St. Louis MO.

The IAV matrix Immobilon (AV) matrix was obtained from Millipore Corp., Milford, MA IDAC means iminodiacetic acid chelating resin, obtained from Pierce, Rockford, IL. Insulin (porcine) was obtained from Eli Lilly, Indianapolis, IN. Leupeptin was obtained from Sigma Chemical Co. St. Louis, MO. L-leuciintiol was obtained from Sigma Chemical Co., St. Louis, MO. Team Marathon RACE was obtained from Clontech, Palo Alto, CA. The oligonucleotides were obtained from Midland Certified Reagent Company, Midland, TX. Pepstatin A was obtained from Sigma Chemical Co. St.

Louius, MO. PMSF (phenylmethylsulfonyl fluoride) was obtained from Sigma Chemical Co., St. Louis, MO. The pruning membranes were obtained from Applied Biosystems, Foster City, CA. PVDF membrane is polyvinylidene difluoride, Millipore Corp., Bedford MA. The RACE cDNA Ready from rat heart (7383-1) was obtained from Clontech, Palo Alto, CA.

The rat skeletal muscle cDNA library (RL3003b) was obtained from Clontech, Palo Alto, CA. PolyA + rat skeletal muscle RNA was obtained from Clontech, Palo Alto, CA. The anion exchange resin of Q contained quaternary ammonium groups on rigid polystyrene / divinylbenzene beads of 15 μm in diameter and was obtained from Pharmacia, Piscataway, NJ. The thermostable DNA and RNA dependent on the Retrotherm DNA polymerase was obtained from Epicenter Technologies, Madison, Wl. The sequenase kits were from United States Biochemical, Cleveland, OH. Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) was obtained from Aldrich, Milwaukee, Wl. The sMBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester) was obtained from Pierce, Rockford, IL. Streptavidin was obtained from Zy ed Corp., South San Francisco, CA. The columns of supelcosil LC-18-DB, 3μm, 2.1x250mm was obtained from Supelco, Corp., Bellefonte, PA. TEA (triethylamine) was obtained from Pierce, Rockford, IL. The 3 'RACE equipment was obtained from Clontech, Palo Alto, CA.

TFA (trifluoroacetic acid) was obtained from Applied Biosystems, Foster City, CA. Thimerosal is the ethyl mercuryothiol sodium salicylate available from Sigma Chemical Co., St. Louis, MO. Tris means tris hydroxyaminomethane. Triton X-100 was obtained from Sigma Chemical Co. , St. Louis, MO. Trypsin (modified sequence degree, porcine) was obtained from Promega, Madison, Wl. WGA stands for wheat germ licitin-sepharose 6MB, which was obtained from Pharmacia, Piscataway, NJ.

Radioactively labeled materials and flashing fluids 125I-protein A (2-10 μCi / μg) was used for Western blot analysis. All isotopes and flashing fluids (Aquasol) were from New England Nuclear, Boston, MA. or Amersham Corporation, Arlington Heights, IL.

General Methods of Peptide Synthesis All peptides including the 15 terminal amino acids COOH terminal of the rat GLUT4 sequence (495-509; [Cys] -Lys-Pro-Ser-Thr-Glu-Leu-Glu-Tyr-Leu-Gly-Pro- Asp-Glu-Asn-Asp (SEW ID NO: 9)) and the fragments thereof (Lys-Pro-Ser-Thr-Glu-Leu-Glu- [Cys] (SEQ ID NO: 10); Thr-Glu -Leu-Glu-Tyr-Leu [Cys] (SEQ ID NO: 11); [Cys] -Gly-Pro-Asp-Glu-Asn-Asp (SEQ ID NO: 12); the peptide GTVap (Phe-Ala) -Ala-Thr-Gln-Phe-Glu-Pro-Leu-Ala-Ala- [Cys] (SEQ ID NO: 8)) and the insulin-derived peptides were synthesized using the solid phase methods in the peptide synthesizer 430A previously described in US Patent No. 5,225,354 to Knowles and Marchesi.All peptides were synthesized with a cysteine residue in the -NH2 or -COOH-thermally identified as [Cys] in the sequence and were covalently linked to? -aminohexyl sepharose 4B or a flourophore to verify the proteolytic cleavage of the insulin-derived peptides The peptides were labeled with sulphydral-specific fluorescein derivatives In this procedure a double molar excess of fluorescein-5-maleimide in dimethyl formamide (40 mg / ml) was added. ml) to the peptide (10mg / ml) in 100mM sodium phosphate, 5mM EDTA, pH 7.1 and incubated for 20 hours at room temperature. The resulting peptide-fluorescein conjugate was purified by CLAP chromatography as described by Knowles and Marchesi, US Patent No. 5,225,354.

Instrumentation The Alia Protein Sequencer, the 120A amino acid analyzer and the 430A peptide synthesizer were from Applied Biosystems, Foster City, CA. The 125I was measured in a Wallac Counter (LKB) 1272 Clinigama. The collection of the CLAP data and the analysis of the tryptic digestion of the 155kD and 165kD proteins was carried out in a Nelson Turbochrome System (Perkin-Elmer Nelson, Norwalk, CT). Mass spectrometry for mass analysis of synthetic peptides and insulin fragments was performed using a laser desorption mass spectrometer Maldi 3 (LD-TOF-MS), with saturated synapinic acid, 0.1% TFA, 50% acetonitrile as the matrix .

General method for protein determination Protein amounts were determined using the bicinchoninic acid (BCA) method as described by the manufacturer (Pierce, Rockford, IL.) Immunoaffinity purification of anti-peptide antibodies Anti-peptide antibodies to GLUT4 and GTVap were purified by immunoaffinity absorption before use in Western blotting or for purification of GLUT4 vesicles. For this purpose, the peptide immunogens containing a single cysteine were coupled to the Sepharose, as described by the following method. The? -aminohexyl Sepharose 4B resin was washed with 50mM Na2P04, lOmM EDTA, pH 7.0. The resin was suspended in a 50% suspension and sMBS (2μmol / ml resin) was added. 10 minutes later at room temperature, the derivatized resin was washed in 50mM Na2P0, 10mM EDTA, pH 7.0, and resuspended in a 50% suspension. The peptide containing cysteine (1-2 g of peptide / ml) was added and allowed to react overnight at room temperature. The resulting peptide-resin conjugate was washed with 0.1M acetic acid and then 50mM Na2P04., EDTA lOmM, pH 7.0 before use. For immunoaffinity purification, the polyclonal antiserum for the individual peptides was diluted 1: 1 with 100mM NaH2P04, 150mM NaCl, pH 7.5 and applied to the corresponding peptide-resin. After a brief wash with the above buffer, the antibody bound by affinity bound with acetic acid was eluted 0. 1M the pH was immediately adjusted to pH 8.0 with Tris 1. OM basic. The purified antibody was dialyzed against and stored in sodium borate 0.1M pH 8.0, with a content of 0.5% sodium azide.

Western blot analysis Prior to Western blot analysis, the proteins were electrophoresed on SDS-PAGE gels (Laemmli, Nature, 277, 680-685, 1970) and transferred to PVDF or Problot membranes by the method of Tobin, et al, PNAS , 76, 4350-4354, 1979. For Western blot analysis of GLUT4 and GTVap, rabbit anti-GLUT4 antibody, purified by immunoaffinity as above, was used at a 1: 5000 dilution (lμg / 5ml); the antibody purified by affinity for GTVap was used at 1: 1000 (lμg / ml). For the detection of the primary antibody, both protein A 125I (lμCi / ml) and goat anti-rabbit alkaline phosphatase (1: 10,000; Promega) were used sequentially. The immunoreactive bands were identified by the proo. of the reaction of the colored alkaline phosphatase and / or autoradiography of the 125I-Protein A, and were quantified by counting 125I-Protein A in a LKB 1272 Clinigamma counter.

Experimental Part and Results: Generation of specific antibodies for GLUT4 A polyclonal rabbit antibody was produced for GLUT4 using a synthetic peptide corresponding to the 15 residual COOH-terminal amino acids of the rat GLUT4 sequence, ie residues 495-509, [Cys] -Lys-Pro-Ser-Thr-Glu-Leu-Glu- Tyr-Leu-Gly-Pro-Asp-Glu-Asn-Asp (SEQ ID NO: 9). The specificity of the antibody to GLUT4 was increased by absorbing the polyclonal serum with 3 peptides (Lys-Pro-Ser-Thr-Glu-Leu-Glu- [Cys] (SEQ ID NO: 10); Thr-Glu-Leu-Glu-Tyr -Leu- [Cys] (SEQ ID NO: 11); [Cys] -Gly-Pro-Asp-Glu-Asn-Asp (SEQ ID NO: 12) covalently linked to γ-aminohexyl Sepharose 4B using the heterobifunctional reagent sMBS The specific antibody for non-absorbed GLUT4 was purified on a 495-509 peptide column and then on Sepharose Protein A. The highly purified and specific GLUT4 antibody was used for Western blotting at a 1: 5000 dilution (lμg of protein / 5 ml of diluent).

Preparation and isolation of rat adipocytes The epididymal fat clusters of 125 g Sprague-Dawley rats were removed and immediately placed in KRBH buffer containing 1% BSA, 2.5mM glucose and 200nM adenosine. The fat was comminuted, collagenase was added at 3 mg / ml, and the mixture was incubated at 37 ° C for 45 minutes with shaking. The dissociated adipocytes were filtered through a 250 micron nylon mesh and washed three times (using moderate centrifugation) with KRBH containing 1% BSA and 200 nM adenosine. The cells were suspended in the wash buffer containing 3% BSA.

Fractionation of rat adipocytes The membrane, PM, HDM and LDM fractions were prepared in a general manner according to Simpson et al., Biochimica et Biophysica Acta, 763, 393-407 (1983). The isolated adipocytes were homogenized in a Wheaton 55ml Teflon pistil homogenizer cooled by 10 strokes. The fat layer was removed after centrifugation at 10,000Xg and the remaining material was rehomogenized. The PM fraction was pelleted at 16,000Xg and the supernatant was recentrifuged to remove any additional PM. The PMs were resuspended and rehomogenized and then purified from mitochondrial DNA and organelles by ultracentrifugation through a 1.12M sucrose pad at 96,000Xg. The MWs were collected from the sucrose pad interface. The HDM sedimented from initial supernatant at 48,000Xg and the resulting supernatant was recentrifuged again to remove any remaining HDM. The LDM was collected on a 1.12M sucrose pad of the HDM supernatant by centrifugation at 212,000Xg. The LDM was collected from the sucrose interface and this step was repeated three times with the LDM collected from additional tubes to concentrate the LDM and remove any contaminating cytosolic proteins.

Immunopurigestion of GLUT4 vesicles The purified GLUT4-specific antibody prepared as above was biotinylated using NHS-biotin in 50mM NaHC03, pH 8.2, for 30 minutes at room temperature. After dialyzing against 20mM Tris, lmM EDTA, pH 8.0, the active biotinylated antibody was recovered by affinity purification on [Cys] -Lys-Pro-Ser-Thr-Glu-Leu-Glu-Tyr-Leu-Gly-Pro- , Asp-Glu-Asn-Asp (SEQ ID NO: 9) covalently linked to inohexyl-Sepharose CL-4B as described above. The immobilon affinity membranes (IAV, 1 cm2) were saturated for 6 hours at room temperature and then overnight at 4 ° C with 500 μg of streptavidin in 0.5M KH2P04, pH 7.5. The residual sites were crowned with 0.44M glutamic acid (6 hours, temperature environment) and the membranes were washed. The biotinylated anti-GLUT4 antibody (50μg / cm2) described above was added in 100mM boric acid, 150mM NaCl, lmM EDTA, pH 7.5, and kept in contact for 6 hours at room temperature. The IAV matrix derived from the resulting antibody was washed 3 times (20 minutes / wash) in TBS, 0.1% Tween 20, and then transferred to TES. The control IAV matrix was prepared with streptavidin and was capped with glutamic acid. LDM was added to the GLUT4 or control IAV matrices. Typically, LDM of 2.4 rats were added to each 1 cm2 of matrix, and incubated overnight at 4 ° C in TES with a 150 mM NaCl content, and subsequently washed 3X in the same buffer. Unbound vesicles were recovered by centrifugation at 212,000xg. The bound vesicular protein was recovered by eluting the matrices with 1.0% SDS in Tris-HCl lOmM, pH 7.4, or in 0.1% Triton X-100 in 20mM Tris, lmM EDTA, pH 7.4. GLUT4 vesicle proteins were frozen in liquid N2 and stored at -80 ° C until further use. Equal volumes of purified GLUT4 vesicles and controls were lyophilized, along with equal protein loads of the recovered unbound LDM vesicles and loaded onto 4-20% SDS-PAGE gels, transferred to PDVF membranes, and stained with Coomassie blue R-250. HE characterized a protein that had a molecular weight of approximately 165kD as specific for GLUT4 vesicles and was partially or completely impoverished from the unbound fraction. Figure 1 qualitatively shows by means of Western blot autoradiography that immunoaffinity absorption using the IAV matrix derived from antibody to GLUT4 results in a specific enrichment of the GLUT4 vesicles compared to the IAV matrix lacking the antibody to GLUT4. The quantification of 125I Protein A indicates an 8-fold enrichment of GLUT4 in purified vesicles by immunoaffinity. Immunoaffinity-purified GLUT4 vesicle proteins can be solubilized using 0.1% Triton X-100 or low concentrations (<1%) of SDS, leaving the antibody bound to the covalently bound streptavidin, to produce a much more protein composition. simple than the total LDM fraction. Figure 2 shows that Coomassie blue staining of the GLUT4 vesicle associated proteins after SD-PAGE by a 165kD and 155kD protein which are uniquely associated with GLUT4 vesicles and not found in the proteins absorbed on the IAV matrix lacking antibodies to GLUT4. Both 165kD and 155kD proteins were depleted of the LDM after absorption with the IAV matrix of GLUT4, impoverished after absorption on the control matrix.

Purification and sequence analysis of the 165kD and 155kD proteins The GLUT4 vesicle proteins were electrophoretically separated on 7-15% acrylamide gels, 20 cm SDS-PAGE, and transferred to Problot membranes. The 165kD and 155kD proteins were identified by Coomassie blue staining. Initial attempts at direct terminal NH2 sequencing of the Problot membrane indicated that the terminal NH2 was blocked. In the subsequent experiments the 165kD and 155kD proteins were separated and identified as previously done, and then digested with trypsin (30: 1).; substrate / enzyme) according to Fernandez et al., Analyt. Biochem., 201, 255-264 (1992). The resulting peptide products were separated on a Supelcosil LC-18-DB, 3μm, 2.1 × 250mm column using a linear gradient of 0.1% TFA to 0.1% TFA, 70% acetonitrile for 3 hours. The effluent was verified at 215 nm and the selected peaks were subjected to sequencing using an ABI 477a Protein Sequencer operated in the gas phase with a 120A online analyzer and a Nelson Turbochrome PE Program. Figure 3 shows the separations by CLAP of the tryptic fragments and indicates the significant similarities between the two proteins. Peptides selected from moderate hydrophobicity and strong UV absorbance were sequenced. The sequences obtained are shown in Table 1. Both peptides were sequenced from the 165kD and 155kD forms and were identical, constituting additional evidence of the requests between these two proteins.

Identification of the GLUT4 vesicle protein of 165kDa as an aminopeptidase The obtained primary sequence was used to search DNA databases including GenBank, EMBL, Nucleic, and GeneSeq, and the PIR, PatchX, and SwissProt protein database. A peptide having 100% homology was found with a triptych fragment of the seroaminopeptidase with that of placental origin in the GeneSeq database. See Table 1.

Table 1 Summary of the sequence data for GTVap-165 and GTVap-155 The underlined residues of fragment # 1 were used to produce high titer rabbit polyclonal antiserum. The underlined residues of fragment # 2 had a 100% homology with a placental aminopeptidase (PLAP) identified above. * Designates the same sequence found in both GTVap-165 and GTVap-155.

Residues in lower case letters denote less solubility, and were assigned the designation Xaa in the claims.

The peptide Phe-Ala-Ala-Thr-Gln-Phe-Glu-Pro-Leu-Ala-Ala- [Cys] (SEQ ID NO: 8) was used to produce polyclonal rabbit antibodies which were subsequently purified by affinity as described above. Those antibodies were referred to as GTVapl and Western blot analysis was used at a 1: 500 dilution particle. Figure 4 shows that affinity purified GTVapl antibodies in Western blot analysis are specific for 165kD and 155kD proteins on GLU.T4 vesicles and also react with a 120kD protein which is less abundant than the first two. . In relation to the controls, purified GLUT4 vesicles are significantly enriched in immunoreactive 165kD and 155kD proteins, as measured by Western blotting. The quantification of 125I Protein A indicated an 18-fold enrichment of the 165kD and 155kD proteins on the purified vesicles by immunoaffinity. To demonstrate the identity of the 165kD and 155kD proteins after removal of the oligosaccharide side chain, the LDM was treated with N-glycosidase F. The LDM purified was solubilized in 0.5% SDS and then buffered in 25mM Hepes, 10mM EDTA, 1.7% β-octylglucoside, pH 7.5 before being digested with 1 unit of N-Glycosidase F (PNGase F) per sample for 12 or 24 hours. hours at 37 ° C. Controls (samples lacking glycosidase) were included for each condition. Figure 5 indicates that the treatment of LDM with N-glycosidase F results in the conversion of both immunoreactive proteins of 165kD and 155kD with a single immunoreactive protein of HOkD. Therefore it is likely that both forms are identical at the protein level with the differences in the number and / or length of oligosaccharide side chains of the glycosylated 165kD and 155kD forms.

The 165kD and 155kD proteins have sequence homology with a previously identified aminopeptidase, and the GLUT4 vesicles are rich in aminopeptidase activity As indicated in Table 1, the estimated sequence of fragment # 2 had a 100% homology with a previously reported plasmid aminopeptidase of placental origin (Tsujimoto et al., EPA Pub # EP 0 535 241 Al).

The synthetic substrate leucine p-nitroanilide was used to confirm that the activity of the aminopeptidase was purified with the GLUT4 vesicles. The GLUT4 vesicles purified by immunoaffinity or the vesicles bound non-specifically to the control IAV membranes were extracted with 20mM Tris, Triton X-100 0.1%, pH 7.5. Equal volumes of extract from the two matrices were assayed for aminopeptidase activity using 1.6 mM leu p-nitroanilide. The results, shown in Figure 6, indicate that the GLUT4 vesicles contain 5 times more aminopeptidase activity than the control IAV matrix. Therefore, the activity of the aminopeptidase co-purifies with the GLUT4 vesicles. Through this application the term Glucose Transport Vesicle aminopeptidase or GTVap is used to designate aminopeptidases associated with GLUT4 vesicles.

Identification of GTVap in membranes in PM and HDM As shown in Figure 7, the GTVap are also found in the PM and HDM fractions by Western blot analysis using the GTVapl antibody. GTVap-165 and GTVap-155 are the predominant forms in HDM and LDM, while those GTVap in PM are much less abundant.

General methods for extracting and testing GTVap from PM, LDM, and HDM membranes The GTVap can be extracted to the maximum (> 95%) of the membrane compartments of LDM, HDM, and PM using low concentrations of detergents. Unless otherwise indicated, the GTVap were solubilized from the GLUT4, LDM, HDM and PM vesicles using 0.1% Triton X-100 in 20mM Tris, pH 7.5 at 4 ° C for 15 minutes. The insoluble material was sedimented at 20,000 X g for 20 minutes and discarded. The enzyme extract (10-20μg of protein) was assayed using 1.6 mM leucine p-nitroanilide in Tris-HCl 20mM, Triton X-100 0.1%, pH 7.5, in a volume of 250μl at 37 ° C. The absorbance of the p-nitroanilide product was measured at 405 nm and quantified by comparison with p-nitroanilide standards. The results were reported as pmoles of p-nitroanilide produced per μg protein / hour at 37 ° C unless otherwise indicated. Alternatively, the results were presented as the change in OD at 405 nm if the protein was too low to be determined (for example, column fractions) or a relative comparison was made.

As shown in Figure 8, PM, LDM, and HDM contain GTVap activity. Although the GLUT4 vesicles showed initially to have aminopeptidase activity, it was necessary confirm that the 165kD and 155kD proteins were actually aminopeptidases. Due to the limited amount of protein obtainable from the purified GLUT4 vesicles or LDM, it was necessary to characterize and purify the HDM enzyme.

Characterization of the activity of the enzyme GTVap To characterize the relative reactivities of GTVap for different amino acid substrates p-nitroanilide, an HDM extract was prepared as described above and incubated with each substrate at a concentration of 1.6mM as described above. As shown in Table 2, the relative activity of GTVap to the synthetic p-nitroanilide amino acid substrates are leucine »proline, alanine > Valine, glycine. To characterize the relative activities of the GTVap to the different amino acid-ß-naphthylamide substrates, a GLUT4 vesicle extract was prepared as described above and each substrate was incubated at a concentration of 200uM as described above. As shown in Table 2A, the relative activity of the GTVap to the synthetic amino acid-ß-naphthylamide substrate are leucine > Lysine > arginine > methionine > Alanine > phenylalanine To determine the optimal pH of GTVap activity, an HDM extract was prepared as above and diluted 1: 9 in 100mM Tris basic, 0.1% Triton X-100 which had previously been adjusted to the values of pH indicated with HCl. After incubation for 15 minutes, a substrate of leucine p-nitroanilide was added and the activity of the enzyme was determined as above. The results shown in Figure 9 indicate that the enzyme had a broad neutral optimum pH.

Table 2 Relative activity of GTVap with various amino acid-p-nitroanilide substrates Amino-acid-p-nitroanilide nols / μg protein / hr leucine 2.9 0.35 proline Alanine 0.33 Valine 0.09 glycine 0.08 Table 2A Relative activity of GLTVap of GLUT4 vesicles with various amino acid-ß-naphthylamide substrates Amino acid-ß-naphthylamide nmoles / 25uL extract / hr leucine 9.5 plant 7.9 arginine ~ 6.5 methionine 2.9 alanine 1.3 phenylalanine 0.9 To determine the temperature stability of the GTVap, the HDM extracts were incubated at various temperatures for 20 minutes before the enzyme analysis as described above. The results shown in Figure 10 indicate a temperature-dependent inactivation with a 25% reduction in activity between 20 ° C and 40 ° C. Between 40 ° C and 50 ° C there is an even greater drop in activity, resulting in a 75% inhibition compared to that of incubation at 20 ° C.

The known aminopeptidases require ion bonds for optimal stabilization of the structure and maximum enzymatic activity. To explore the ionic requirements for the GTVap, the extracts of the HDM with Triton X-100 were incubated with several ions for 15 minutes at 37 ° C before the addition of the substrate leu-p-nitroanilide. The results, shown in Figure 11, demonstrate that particular ions can both activate and inhibit enzymatic activity. It is also evident that the enzyme has significant activity (approximately 55% of maximum activity) without the addition of divalent cations. Zinc, cobalt and manganese optimally activate at μmolar concentrations and inhibit immortal concentrations. Calcium and magnesium also partially activate, but at high mmolar ion concentrations. The lithium and potassium ions have no effect on the enzymatic activity between lμm and lOmM. It was evident during the initial investigations of the GTVap that the substrate of Triton X-100 loses enzymatic activity with time at 4 ° C. An attempt was made to stabilize the enzymatic activity by adding the specific ions that previously showed to activate the enzyme. Immediately after the HDM extraction routine the extracts were diluted in 20mM Tris-HCl, pH 7.5 containing the DTT ions at concentrations previously shown increase enzymatic activity. At several times before at 4 ° C, aliquots were removed and the activity of GTVap was determined. The results, shown in Figure 12, indicate that calcium stabilizes enzyme activity against time-dependent inactivation. Zinc, cobalt and magnesium, which previously showed increased activity, do not stabilize the enzyme. A number of protease inhibitors were studied to examine their inhibitory effect on GTVap. Enzyme determination methods were described above with the exception that various concentrations of the inhibitors were added to the HDM enzyme extracts 15 minutes before the addition of the leu-p-nitroanilide substrate. The data were presented in relation to the inhibitor-free control which had a 100% activity. The following inhibitors, with the maximum tested concentration shown in parentheses, had no effect on GTVap activity using the leucine-p-nitroanilide substrate: diisopropyl fluorophosphate (5mM), PMSF (lmM), benzamidine (lOmM), leupeptin (5mM). ), EDTA (5mM). It was found that the zinc chelators, phenanthroline and dipyridyl, were > 98% inhibitors a > 0.2mM, as shown in Figure 13. Leu-Leu-Phe-chloromethyl ketone (LLPAC), a known inhibitor of calpain, has an IC5o of GTVap of 1 mM. Dithiothreitol (DTT) has a 30% inhibition at 5 mM but increases the enzymatic activity by 20% at 0.2 mM. A number of aminopeptidase inhibitors identified above were tested on GTVap. The most effective inhibitor was leutiol with a CI5o of 2 uM. Amastatin has a CI5o of 0.35 mM and bestatin and actinonin had an IC50 of > lmM As shown in Figure 14.

Purification of HDTV 165, 155 and 120 GTVap The strategy for the purification of the enzymes of GTVap was formulated based on the activation of the enzyme relatively fast time dependent. The purification scheme employs two affinity purifications (WGA and IDAC) followed by anion exchange chromatographic separation. Calcium ions were used to maintain optimal stability when possible. The purified HDM at a concentration of 1 mg / ml was solubilized in 20 mM Tris-HCl, 0.1% Triton X-100 with a content of 2 mM CaCl 2, 150 μM PMSF, 1 mM DFP, 1 M idine benza, leupeptin 2 μM, and pepstatin A l μM. It was found that this extraction solubilizes 96% of the total aminopeptidase activity and 77% of the total protein of the HDM fraction. The insolubilized membrane was sedimented to 48, 000Xg for 20 minutes and discarded. The solubilized extract was incubated in batches with wheat germ agglutinin (WGA) -Sepharose. In particular, 1 ml of wheat germ lectin-packed Sepharose 6MB resin, referred to as WGA, was added per 10 mg of HDM protein and kept rotating overnight at 4 ° C. The resin was washed with the above buffer CaCl2 and eluted with 5.0 ml of 0.5 M N-acetylgucosamine. Approximately 93% of the total aminopeptidase activity was bound and could be eluted from the WGA column using 0.5 M N-acetylglucosamine. The fraction purified with WGA could be applied directly to the Resource Q column of anion exchange. Typically, the entire fraction purified with WGA was applied to 1 ml of a Resource Q anion exchange resin column at 4 ° C. A linear gradient of 0-0.5 M NaCl in 20 mM Tris, 0.1% Triton X-100, pH 7.8 at 4 ° C was used for the elution. All fractions were tested for aminopeptidase activity and for Western blot reactivity with the GTVapl antibody. As shown in Figures 15a and 15b, this ion exchange method separates the 120 kD immunoreactive protein from the forms GTVap-155 and GTVap-165 kD. The Western blot profile of Resource Q using identifications with GTVapl antibodies of the 120 kD protein and the GTVap-155 kD and 165 kD proteins shows a excellent correlation with the activity of the aminopeptidase. The first acute elution peak of the Resource Q column is GTVap of 120 with the largest peak of subsequent elution being first that of GTVap-155, followed closely by the GTVap of 165. Alternatively, the fraction purified with WGA it could also be applied directly to a column of iminodiacetic acid preloaded with zinc. In particular, the fraction purified with WGA was passed immediately downwards from an iminodiacetic acid chelation column (IDAC) of 0.5 ml, previously loaded with ZnS0410 mM in 20 mM Tris. Triton X-100 0.1%, pH 7.5, according to the manufacturer's recommendation. After application of the WGA eluate, the column was washed with 10 column volumes of 20 mM Tris, 0.1% Triton X-100, pH 7.5. The column was then eluted with 10 mM EDTA in 20 mM Tris, 0.1% Triton X-100, pH 7.5. Approximately 93% of the aminopeptidase activity was bound to the IDAC and could be eluted with 10 mM EDTA. The fractions that flowed through the IDAC and that were bound to the IDAc were subjected to separate chromatography on 1 ml of a Resource Q anion exchange resin as described above. The results, shown in Figure 16, show that the IDAC column loaded with zinc binds all the GTVap-120, while the Most of the GTVap-155 and GTVap-165 flow through it. This observation was confirmed by the Western blot analysis of those fractions (data not shown). Since zinc binding probably illustrates the coordination of the histidyl residues of the protein with the bound zinc ions, it may be that the heavily glycosylated GTVap may be spherically prevented from binding to the immobilized zinc. The fraction of GTVap-155 and GTVap-165 that binds to zinc columns can be dimerized with GTVap-120 and may not interact directly with zinc. Previous biophysical studies on other aminopeptidases suggest that these enzymes may exist as dimers. In summary, GTVap-120, GTVap-155, and GTVap-165 are enzymatically active, all react with the GTVapl antibody and can be enriched and separated by the purification scheme described above.

GTVap translocates to the plasma membrane in response to insulin It is known that GLUT4 translocates from an intracellular vesicular compartment to the plasma membrane after stimulation with insulin. To determine if GTVap also translocates, they were stimulated with insulin adipocytes dissociated from pieces of rat epidimal fat prepared as described above. Insulin (10 nM) was added to the recently prepared adipocytes which were kept for 20 minutes at 37 ° C. The adipocytes were then washed in TES buffer. The insulin-stimulated and control adipocytes were fractionated in the subcellular compartments and analyzed for the enzyme GTVap (Western spotting) as described above. Figure 17 indicates that GTVap translocates to the plasma membrane in response to insulin stimulation as evidenced by increased reactivity by Western blots. Figure 18 indicates that the enzymatic activity of GTVap increases to 29% in the plasma membrane after insulin stimulation.

Escrow of the GTVap of insulin and synthetic peptide substrates HDM (1.8 mg / ml) was incubated at 37 ° C with 120 μg / ml porcine insulin in 50 mM sodium borate, pH 7.5, containing the following proetase inhibitors that previously showed inhibitory effect on GTVap: 2 mM benzamidine, leupeptin 2 uM, 1 μM pepstatin, 1 mM DFP, and 150 μM PMSF. The reaction was finished after several times of incubation by centrifugation of aliquots at 200,000Xg to remove the HDM. The products of the insulin were detected by mass spectrometry or were first subjected to chromatography on a Beckman Spherisorb C 18 column of 2.1x15 to 0.25 ml / min. in a linear gradient of acetonitrile from 27% to 31% in 1% TFA adjusted to pH 3.0 with TEA for 4 hours, before the mass spectrometry analysis. Using this method both intact HDM and extracted with Triton X-100 showed to have aminopeptidase activity towards insulin. As shown in Figures 19a, b, and c, the mass spectroscopic analysis indicated that the N-terminal residual phenylalanine and valine of insulin subunit B were sequentially removed from the intact insulin molecule.

Identification of a nucleic acid sequence coding for GTVap Two degenerate oligonucleotide collections were obtained, one of which encoded for the first 7 amino acids of the peptide and the other of which encoded for the reverse complement of the last 7 amino acids. Those oligonucleotide collections were used to effect the polymerase chain reaction with AmpliTaq DNA polymerase on the rat skeletal muscle cDNA generated from polyA + rat skeletal muscle RNA using the 3 'RACE kit, or by reverse transcription followed by the chain reaction of the polymerase using the retro-polar DNA polymerase on polyA + rat skeletal muscle RNA. From those reactions, short regions of the unambiguous DNA sequence coding for the peptides on which the oligonucleotides were based are obtained. The use of those unambiguous nucleic acids labeled with 32P as probes, a cDNA library was selected from • Rat skeletal muscle gtll lambda and 25 clones that code for GTVap were identified. Three of these, identified as 5.3, 10.1, and 12.1, were sequenced with a final sequence based on the sequence of clone 12.1 since both clones 5.3 and 10.1 contained introns. Based on the nucleic acid sequence near the 5 'and 3' ends of clone 12.1, additional oligonucleotide probes were designed to effect the 3 'and 5' RACE using the Marathon RACE kit. Seven reaction products were identified at the 3 'end, and two of those identified as KC44 and KC45 were sequenced to obtain the 3' end of the cDNA described in Figure 20 (SEQ ID Nos. 15 and 16). Two reaction products were identified by the Marathon RACE 5 'reaction and the sequence was obtained complete for introns 334 and the partial sequence for clone 331. A 5 'marathon race reaction was performed based on the sequence of nucleic acid near the 5' end and that of clone 334 using the RACE product of the reaction 5 'Marathon RACE initial com pattern. These latter results from additional clones of which three clones, identified as clones 2, 3 and 5 were sequenced. Taken together, these sequences make the complete nucleic acid sequence of Figure 20 (SEQ ID Nos 15 and 16). The sequencing of all the clones was carried out using Sequenase with the exception of clone 334 which was sequenced by the LARK Sequencing Technology, Houston, TX. The sequence was noticed using Assemblylign (Laboratory Research Products, Eastman Kodak Company, New Hacen, CT) and the Wisconsin Package (Genetics Computer Group, Madison, Wl). The start codon was identified using the first ATG in the nucleic acid sequence that allowed the initiation of translation [Kozak, J. Cell Biol., 108, 229-241 (1989)]. The long version of the mature protein (SEQ ID NO 15) contains 1026 amino acids and has an estimated molecular weight of 117239. The nucleic acid sequence shows a high degree of similarity to several unidentified expressed sequence tags including accession numbers R47032 and H08895. R47032, which was isolated from noncalcified rat incisor tissue, is identical to GTVap in 90 nucleotides, also diverges at both ends. It is likely to code for a form of the GTVap not divided or divided alternately. H08895, which was isolated from the human brain, is 88.5% identical to GTVap in 382 nucleotides, the full length of this tag sequence tag expressed. It is likely that H08895 codes for a part of the human GTVap. The estimated protein is 32.5% identical to rat N-aminopeptidase and sample > 20% identity with other aminopeptidases. This level of protein identity corresponds to the nucleic acid identity of 50% or 60% over more than 1000 nucleotides.

Identification of a Conduction Sequence for a Sequence of Retention Insensitive to Insulin Since GTVap and GLUT4 are found in GLUT4 vesicles and respond to insulin as evidenced by their simultaneous translocation of the plasma membrane, it was conceivable that both proteins start from a common structural motif (retention sequence). The structural reason that gives this property to these proteins is probably found in other proteins that are translocated in an insulin-sensitive way. In an attempt to identify an insulin-sensitive retention sequence, the GTVap sequence of the estimated protein was compared to the protein sequences of the cytoplasmic GLUT4 domains using the Wisconsin Package computer program (Genetics Computer Group, Madison, Wl). Significant homology was evident in the two adjacent segments of the cytoplasmic domain of the GTVap (SEQ ID Nos 17 and 18) and with the inverted cytoplasmic domain (C-N reading) of GLUT4 (SEQ ID NO 19). This consensus sequence is shown by the sequence identified as SEQ ID Nos. 20 and 21. The other embodiments of the invention will be apparent to those skilled in the art from the consideration of this specification or the practice of the invention described herein. It is intended that the specification and examples be considered as exemplary only, with the scope and spirit of the invention indicated by the following claims.

SEQUENCE LIST (1. GENERAL INFORMATION: (i) APPLICANT: Knowles, W. J .; Guralski, D .; Haigh, W .; Letsinger, J. T .; Clairmont, K .; and Hart, J. (ii) TITLE OF THE INVENTION: Glucose Transporter Vesicle Aminopeptidase (iii) SEQUENCE NUMBER: 21 iiv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Pamela A. Simonton, c / o Bayer Corporation (B) STREET: 400 Morgan Lane (C) CITY: West Haven (D) STATE: Connecticut (E) COUNTRY: United States ( F) C. P. : 06516 | v) LEGIBLE FORM IN COMPUTER: (A) TYPE OF MEDIUM: Disk, 3.5 inches, with storage capacity of 1, 300 Kb (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Macintosh System 7.1 (D) PROGRAM: Word Perfect 3.0a (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: 09/19/95 (C) CLASSIFICATION: preliminary class 435 (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 08 / 309,232 (B) SUBMISSION DATE: 09/20/94 (C) CLASSIFICATION: preliminary class 435 (viii) INFORMATION FROM THE MANDATORY / AGENT (A) NAME: William F. Gray (B) REGISTRATION NUMBER: 31018 (C) REFERENCE NUMBER / FILE: MWH 323P1 (ix) INFORMATION BY TELECOMMUNICATION: (A) TELEPHONE: (203) 937-2712 (B) TELEFAX: (203) 931-5492 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: eleven amino acids (B) TYPE: peptide (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein fragment (A) Description: polypeptide of tryptic digestion of full-length protein (v) TYPE OF FRAGMENT: internal fragment (vi) ORIGINAL SOURCE: protein purified by affinity from- (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (F) TYPE OF TISSUE: adipose (ix) CHARACTERISTICS: (A) NAME / KEY: GTVap fragment p94 (C) IDENTIFICATION METHOD: protein sequencing (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: Phe Ala Ala Thr Gln Phe Glu Pro Leu Ala Ala 5 10 (3) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: nineteen amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: protein fragment (A) Description: polypeptide of tryptic digestion of full-length protein (v) TYPE OF FRAGMENT: internal fragment (vi) ORIGINAL SOURCE: protein purified by affinity from- (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (F) TYPE OF TISSUE: adipose (ix) CHARACTERISTICS: (A) NAME / KEY: GTVap fragment p85 (C) IDENTIFICATION METHOD: protein sequencing (D) OTHER INFORMATION: partial comparison of GeneSeq partial database (access # GSP: R28142; EP 0 535 241 Al) identified as leucine aminopeptidase of placental origin. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: lie Leu Gln Asn Gln lie Gln Gln Gln Thr Arg Thr Asp Glu Gly Xaa 5 10 15 Pro Xaa Met (4) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 33 nucleotides (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other nucleic acid: Synthetic oligonucleotide probe based on the peptide sequence (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TTYGCNGCNA CNCARTTYGA RCCNYTNGCN GCN 33 (5) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 33 nucleotides (B) TYPE: nucleic acid (C) HEBRA: 'simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other nucleic acid: Synthetic oligonucleotide probe based on the peptide sequence (iii) HYPOTHETICAL: yes (iv) ANTICIPATION: yes (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: NGCNGCNARN GGYTCRAAYT GNGTNGCNGC RAA 33 (6) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: fifteen amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: protein fragment (A) Description: polypeptide of tryptic digestion of the full-length protein (v) TYPE OF FRAGMENT: internal fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (F) TYPE OF TISSUE: adipose (ix) CHARACTERISTICS: (A) NAME / KEY: GTVap fragment p85 (C) IDENTIFICATION METHOD: protein sequencing (D) OTHER INFORMATION: amino acids positively identified in the GTVap protein sequence; used for the design of the oligonucleotide probes (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: He Leu Gln Asn Gln He Gln Gln Gln Thr Arg Thr Asp Glu Gly 5 10 15 (7) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 45 nucleotides (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other nucleic acid: Synthetic oligonucleotide probe based on the peptide sequence (iii) HYPOTHETICAL: yes (iv) ANTICIPATION: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: ATHYTNCARA AYCARATHCA RCARCARACN MGNACNGAYG ARGGN 45 (8) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 45 nucleotides (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Other nucleic acid: Synthetic oligonucleotide probe based on the peptide sequence (iii) HYPOTHETICAL: yes (iv) ANTICIPATION: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: NCCYTCRTCN GTNCKNGTYT GYTGYTGDAT YTGRTTYTGN ARDAT 45 (9) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: twelve amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: synthetic peptide (A) Description: synthetic peptide designed from the GTVap p94 fragment and modified with a terminal carboxy cysteine (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Phe Ala Ala Thr Gln Phe Glu Pro Leu Ala Ala (Cys) 5 10 (10) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: sixteen amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: synthetic peptide (A) Description: synthetic peptide designed from amino acids 495-509 of the GLUT4 protein sequence and modified with an amino terminal cysteine [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: (Cys) Lys Pro Ser Thr Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp 5 10 15 (11) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: eight amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: synthetic peptide (A) Description: synthetic peptide designed from amino acids 495-501 of the GLUT4 protein sequence and modified with a terminal carboxy cysteine (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: Lys Pro Ser Thr Glu Leu Glu (Cys) 5 (12) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: seven amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: synthetic peptide (A) Description: synthetic peptide designed from amino acids 498-503 of the GLUT4 protein sequence and modified with a cysteine. carboxyl terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: Thr Glu Leu Glu Tyr Leu (Cys) 5 (13) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: seven amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: Synthetic peptide (A) Description: Synthetic peptide designed from amino acids 504-509 of the GLUT4 protein sequence and modified with an amino terminal cysteine (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: (Cys) Gly Pro Asp Glu Asn Asp 5 (14) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: twelve amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: protein fragment (A) Description: polypeptide of tryptic digestion of the full-length protein (v) TYPE OF FRAGMENT: internal fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (F) TYPE OF TISSUE: adipose (ix) CHARACTERISTICS: (C) IDENTIFICATION METHOD: protein sequencing (D) OTHER INFORMATION: GTVapl peptide sequence that includes the assigned residues with less total confidence (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: Phe Ala Ala Thr Gln Phe Glu Pro Leu Ala Ala arg * • 5 10 (15) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: nineteen amino acids (B) TYPE: peptide (ii) TYPE OF MOLECULE: protein fragment (A) Description: polypeptide of tryptic digestion of full-length protein (v) TYPE OF FRAGMENT: internal fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (F) TYPE OF TISSUE: adipose (ix) CHARACTERISTICS: (C) IDENTIFICATION METHOD: protein sequencing (D) OTHER INFORMATION: GTVap2 peptide sequence that includes the assigned residues with less total confidence; see OTHER INFORMATION on SEQ ID NO: 2 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: He Leu Gln Asn Gln He Gln Gln Gln Thr Arg Thr Asp Glu Gly thr 5 10 15 Pro asn Met (16) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 3388 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA; (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (D) DEVELOPMENT STAGE: adult (F) TYPE OF TISSUE: skeletal muscle (vii) ORIGINAL SOURCE: (A) LIBRARY: Clontech rat skeletal muscle cDNA library in GT11 and lambda and rat skeletal muscle isolated mRNA (B) CLONA: 5.3 (lambda gtll library), clones 5,334 and KC44 product of the PCR. (ix) CHARACTERISTICS: (A) NAME / KEY: The cDNA includes the complete coding region for GTVap, long version (C) IDENTIFICATION METHOD: nucleic acid hybridization (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CTCTCOG? OT AG? AAGC Ta GOGCOCTOOO CTGCTGAGGA CCCGC? CCGG GCG ?? O ATO 59 1 G? G? CC TTT ACC? AT GAT CG? CTT CAG CTT CCA AGG ?? T ATG ATT GA? 107 Glu Thr Pha Thr? Sn Aßp Arg Leu Gln Leu Pro Krg Aßn Mee 11 * Glu 5 10 1S AAC AGC ATG TTT OAA GAA GAA CCA GAT GTG OTA G? TTA GCC AAA G ?? 155 Aßn Ser Mee Phe Glu Glu Glu Pro? ßp Val Val Aßp Leu Ala Lyß Glu 20 25 30 CCT TGT TTA C? T CCT CTG OAA CCT GAT GJ-IA GTT GAA TAT OAO CCC CGA 303 Pro Cyß Lau Hiß Pro Leu Glu Pro Aßp Glu Val Glu Tyr Glu Pro Arg 35 40 45 GGT TCG? GG CTT CTG GTG AO? GOT CTT GGT GAG CAT GAO ATO GAT GAG 251 Gly Ser Arg Leu Leu Val Arg Gly Leu Gly Glu Hiß Glu Met Aßp Glu 50 SS 60 65 GAT OAA GAO GAT TAT OAO TCA TCT GCC AAO CTG CTO GGC ATO TCC TTC 299 Asp Glu Olu Asp Tyr Glu Ser Sar Wing Lyß Leu Leu Oly Met Ser Phe 70 75 80 'ATG AAC AGA AOC TCA GGC CTT CGG ?? C? GT OCA AC? OOC TAC AGO CAG 347 Met Asn Arg Ser «ßr Gly Leu? Rg Aßn Ser Wing Thr Gly Tyr? Rg Gln 85 90: JS AGT CCA GAT GGG ACT TGT TCA GTA CCC TCT GCC? GG? CC TT? GTA ATC 395 Ser Pro? ßp Gly Thr Cy »Ser Val Pro Ser Ala Arg Thr M? Val lie 100 105 110 TOT GTT TTT GTC ATT GTG GTT GCT GTC TCT GTA ATC ATO «TG? TT TAT 443 Cys Val Phe Val Val Val Val Val Val Val Val Val Met Met Val He Tyr 115 120 125 CTA CTG CCT AGA TOT ACC TTT ACC AAA OAA GGC TGC CAC / AA AC? ?? C 491 Leu Leu Pro? Rg Cy »Thr Phe Thr Ly» Glu Qly Cy »Hiß l.yß Thr? ßn 130 135 140 145 CAG TC? GCA GA? CTC ATT CAG CCG? TT GCT? C? J »VAC GCO? AA OTO TTT 539 Gln Ser Wing Glu Leu lie Gln Pro Xlß Wing Thr Asn Gly Lyß Val Phe 150 155 160 CCA TGG OCA CAA ATT AGO CTT CCC ACT OCC ATT ATT CCT CA? CCC TAT 587 Pro Trp Wing G n He Arg Leu Pro Thr Wing He He Pro Gln? Rg Tyr 165 170 175 GAA CTT AGC CTA C? T CCA AAC CTA ACC TCA ATO AC? TTC AGG GGT TCT 635 Glu Leu Ser Leu Hi? Pro? Sn Leu Thr Ser Met Thr Phe? Rg Gly South 180 IBS 190 GTG AC? TT TCA CTT CAG GCT CTT CAÁ GAT ACÁ COO GAC ATC ATT CTC 683 Val Thr He Smr Leu Gln? The Leu Gln? ßp Thr? Rg? ßp He He Leu 195 200 205 CAT AOC AC? OOA CAT A? T? TT TC? AßT OTO AC? TTT ATO TCO OCO OTT 73 Hiß Ser Thr ßly Hiß Aßn Ser Ser Val Thr Phe Met Sar Val Val 210 215 220 225 TCC AGT CAA GAA AAA CAÁ GTT GAA ATT CTG OAA TAT CCA TAT CAT GAA 779 Ser Ser Gln Glu Lys Gln Val Glu He Leu Glu Tyr Pro Tyr Hiß Glu 230 235 240 CAA ATC GCC GTT GTT OCC CCT GAA AOC CTT CTA ACA GGA GC AAT TAT 9 Gln He Ala Val Val Ala Pro Glu Ser Leu Leu Thr Gly Asn Tyr 245 250 2Ü5 ACC TTO AAG AT? OA? T? T TC? GCA AAT ATA TCT AAC TCT T? C T? T OOO 875 Thr Leu Ly »He Glu Tyr Ser Ala Aßn He Ser Aßn Ser Tyr Tyr Gly. 260 265 270 OGT TAT GGC ATC ACC T? C? CA GAT AAA AOT AAT GAG AA? A? C? AC TTT 923 n Phe Tyr Gly He Thr Tyr Thr Asp Lyß Ser? ßn Glu Lyß Lyß? ßn Phe U 275 280 285 GC? GC? CT CAO TTT GA? CCT TTO GC? GC? AOA TCT GCT TTT CCT TGT 971? The Wing Thr Gln Phe Glu Pro Leu Wing Wing Arg Ser? The Phß Pro Cyß 290 295 300 305 TTT G? T GAA CC? OC? TTT AAO GCC ACA TTT ATC ATC AAG ATO ACÁ? OO 1019 Phe Asp Glu Pro Wing Phe Lys Wing Thr Phe He He Lys Hit Thr Arg 310 315 320 GAT GAC CAC CAT ACT OCA TTA TCA A? T ATG CCT A? O? AG TCA TCA GTC 1067 Aßp Glu Hi? Hi? Thr Ala Leu Ser? sn Mat Lyß Ly Ser Ser Val 32S 330 33S CCT? GAA GAA GOA CTT ATT CAA GAT TTT TCT OAA? OT OTO AAA 1115 5 Pro Thr Glu Glu Gly Leu He G n Aßp Glu Phß Ser Glu Ser Val Lyß 340 345 350 ATG AGC ACÁ TAC CTO GTT GCT TTC ATT GTA GGG GAO ATG AGG ?? C CTG 1163 Met Ser Thr Tyr Leu Val? Phe He Val Gly Glu Met? Rg? ßn Leu 355 360 365 AGT CAG GAT GTA AAC GGO ACT CTG GTT TCT GT? T? T GCT GT? CCA GAA 1211 Ser Gln Aßp Val Aßn Gly Thr Leu Val Ser Val Tyr Ala Val Pro Glu 370 375 380 385 AAA ATT GAT C ?? OTT T? C CAT GCC TTs GAC ACA ACT GTA AAG CTC CTT 1259 Lys He Asp Gln Val Tyr Hiß? Leu Aßp Thr Thr Val Lyß Leu Leu 390 395 400 GAO TTT TAT CAÁ A? T T? C TTT O ?? ATT CAÁ CT CCA CTA AAO AAA TTG 1307 Glu Phe Tyr Gln Aßn Tyr Phß Glu He Gln Tyr Pro Leu Lyß Lyß Leu 40S 410 415 GAT CTO GTO GCC ATT CCT GAC TTT GAA GCA GGA GCA ATG O? A AAT TGO 1355? Sp Leu to Ale He Pro? ßp Phe Glu? the Gly Wing Met Glu Aan Trp 420 425 430 GGC CTQ CTT ACO TTC COA GA? GAO ACT CTT CTO TAT GAC AAT '3CC ACT 1403 Gly Leu Leu Thr Phe Arg Glu Glu Thr Leu Leu Tyr Aßp? ßn? The Thr 435 440 445 TCT TC? OTA GCA OAT AGA AAA CTO GTC? CT AAA ATC ATC GCT CAC OAO 1451 Being Val? La? Ap Arg Lys Leu Val Thr Ly? He He? La iiia Glu 450 455 460 465 CTG GCA CAT CAO TGG TTT GOT AAT CTG GTT ACÁ ATO CAO TOG TGO A? T 1499 Leu Wing Hiß G n Trp Phe Gly Asn Leu Val Thr Met Gln Trp Trp? An 470 475 480 GAC CTG TGO CTA ?? CG? A GGC TTT GCC? CT TTC? TG O? G TAT TTC TCT 1547 Asp Leu Trp Leu Aßn Glu Gly Phe Wing Thr Phe Mee Glu Tyr Phe Ser 485 490 495 GTG GAA AAA ATA TTC? A? GAO CTC A? C AGT TAT GA? GAC TTC TTA G? T 1595 Val Clu Lyß He Phß Lyß Glu Leu Aßn Ser Tyr Glu Aßp Phe Leu? ßp 500 505 510 GCT COA TTT AAA? CC ATG AGO JUU ». OAT TCC TTO A? T TCO TCT C? T CCA 1643 Wing Arg Phe Lyß Thr Met Arg Lyß Aßp Ser Leu Aßn Smr Ser H: iß Pro 515 520 525 ATA TCA TCA TCT GTT CAO TCA TCA GAA CAÁ AT? GA? GA? ATO TTT GAT 1691 Be Ser Be Ser Val Gln Smr Ser Glu Gln He Glu Glu Met Phe Aßp 530 535 540 545 TCT CTT TCG TAT TTT AAO CAG GGA GCG TCT CTC TTO TTG ATO TTO AAA 1739 Be Leu Be Tyr Phe Lyß Gln Gly Wing Be Leu Leu Leu Met Leu Lyß 550 555 560 AGT TAC CTT AGT GAA GAC? Iu TTT CAG CAT OCC ATC ATT CTT TAC CTO 17ß7 Ser Tyr Leu Ser Glu Asp Val Phe Gln His Ala He Ha Leu Tyr Leu 555 570 575 CAC? AT CAC AGC TAT OCA GC? ? TT CAA AGC GAT GAC CTC TOO OR? C? GC 1835 Hia Even Hi »Be Tyr? The? The He Gln Ser? ßp? ßp Leu V * m Aßp Ser 580 585 590 TTC A? TG? G GTC ACÁ OOC? ? A ACT CTA GAT OTA AAG AAA ATO ATO A? A 1883 Phe? ßn Glu Val Thr Gly Ly? Thr Leu Asp Val Lys Ly? Met Met Ly? 595 600 605 ACC TOG? CC CTA CAG AA? GG? CC TTC? TTA GTG ACC GTC CAO AO? A? G 1931 Thr Trp Thr Leu Gln Lyß Cly Phß Pro Leu Val Thr Val Oln? Rg Lyß 610 615 620 625 GGG ACT GAG CTT CTT CAÁ CAÁ GA? AGA TTT TTT CCA AGC ATO C ?? 1979 Gly Thr ßlu Leu Leu Leu Gln Gis »Olu? Rg Phe Phe Pro Ser Met Gln 630 635 640 DC? GAA ATT CAG GAT TC? GAT ACA AGC CAC CTT TOG CAT ATT CCA ATA 2027 Pro Glu He Gln Asp Smr Asp Thr Ser His Leu Trp Hiß He Pro He 645 650 65S TCC TAT GTC ACT GAT GGA AGA A? C TAT TCA GAA TAT COA TCA GTT TCA 075 Ser Tyr Val Thr Aßp Cly Arg Asn Tyr Ser Glu Tyr Arg Ser Val Ser 660 665 670 CTA CTG GAC AAG AAA TCA GAT GTC ATC A? T CTT? C? G ?? CAÁ GTA C ?? 2123 Leu Leu Asp Lys Lyß Ser Asp Val He Asn Leu Thr Glu 31n Val ßln 675 6T0 685 TGG GTC AA? GTC AAT ACÁ AAC ATO ACG GOC T? C T? C? TT OTT CAC TAT 2171 Trp Val Lys Val Aßn Thr Aßn Met Thr Gly Tyr Tyr He Val Hiß Tyr f > 90 69S 700 705 GCT CAT GAT GGC TGG GCA GCT CTA ATC A? C C? G TTA AAA? GA A? T CCC 2219 Ala His Asp Gly Trp? The? The Leu He? ßn Gln Leu Ly? J rg A? N Pro 710 715 720 TAT GTT CTG AGT G? C AA? GAC COA GCC AAC CAC ATC AAT J * C ATC TTT 2267 Tyr Val Leu Ser? ßp Lyß Aßp Arg Ala Aßn Leu He Aßn Asn He Phe 725 730 V35 GAA CTT GCA GGC CTT GGC AAA GTG CCT CTT CAG ATO OCA TTC GAT TTG 2315 Glu Leu Wing Gly Leu Gly Lyß Val Pro Leu Gln Met the Phe Aßp Leu 740 745 750 ATT GAC TAC CTT AGA AAT GAG ACC CAC ACT GCA CCT ATC? CT GA? GCC 2363 He? Ap Tyr Leu Arg Aßn Glu Thr His Thr Wing Pro He Thr Clu Wing 755 760 765 CTG TTC CAG ACT GAC CTC ATC TAT AAT CTC CTO GAA AA? CTG GG? C? C 2411 Leu Phe Gln Thr Asp Leu He Tyr Asn Leu Leu Glu Ly? Leu Oly Hi? 770 775 780 7B5 ATG GAC CTG TCC TCA AGA TTA GTO ACC AGA GTA CAT AA? TPG CTC CAG 2459 Met Acp Leu Ser Ser Arg Leu Val Thr? Rg Val Hiß Lyß L »» u Leu Oln 790 795 800 A? C CAÁ ATC CAG CAG CAG AC? TGO? CA GAT GAA GGC ACÁ CC? TCC ATO 2507 Asn Gln He Gln Gln Gln Thr Trp Thr Asp Glu Gly Thr Piro Smr Met CGA GAG CTT CGß TCA GCC TTO CTO GAA TTT OCC TOC © CC C? C AOC CTA 2555? Rg Glu Leu? Rg Ser? Leu Leu Glu Phe Ala Cy »Wing H ß Ser Leu 820 825 830 GAG AAC TGT ACC ACT ATO GCC ACÁ A? O CTG TTT GAT GGT IK-G ATO GCA 2603 Glu Asn Cyß Thr Thr Mßt Wing Thr Lyß Leu Phe Aßp Gly Trp Met Wing 835 840 845 TCA AAT GOA ACT CAO AGC CTG CCG ACT GAC GTC ATO ACC ACT GTG TTC 2651 Be Aßn Gly Thr Gln Ser Leu Pro Thr Aßp Val Met Thr Thr Val Phß 850 855 860 865 AAA GTT GGA GCA AG? ? CC GAG AA? GGC TGG TTG TTC CTC TTT AGC ATG 2699 Lya Val Gly Ala Arg Thr Glu Lys Oly Trp Leu Phe Leu Phe Ser Mßt 870 875 880 TAC TCC TCC ATG GGC TCT GA? GCA GAO A? G GAT A? A ATA CTT GA? GCC 2747 Tyr Ser Ser Met Oly Ser Glu Wing Glu Lyß Asp Lyß He Leu Glu Wing 885 890 8) 5 CTG GCC AGC TCA GCO GAT GC? CAT AAA CTT TAC TGG TT? ATG AA? GT 2795 Leu Wing Ser Wing Asp Wing Hi »Lyß Leu Tyr Trp Leu M«? T Lyß Ser 900 905 910 AGC CTT GAT GOT AT AT ATC COG ACG? CAG AAO TTO TCA CTT ATC ATT 2843 Be Leu? ßp Gly Aßp He He Arg Thr Gln Lyß Leu Ser Litu He He 915 920 925 AGA ACÁ GTG GGC AGA CAG TTT CCT GGA CAT TTO CTG GCA TGO GAT TTT 2891 Arg Thr Val Gly Arg G n Phe Pro Gly Hiss Leu Leu? The Trp Aßp Phe 930 935 940 945 GTT AAG GAA A? C TOG? AT? AG CTT GTA CAT AAG TTC CAT CTG GGC TCC 2939 Val Lyß Glu Asn Trp Asn Lys Leu Vml Hiß Lyß Phe His Leu Gly Ser 950 955 960 TAT ACC ATT CAA AGC ATT GTT GCT GOA TCT ACT CAC TTA TTT TCA ACG 2987 Tyr Thr He Gln Ser He Val? La Gly Ser Thr Hi? Leu Phu Ser Thr 965 970 97! >; G ACÁ CAT TTA TCT GAO GTC CAG GAA TTC TTC G ?? AAT CAO TC? G? G 3035 Lyß Thr His Leu Ser Glu Val Gln Glu Phe Phe ßlu Aan G n Ser ßlu 980 985 990 GCA ACC TTG CAG CTT CGG TGT GTT CAG GAO GCC TTC GAA CTG ATT G? G 3083 Wing Thr Leu Gln Leu Arg Cy »Val Gln Glu Ala Phe Glu Val He Glu 995 1000 1005 CTG AAT ATC CAG TGG ATO GCC AGG AAT CTO? AA ACT CTG ACÁ CTG TOO 3131« u? An He Gln Trp Met Ala? Rg Aan Leu Lyï Thr Leu Thr Leu Trp 1010 1015 1020 1025 CTG TAGCCCTC? C AOCTGATCTT CCGGTGCCCA TGGCTCTCCT GCTTTTOCA? 184 Leu AGGTTG? GTG ?? GOCCOOCC TGCT? CTGAO TTOTTTOCAC TGTT? GGA C l ?? GTTAOCTC 32 4 AGGGCCCA? T TOT? TTTTTC? T? TCTTTTC TOAA? TOTCC TTAGGCGOT? «TTATTTATT 3304 ? CAAAATTAT ATTC? CCTOT ACOTC ?? CC? TCTAC? AT? A C? GTQA? CAC CíTOCCCOCGC 3364 CCCCCCCTCOA OCCCT? TAßT G? GT 3 88 (17) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 3385 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA; (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus norvegicus (B) CEPA: Sprague-Dawley (D) DEVELOPMENT STAGE: adult (F) TYPE OF TISSUE: skeletal muscle Vii) IMMEDIATE SOURCE: (A) LIBRARY: Clontech rat skeletal muscle cDNA library in GT11 lambda and rat skeletal muscle isolated mRNA (B) CLONA: 12.1 (lambda gtll library), clones 5,334 and KC44 product of the PCR 10 (ix) FEATURES: (A) NAME / KEY: includes the complete cDNA that codes for the short version of the GTVap. (C) IDENTIFICATION METHOD: Hybridization of nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: CTCTCGGAGT ACAAAGCTTG GOGCGCTGGO CTOOTOAGCA CCCGC? OCGO 3CO ?? O ATO 5 Met 1 - ° GAG ACC TTT? CC AAT OAT CG? CTT CAO CTT CCA AGO A? T ATO ATT OA? 107 Glu Thr Phe Thr? ßn Aßp Arg Leu Gln Leu Pro? Rg? An Met He Glu 5 10 15 AAC AGC ATG TTT GA? OAA GA? DC? G? T OTO GT? O? T TTA OCC AA? OAA 155 Aßn Smr Met Phe Glu Glu Glu Pro Asp Val Val Asp Leu Ala Lya Glu 20 25 30 CCT TGT TTA CAT CCT CTG GA? CCT G? T GA? GTT GAA TAT OAO CCC COA 203 Pro Cy »Leu Hiß Pro Leu Olu Pro Aßp Glu Val Glu Tyr Glu Pro? Rg 35 40 45 GGT TCG AGG CTT CTO OTG AG? GGT CTT GGT GAO CAT GAG ATO GAT GAG 251 Gly Ser Arg Leu Leu Val Arg Gly Leu Gly Glu Hiß Glu Met Aßp Olu 25 50 55 60 65 G? T GAA GAG GAT TAT OAO TCA TCT OCC A? Q CTO CTO OOC ATO TCC TTC 299 Asp G? Glu? Sp Tyr Olu Ser Ser? The Ly? Leu Leu Gly Met Ser Phe 70 75 BO ATG AAC AGA AGC TCA GGC CTT CGO AAC AGT GC? AC? GGC TAC AGO CAO 347 Mee Aßn Arg Ser Gly Leu Arg Aßn Be Wing Thr Gly Tyr? Rg Gln 85. 90 95 AGT CCA GAT GGG ACT TOT TCA GTA CCC TCT GCC AGO ACC TTA OTA ATC 395 Ser Pro A »p Oly Thr Cyß Ser Val Pro Ser? La? Rg Thr Leu Val He 100 105 110 TGT CTT TTT CTC? TT OTO GTT GCT GTC TCT OT? ? TC ATO OTO? TT T? T 443 Cys Val Phe Val Val Val? Val Val Val Met Met Val Tyr 115 120 125 CT? CTG CCT AGA TGT ACC TTT ACC AA? GA? GGC TGC CAC AAA ACÁ A? C 491 Leu Leu Pro? Rg Cy? Thr Phe Thr Lys Glu Gly Cy? Hi »Ly? Thr? ßn 130 135 140 145 CAG TC? GC? GA? CTC? TT CAO CCG ATT GCT ACÁ A? C GGG ??? GTO TTT 539 G n Ser? The Glu Leu He Gln Pro He? The Thr? ßn Gly Lye Val Phe 150 155 160 CCA TGG GCA C ?? ? TT? GG CTT CCC ACT GCC ATT ATT CCT CAA COC TAT 587 Pro Trp Wing Gln He? Rg Leu Pro Thr Wing He He Pro ßln Arg Tyr 165 170 175 CAÁ CTT AGC CTA C? T CCA A? C CT? ? CC TCA ATG AC? TTC AOG 03T TCT 635 Glu Leu Ser Leu Hiß Pro Aan Leu Thr Ser Met Thr Phe? Rg Oly Ser 180 185 190 GTO? C? ? TT TC? CTT CAO OCT CTT C ?? OAT AC? COO GAC ATC ATT CTC 683 val Thr He Ser Leu Gln Ala Leu Gln Aßp Thr Arg Aßp He He Leu 195 200 205 CAT AGC ACÁ GGA CAT AAT ATT TCA AGT GTO ACÁ TTT ATG TCO GOO OTT 731 Hiß Smr Thr Gly Hiß Aßn He Ser Ser Val Thr Phe Mßt «er?« A Val 210 215 220 225 TCC AGT C ?? GAA? AA CAA GTT GAA ATT CTG GAA TAT CC? T? T C? T OA? 779 Smr Ser G n Glu Lya Gln Val Glu He Leu Glu Tyr Pro Tyr Hi »Olu 230 235 240 CAÁ ATC GCC GTT GTT GCC CCT G ?? AGC CTT CTA ACA CGA CAC AAT TAT 827 Gln He Wing Val Val? Pro Glu Ser Leu Leu Thr Gly Hi? A? N Tyr 245 250 255 ACC TTG AAG ATA GAA TAT TCA GCA AAT ATA TCT AAC TCT TAC TAT GOO 875 Thr Leu Lyß He Glu Tyr Ser Wing Asn He Smr Aßn Ser Tyr Tyr Oly 260 265 270 TTT TAT GGC ATC? CC TAC ACÁ GAT AAA AOT AAT GAG AAA A? G AC TTT 923 Phe Tyr Gly He Thr Tyr Thr Asp Lys Ser Aan Clu Lyß Lyß Aßn Phß 275 280 285 OCA OCA ACT CAO TTT GA? CCT TTO GCA GCA AGA TCT OCT TTT CCT TOT 971 Wing Wing Thr Gln Phe Glu Pro Leu Wing Wing Arg Ser? La Phe! »Cyß 290 295 300 305 TTT GAT G? A CC? GC? TTT AAO GCC ACA TTT ATC AT ATCC TO AAAGO? ? TTCC? ACC? A AOGGG 1019 Phe Asp Glu Pro Ale Phe Lyß Wing Thr Phe He He Ly »He Vhr Arg 310 315 220 GAT GAG CAC C? T? CT GC? TT? TCA AAT ATO CCT A AAAOG A A ?? GC T TCC ?? I 1CCA? G GTTCC 1067? ßp Glu Hia Hia Thr? The Leu Ser Asn Met Pro Ly? Ly »Ser Ser Val 325 330 335 CCT ACÁ GA? GAA GG? CTT ATT CAA GAT GAO TTT TCT OAA AOT OTO AA? 1115 Pro Thr Glu Glu Gly Leu He Gln Aßp Olu Phe Ser Glu Ser Val Lyß 340 345 350? TG AGC ACÁ TAC CTG GTT OCT TTC ATT GTA GOO CAG ATO AGO A >; \ C CTG 1163 Met Ser Thr Tyr Leu Val Wing Phe He Val Gly Glu Met? Rg Even Leu 355 360 365 AGT CAG GAT GTA AAC GGG ACT CTG GTT TCT GTA TAT GCT GTA CCA GAA 1211 Ser Gln Asp Val Aßn Gly Thr Leu Val Ser Val Tyr Ala Val Pro Glu 370 375 380 385 AAA ATT GAT CAA GTT TAC CAT GCC TTG GAC ACA ACT GTA AAG CTC CTT 1259 Lya He Asp Gln Val Tyr His Wing Leu Asp Thr Thr Val Lys Lau Leu 390 395 400 GAG TTT TAT C ?? ?? T TAC TTT GAA ATT CAÁ TAC CCA CTA AAG A ?? TTO 1307 Glu Phe Tyr Gln Aan Tyr phß Glu He Gln Tyr Pro Leu Lys Lys Leu 405 410 415 GAT CTO GTG GCC ATT CCT GAC TTT GA? GCA GG? GC? ATO GAA ?? T TOO 1355 Asp Leu Val? The He Pro? S Phe Glu? The Gly Wing Met Qlu? ßp Trp 420 42S 430 GGC CTG CTT? CG TTC CGA OA? GAG ACT CTT CTG TAT GAC AAT GCC ACT 1403? And Leu Leu Thr Phe Arg Glu Glu Thr Leu Leu Tyr? Sp? ßn A it Thr 435 440 445 TCT TCA GTA GCA GAT AG? AA? CTG GTC- ACT AAA ATC ATC GCT CAC! GAO 1451 Ser Val Val Al Aßp Arg Lyß Leu Val Thr Lya He He Ala Hia Glu 450 455 460 465 CTO GCA CAT CAG TGG TTT GGT AAT CTO GTT? C? ATO CAO TOO TGG AAT 1499 Leu? La Hi? Gln Trp Phe Gly? ßn Leu Val Thr Met Gln Trp Trp Asn 470 475 480 GAC CTO TGO CTA AAC GAA GGC TTT GCC? CT TTC ATG GAG TAT TCT 1547 Aßp Leu Trp Leu Asn Glu Gly Phe? Thr Phe Met Glu Tyr Phe Ser 485 490 495 GTG GA? AAA ATA TTC A ?? G? G CTC AAC AGT TAT GA? G? C TTC TTA GAT 1595 Val G u Lys He Phe Lya Olu Leu Aßn Ser Tyr Olu? »P Phe Leu Aßp 500 505 510 GCT CGA TTT AAA ACC ATO? GG ?? A OAT TCC TTO? AT TCG TCT C? T DC? 1643 Wing Aro Phe Ly »Thr Met Aro Ly»? ßp Ser Leu? ßn Ser Smr Hi? Pro 515 520 525 ATA TCA TCA TCT OTT CAO TCA TCA GA? C? A AT? O? A OR? A ATO TTT O? T 1691 I Ser Ser Ser Val Gln Ser Ser Glu Gln He Glu Glu Met Phe? Sp 530 535 540 545 TCT CTT TCG T? T TTT? AO OGA OCG TCT CTC TTG TTG ATG TTO A ?? ? OT 1739 Be Leu Be Tyr Phß Lyß Gly Wing Be Leu Leu Leu Met Leu Lya Be 550 S5S 560 TAC CTT AGT GAA GAC GTO TTT CAG CAT GCC ATC ATT CTT TAC CTO C? C 1787 Tyr Leu Ser Glu Aßp Val Phe Gln His Wing He He Leu Tyr Leu Hiß 565 570 575 AAT CAC AGC TAT GCA GC? ATT CA? ? GC G? T G? C CTC TGO GAC AGC TTC 1835 Asn Hi? Ser Tyr Ala Wing Gln Ser A? P? Sp Leu Trp Asp Smr Phe 580 58S 590 AAT GAO GTC ACA GGC AA? ACT CTA OAT GTA AAG AAA ATG ATG AAA ACC 1883 Asn Glu Val Thr Gly Lys Thr Leu Asp Val Lys Lys Met Met Lyß Thr 595 600 605 TGG ACC CTA CAG AAA GGA TTC CCA TTA GTG ACC GTC CAG AGA A? GGGG 1931 Trp Thr Leu Gln Lyß Gly Phe Pro Leu Val Thr Val Gln Arg Lyß Qly 610 615 620 625 ACT GAG CTT CTT CTA CAÁ CA? GAA AGA TTT TTT CCA AGC ATO CAA CCA 1979 Thr Glu Leu Leu Gln Gln 'Glu Arg Phe Phe Pro Ser Met Without Pro 630 635' 640 GA? ATT CAG GAT TCA GAT ACA AGC CAC CTT TGG CAT ATT CCA ATA TCC 027 Glu He G n Asp Ser Asp Thr Ser His Leu Trp Hiß He Pro He Ser 645 645 655 TAT GTC ACT GAT GGA? G? AAC TAT TCA GA? TAT CGA TCA GTT TC? CTA 2075 Tyr Val Thr Asp Gly Arg Aßn Tyr Ser Glu Tyr Arg Ser Val Ser Leu 660 665 670 CTG GAC A? G AAA TCA G? T GTC ATC AAT CTT ACÁ GAA CAA GTA < : ?? TGG 2123 Leu Aßp Lyß Lyß Ser Aßp Val He Aßn Leu Thr Glu Qln Val (A Trp 675 680 685 GTC AAA GTC AAT A A C C ATG ACG G C T C T C C ATT GT CAC VAT OCT 2171 Val Lyß Val Aan Thr Aßn Met Thr Gly Tyr Tyr He Vßl Hiß Tyr Wing 690 695 700 705 CAT GAT GGC TGG GCA GCT CTA ATC AAC CAO TTA AAA AG? AAT CCC TAT 2219 Hiß Asp Gly Trp Ala Ala Leu He Aßn Gln Leu Lyß Arg Aßn ro Tyr 710 715 T20 GTT CTG AGT GAC A? A GAC COA OCC AAC CTO ATC AAT AAC ATC ITT GA? 2267 Val Leu Ser Asp Lyß? ßp? Rg Ala? ßn Leu He Aßn Aßn He Phe Glu 725 730 735 CTT OCA GOC CTT GOC AAA OTO CCT CTT CAO ATG GC? TTC G? T TTO? TT 2315 Leu Wing Gly Leu Gly Lyß Val Pro Leu Gln Met Wing Phe Aßp Leu Zl «740 745 750 GAC TAC CTT AG? A? T GAO ACC CAC ACT GCA CCT ATC ACT OA? GCC CTO 2363 Asp Tyr Leu? Rg? ßn Glu Thr Hiß Thr? The Pro He Thr ßlu? The Leu 755 760 765 TTC CAO ACT QAC CTC ATC T? T? AT CTC CTO OA? A ?? CTG OO? OU7 ATO 2411 Phe Gln Thr? Ap Leu He Tyr? ßn Leu Leu Olu Lyß Leu Oly Hiß Met 770 775 780 785 GAC CTG TCC TCA AG? TT? GTO ACC AGA GTA CAT A ?? TTO CTC C \ Q ?? C 2459 Aßp Leu Ser Ser Arg Leu Val Thr Arg Val Hiß Lyß Leu Leu Gln Aßn 790 795 830 CAÁ ATC CAO CAG CAO AC? TCG? CA GAT GA? GGC ACÁ CCA TCC ATO COA 2507 Gln He Gln Gln Cln Thr Trp Thr Asp Glu ßly Thr Pro Ser M.Jt? Rg 805 810 815 GAG CTT COO TCA GCC TTO CTO GA? TTT GCC TOC GCC C? C? GC CP? CAO 2SS5 Glu Leu Arg Ser? Leu Leu Olu Pha Ala Cy?? Hi? Ser Ltiu Olu 820 825 830 AAC TGT ACC ACT ATO GCC AC? AAG CTG TTT GAT GGT TGG ATG GC? TC? 2603 Asn cyß Thr Thr Met Ala Thr Lys Leu Phß Aßp Gly Trp Met? Ser 835 840 845 AAT GG? ACT CAO? OC CTG CCG ACT CAC OTC ATO ACC ACT GTO TC? AA 2651 Aan Gly Thr Gln Ser Leu Pro Thr Asp Val Mee Thr Thr Val? He Lyß T50 855 860 865 GTT GGA GC? AGA ACC GAO A? A GGC TGQ TTO TTC CTC TTT AGC ATO TAC 2699 Val Gly Ala Arg Thr Glu Lyß Gly Trp Leu Phe Leu Phß Ser Met Tyr 870 875 (180 TCC TCC ATO GOC TCT GAA GCA GAO A? G GAT AAA ATA CTT GA? CCC CTG 2747 Being Smr Met Gly Being Glu? The Clu Lys Asp Lya He Leu Glu? The Leu 885 890 895 GCC AGC TCA GCG GAT GC? C? T A ?? CTT TAC TOG TTA ATO AAA? OT AGC 2795 Wing Being Wing Asp? Hi? Lys Leu Tyr Trp Leu Met Ly? Ser Ser 900 905 910 CTT GAT GGT GAT ATC ATG CGG? CA C? G AAO TTG TCA CTT ATC? 'TT AOA 2843 Leu Asp Gly? «P He He Arg Thr Gln Lyß Leu Ser Leu He I e Arg 915 920 925 ACÁ GTG GGC AO? CAO TTT CCT GGA CAT TTG CTO GCA TOC GAT TTT GTT 2891 Thr Val Gly Arg Gln Phe Pro Gly His Leu Leu Wing Trp? Ap Phe Val 930 935 940 945 AAQ GAA AAC TGG AAT AAO CTT GTA CAT AAG TTC CAT CTO GOC TCC T? T 2939 Lys Glu Asn Trp Asn Lys Leu Val Hiß Lya Phe His Leu Gly Ser Tyr 950 955 960 ACC ATT CA? AOC ATT GTT GCT GOA TCT ACT CAC TTA TTT TCA AC3 AAG 2987 Thr He Gln Ser He Val Wing Gly Ser Thr His Leu Phe Smr T r Lyß 965 970 975 AC? CAT TTA TCT GAG GTC CAG GA? TTC TTC OR? A A? T CAO TC? GAO GCA 3035 Thr Hiß Leu Ser Glu Val Gln Glu Phe Phe Glu Aßn Gln Ser Glu Wing 980 985 990 ACC TTO CAG CTT CGG TOT GTT CAG GAO GCC TTC O? A GTG ATT OAC¡ CTO 3083 Thr Leu Gln Leu Arg Cys Val Gln Glu Ala Phe Glu Val He Glu Leu 995 1000 1005 AAT ATC CAG TGG ATO GCC AOO? AT CTG? A? ACT CTG AC? CTO TGO CTO 3131 Even He Gln Trp Met Wing Arg Aßn Leu Lys Thr Leu Thr Leu Trp Leu 1010 1015 1020 1025 TAGCCCTCAC AGCTGATCTT CCGGTGCCC? TGGCTCTGCT OCTTTTGCAA AGGTTG? GTO 3191? AGGCCGGCC TGCTACTG? G TTGTTTGCAC TGTTAOG? TC TAGTTAGCTC? GGQOCC? AT 3251 TOTATTTTTC ATATCTTTTC TGAAATGTCC TTAGOCGGTA GTTATTTATT? C? A? TTAT 3311 ATTC? CCTGT ACGTCA? CCA TCT? C? AT? A CAOTO? AGAC CTGCCCGCCC OGCCCJCTCGA 3371 GCCCTATAGT GAOT 33 B5 (18) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 18 amino acids (B) TYPE: amino acid (ii) TYPE OF MOLECULE: (A) Description: - peptide fragment of GTVap with homology with the second repeat in the GTVap (Seq ID No. 18) and Glut4 (Seq ID No. 19) and which is provably involved in retention and classification of an insulin sensitive compartment. (iii) HYPOTHETICAL: yes (ix) CHARACTERISTICS: (A) NAME / KEY: retention signal for the insulin-sensitive compartment (C) IDENTIFICATION METHOD: similarity with another sequence in GTVap and the sequence in Glut4 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: His Pro Leu Glu Pro Asp Glu Val Glu Tyr Glu Pro Arg Gly Ser Arg 5 10 15 Leu Leu (19) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 17 amino acids (B) TYPE: amino acid (ii) TYPE OF MOLECULE: (A) Description: - peptide fragment of GTVap with homology with the first repeat in GTVap (Seq ID NO 17) and Glut4 (Seq ID NO 19) and that is provably involved in the retention and classification of an insulin-sensitive compartment. (iii) HYPOTHETICAL: yes (ix) CHARACTERISTICS: (A) NAME / KEY: retention signal for the insulin-sensitive compartment (C) METHOD "IDENTIFICATION: similarity with another sequence in GTVap and the sequence in Glut4 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: His Glu Met Asp Glu Asp Glu Glu Asp Tyr Glu Be Ser Ala Lys Leu 5 10 15 Leu (20) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 21 amino acids (B) TYPE: amino acid (ii) 'TYPE OF MOLECULE: (A) Description: -glut4 peptide fragment with homology with two repeats in GTVap (Seq ID NOs 17 and 18), and which is probably involved in the retention and classification of a compartment sensitive to insulin. (iii) HYPOTHETICAL: yes (ix) CHARACTERISTICS: (A) NAME / KEY: Retention signal for an insulin sensitive compartment (C) IDENTIFICATION METHOD: similarity with the sequences in GTVap (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: Leu Leu Glu Gln Glu Val Lys Pro Ser Thr Glu Leu Glu Tyr Leu Gly 5 10 15 Pro Asp Glu Asn Asp 20 (21) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 14 to 22 amino acids (B) TYPE: amino acid (ii) TYPE OF MOLECULE: (A) Description: - peptide. (iii) HYPOTHETICAL: yes (ix) CHARACTERISTICS: (A) NAME / KEY: forward consensus of the insulin-sensitive retention sequence common to GTVap and GLUT4 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: Xxaa Xaa (X1aa) 3 (Xaa) 2-3 Tyr Glu (Xaa) 1-3 Ser (Xaa) o-? X2aa (Xaa) < M Leu Leu (22) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 14 to 22 amino acids (B) TYPE: amino acid (ii) TYPE OF MOLECULE: (A) Description: - peptide. (iii) HYPOTHETICAL: yes (ix) CHARACTERISTICS: (A) NAME / KEY: reverse consensus of the insulin-sensitive retention sequence common to GTVap and GLUT4 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: Leu Leu (Xaa) 0-4 X2aa (Xaa) 0-? Ser (Xaa) 1-3 Glu Tyr (Xaa) 2-3 (X1aa) 3 Xaa Xxaa It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (31)

1. A protein, characterized in that (a) is a component of the vesicles that contain GLUT4 in the natural state; (b) it has aminopeptidase activity; (c) has a molecular weight of about 110 kD in its deglycosylated form; (d) comprises the amino acid sequences Phe-Ala-Ala-Thr-Gln-Phe-Glu-Pro-Leu-Ala-Ala (SEQ ID NO: 1) and Ile-Leu-Gln-Asn-Gln-Ile-Gln -Gln-Gln-Thr-Arg-Thr-Asp-Glu-Gly-Xaa-Pro-Xaa-Met (SEQ ID NO: 2); and (e) reacts with the antibodies raised against the peptide identified as (SEQ ID NO: 1); and muteins of it.

2. The protein according to claim 1, further characterized in that it is encoded by the cDNA sequence shown as (SEQ ID NOs 15 and 16) and is essentially identical to the sequence of the protein estimated for GTVap (SEQ ID No. 15). and 16).

3. The protein according to claim 1, further characterized in that (a) has optimal activity at neutral pH; (b) its relative activity towards synthetic p-nitroanilide amino acid substrates is: leucine »proline, alanine > valine, glycine; (c) its activity is modulated by divalent ions of Co, Zn, Mg, Mn, and Ca; (d) the minimum inactivation temperature is between 40 ° C and 50 ° C; (e) its activity is stabilized by Ca ions; (f) its activity is reduced by phenanthroline, dipyridyl, leutiol, amastatin, actinonin, and bestatin; (g) has at least three glycosylated forms having molecular weights of approximately 165 kD, 155 kD, and 120 kD, respectively.

4. The protein according to claim 3, further characterized in that its relative activity towards the synthetic β-naphthylamide amino acid substrates is: leucine > Lysine > arginine > methionine > Alanine > phenylalanine

5. A method for purifying GTVap and separating the glycosylated species therefrom, characterized in that it comprises the following steps: (a) extracting the GTVap from at least one source; (b) contacting the GTVap extract with lectin affinity resin to absorb the glycosylated GTVap; (c) eluting the glycosylated GTVap from the lectin affinity resin; (d) contacting the eluate of the lectin affinity resin with a chelation chromatography resin comprising a metal ion that acts simultaneously with the resin and the GTVap; (e) collecting unbound material from the chelation chromatography resin; (f) eluting the bound material from the chelation chromatography resin; (g) separately contacting the fractions containing the unbound and eluted GTVap from the chelation chromatography resin with anion exchange resin; (h) eluting the bound GTVap species from the anion exchange resin; and (i) detecting the GTVap species separated from the anion exchange resin.

6. The method according to claim 5, characterized in that the step of extracting the GTVap is carried out using a mixture of at least one metal ion stabilizer and a detergent.

7. The method according to claim 6, characterized in that the stabilizer is a calcium ion.

8. The method according to claim 5, characterized in that in the step of contacting the eluate the lectin affinity resin with a chelation chromatography resin, the zinc ion is used as the metal ion.

9. The method in accordance with the claim 5, characterized in that the step of separately contacting the fractions containing the non-bound and eluted GTVap of the chelation chromatography resin with anion exchange resin, the anion exchange resin contains quaternary ammonium groups.

10. A method for identifying modulators of GTVap activity, characterized in that it comprises the following steps: (a) providing GTVap or the material containing GTVap having a testable amount of enzymatic activity; (b) incubating the GTVap or the material containing GTVap with a test substance to be assayed for its ability to modulate the activity of the GTVap; (c) adding a GTVap substrate; (d) verify the activity of the GTVap as a function of time; and (e) determining the modulating effect of the test substance on the GTVap.

11. The method according to claim 10, characterized in that the GTVap or the material containing GTVap having an assayable amount of enzymatic activity is obtained from adipose tissue, skeletal muscle tissue, cardiac muscle, cell lineages derived from those tissues, or from a recombinant source.

12. The method according to claim 10, characterized in that in the step of adding a GTVap substrate, leucin paranitroanilide or a polypeptide substrate is employed.

13. An antibody specific for GTVap, produced using substantially pure GTVap or a fragment thereof.

14. A method for determining GTVap in biological material, characterized in that it comprises the following steps: (a) preparing a specimen of biological material to optimally expose the immunoreactive epitopes; (b) incubating the specimen or an extract thereof with the antibody specific for GTVap; (c) removing unbound antibodies from the specimen; and (d) quantify the antibodies bound to the GTVap in the specimen.

15. The method according to claim 14, characterized in that the incubation step of the antibody specific for GTVap is the antibody GTVapl.

16. An oligonucleotide probe, characterized in that it is specific for a nucleic acid sequence encodes a segment of the GTVap.

17. The oligonucleotide probe according to claim 16, characterized in that the oligonucleotide is selected from the group consisting of oligonucleotides having the following sequences: 5 'TTY GCN ACN CAR TTY GAR CCN YTN GCN 3' GCN (SEQ ID NO: 3 ); 5 'NGC NGC NAR NGG YTC RAA YTG NGT NGC NGC RAA 3' (SEQ ID NO: 4); 5 'ATH YTN CAR AAY CAR ATH CAR CAR CAR ACN MGN ACN GAY GAR GGN 3' (SEQ ID NO: 6) 5 'NCC YTC RTC NGT NCK NGT YTG YTG YTG DAT YTG RTT YTG NAR DAT 3 '(SEQ ID NO: 7) and fragments thereof; wherein N is A, T, C, G, or I (inosine); R is A or G; And it is C or T; M is A or C; "K is G or T; S is C or G; W is A or T; H is A, C, or T; B is C, G, or T; V is A, C, or G; and D is A, G, or T.

18. A method for determining a nucleic acid sequence encoding a segment of the GTVap in a biological specimen, characterized in that it comprises the following steps: (a) preparing a biological specimen for analysis of the nucleic acid material: (b) incubating the nucleic acid material of the biological specimen with an oligonucleotide probe specific for a nucleic acid sequence encoding a segment of GTVap; (c) removing the unbound oligonucleotide probe from the nucleic acid material; (d) determining the oligonucleotide probe bound to the nucleic acid sequence encoding a segment of the GTVap.

19. A nucleic acid, characterized in that it essentially consists of a nucleic acid encoding a GTVap having the amino acid sequence described in Figure 20 (SEQ ID Nos. 15 and 16) or a nucleic acid capable of hybridizing to the complement thereof under stringent conditions and coding for a GTVap.

20. The purified nucleic acid according to claim 19, characterized in that the purified nucleic acid is present in a vector.

21. The purified nucleic acid according to claim 19, characterized in that the purified nucleic acid is present in a cell.

22. The purified nucleic acid according to claim 19, characterized in that the nucleic acid is under the transcriptional control of a heterologous promoter.

23. A homogeneous cell population, characterized in that each of the cells comprises a cloned nucleic acid encoding a GTVap having the amino acid sequence described in Figure 20 (SEQ ID Nos, 15 and 16) or a nucleic acid capable of hybridizing to the complement it under strict conditions that codes for the GTVap.

24. The homogeneous cell population according to claim 23, characterized in that each cell is a mammalian cell.

25. A method for producing GTVap characterized in that it comprises the step of: culturing the cells according to claim 23 in medium to form a population of cells expressing GTVap, and purifying the GTVap from the cells or from the culture medium.

26. A polypeptide, characterized in that it encodes the retention sequence of a protein, and is selected from the group consisting of a first polypeptide comprising the following residual amino acid sequence: -X1aa-Xaa- (Xxaa) 3- (Xaa) 2.3-Tyr -Glu- (Xaa)? -3-Ser- (Xaa) 0 -? "X2aa- (Xaa) 0-4-Leu-Leu- (SEQ ID NO: 20), and a second polypeptide, comprising the following sequence of residual amino acids: -Leu-Leu- (Xaa) 0-4 ~ X2aa- (Xaa) 0 -? - Ser- (Xaa)? _3-Glu-Tyr- (Xaa) 2-3- (Xxaa) a-Xaa -X ^ a- (SEQ ID NO: 21 *), where Xaa represents any residual amino acid, Xaa represents Pro, Glu, or Asp, and X2aa represents Arg or Lys, and muteins thereof.

27. The polypeptide according to claim 26, characterized in that the retention sequence is: His Pro Leu Glu Pro Asp Glu Val Glu Tyr Glu Pro Arg Gly Ser Arg Leu Leu (SEQ ID NO 17); His Glu Met Asp Glu Asp Glu Glu Asp Tyr Glu Ser Be Ala Lys Leu Leu (SEQ ID NO 18); o Leu Leu Glu Gln Glu Val Lys Pro Ser Thr Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp (SEQ ID NO 19).

28. A method for identifying an insulin-sensitive retention sequence of a protein other than GTVap whose classification is regulated by insulin characterized in that it comprises determining the amino acid sequence of such a protein, comparing the sequence with the amino acid sequence of the polypeptide set forth in claim 26 and identify an amino acid sequence that contains or is homologous to the amino acid sequence of the polypeptide.

29. A method for identifying an intracellular sorting protein or an active fragment thereof which binds to an insulin-sensitive retention sequence of a second protein, the method is characterized in that it comprises: a) incubating the intracellular sorting protein with the sequence of retention under favorable conditions for the binding between the sorting protein and the retention sequence, and b) identify the specifically bound sorting protein.

30. A method to identify a compound capable of altering the intracellular classification of at least one of GLUT4 and GTVap, the method is characterized in that it comprises: a) incubating an intracellular sorting protein or active fragment thereof with the polypeptide retention sequence according to claim A under favorable conditions for binding between the sorting protein and the sequence of retention, in the presence and absence of a test compound; b) quantify the specifically bound complexes of the sorting protein and the retention sequence; and c) comparing the degrees of complex formation in the presence and absence of a test compound.

31. A method for treating insulin resistance syndromes, characterized in that it comprises administering to a subject exhibiting an insulin resistance syndrome an effective amount of a GTVap modulator in a pharmaceutically acceptable carrier.