JPH06184199A - Target protein of low-molecular weight g protein rab3a p25 - Google Patents

Abstract

PURPOSE:To obtain an amino acid sequence in the target protein of a low- molecular weight G protein rab3A p25 and a DNA base sequence capable of coding the amino acid sequence thereof. CONSTITUTION:The target protein of a low-molecular weight G protein rab3A p25 is extracted and isolated from a bovine cerebral membrane to determine the partial amino acid sequence thereof. An oligonucleotide synthesized on the basis thereof and a bovine cDNA library are used to determine the DNA base sequence capable of coding the whole amino acid sequence thereof.

Description

Detailed Description of the Invention

[0001]

[Industrial application] The present invention relates to the target protein of the low molecular weight G protein rab3A p25 and the DNA encoding it.
It is effectively used in the field of medicine.

[0002]

2. Description of the Related Art Living cells respond to hormones, neurotransmitters and the like, and exchange physiological information with each other to express physiological functions. In cell signal transduction, factors are first bound to surface receptors of target cells, and then receptors across the cell membrane are activated to mediated via internal effector molecules, and the signal transduction is completed through many processes. In recent years, breakthroughs have been made in this information transduction mechanism, and GTP-binding proteins (abbreviated as G proteins below) function as transducers that transmit receptor stimulation to effector molecules, resulting in hormone secretion, muscle contraction, and recognition. It is known to play a central role in cell activities leading to More recently, another type of G has been involved in the reaction process after stimulation transmission of cell membrane receptors.
It became clear that proteins were involved. This G protein is a group of low molecular weight G proteins having a molecular weight of about 20,000, which is different from the group of G proteins functioning as a transducer of cell membrane receptors. In other words, regarding the secretory mechanism of cells, when the secretory protein synthesized in the cytoplasm is secreted to the outside of the cell, the secretory reaction of vesicle formation, fusion of vesicles with cell membrane, and extracellular release of secretory protein It was found that the low molecular weight G protein is deeply involved (Taka
i, Y. et al., Int. Rev. Cytol., 133, 187-230, 199
2).

So far, ras (Ha-, Ki-, N-),
Genes encoding more than 40 kinds of low molecular weight G proteins such as rho (A, B, C), ral, and rab (1-4) were cloned from mammalian tissues to yeast, and the proteins of these genes were also purified sequentially. There is. The present inventors separated 15 kinds of low molecular weight G proteins from bovine cerebrum and succeeded in homogenizing them. Among these, a new low molecular weight G protein is rab3A p25 (Ki
kuchi, A. et al., J. Biol. Chem., 263, 2897-2904,
1988). rab3A p25 belongs to the rab subfamily, which is a subfamily of small G proteins similar to ras p21.
This rab family has been shown to be involved in the regulation of intracellular vesicle transport such as secretory response and endocytosis (Balch, WB et al., Trends Biochem. Sci., 1
5,473-477, 1990). rab3A p25 is present in the presynapse, and is especially distributed in presynaptic membranes and synaptic vesicles near the active zone.
It is becoming clear that it regulates neurotransmitter release from presynapses (Mizoguchi, A. et al., JB
iol. Chem., 265, 11872-11879, 1990). rab3A p25 has
There are inactive type of GDP binding type and active type of GTP binding type,
The exchange reaction from GDP-bound type to GTP-bound type controls the conversion from GDP-bound type to GTP-bound type.
It is controlled by rotein (GEP). We have rab
As a GEP that acts on 3A p25, smg p25 GDI (GDP dissociation inhibitory protein) was purified from the soluble fraction of bovine cerebrum to determine its primary structure. Furthermore, we have found that smg p25 GDI
However, it not only regulates the GDP / GTP conversion reaction of rab3A p25 but also translocates it from the membrane fraction to the soluble fraction of rab3A p25.
It has been shown that it regulates cations and also acts on rab11 p24 and SEC4 p24 belonging to the rab family. Based on the findings to date, the present inventors have
The mechanism of action of rab3A p25 was considered as follows. GT
P-linked rab3A p25 forms a complex with smg p25 GDI, exists in the synaptic cytoplasm, and dissociates with rab GDI.
rab3A p25 is converted to GTP-bound form and binds to target proteins on synaptic vesicles. This GTP-bound rab
The synaptic vesicle bound to 3A p25 targets and fuses to the synaptic membrane.

The vesicle transport pathway is very tightly controlled, and many low molecular weight G proteins are involved in vesicle transport, which is necessary for different vesicles to target different acceptor membranes. Is estimated to be
However, the target protein has not yet been isolated and its structure has been confirmed, and confirmation of the structure of the target protein has become an important issue for elucidation of the secretory mechanism and relationship with disease.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides an amino acid sequence and a cDNA encoding the same by isolating and structurally analyzing the target protein of the low molecular weight G protein rab3A p25.

[0006]

[Means for Solving the Problems] In view of the above situation, the present inventors found a protein that binds to rab3A p25 from a bovine cerebral membrane fraction and partially purified it (Sirataki, H., et al., J. B.
iol. Chem., 267, 10946-10949, 1992). This protein has a molecular weight of 85,000 and binds to GTP-bound rab3A p25,
Low affinity for GDP-bound rab3A p25. Also c-ki-
It does not bind to low molecular weight G proteins such as ras p21, rhoA p21, smg p21 and rab11 p24. Furthermore, this protein is GAP
(GTPase activity promoting protein), GDI (GDP dissociation suppressing protein that suppresses GDP / GTP exchange reaction), and GDS (GDP dissociation promoting protein that promotes GDP / GTP exchange reaction) are not active. It is likely to be a target protein.

Therefore, the inventors of the present invention have eagerly tried to analyze the nucleotide sequence encoding this target protein, and have completed the present invention by elucidating the entire structure. As a result of primary structure analysis, the target protein of rab3A p25 of the present invention is a novel protein consisting of 704 amino acids and having a molecular weight of 77 and 976, and the present inventors named it rabphilin-3A. Hereinafter, this target protein is described as rabphilin-3A. That is, the present invention is rab
The present invention relates to the entire amino acid sequence of philin-3A, the DNA encoding it, and the anti-rabphilin-3A antibody. rabphi
From the function of lin-3A, it is expected to be applied as a reagent for elucidating the neurotransmitter secretion mechanism and pathology of various brain diseases, and further for treatment and diagnosis of the same diseases. The present invention will be described in detail below.

(1) Purification of rabphilin-3A Our method (Sirataki, H., et al., J. Biol. C
him., 267, 10946-10949, 1992), a crude membrane fraction is obtained by cholic acid extraction using bovine cerebrum as a raw material. This crude membrane fraction was used as a buffer containing cholate in buffer A (20 mM HEPES-NaOH, pH
7.4, 5 mM MgCl ₂ , 1 mM dithiothreitol) is used for purification with a Q-Sepharose column by a salt gradient extraction method. Use CHAPS-containing Buffer A for the obtained target fraction
A highly purified product can be obtained by purification with a hydroxyapatite column using a Mono Q column, CHAPS and Buffer A containing KH ₂ PO ₄ and then 5-20% sucrose density gradient ultracentrifugation. Finally, this high-purity rabphilin−
Fractionate 3A by SDS-PAGE and transfer to a PVDF membrane (polydenylidenedifluoride membrane). After transfer, it was stained with Ponceau S and the band corresponding to rabphilin-3A was cut out and isolated.

(2) Method for identifying rabphilin-3A In the above purification process, rabphilin-3A is identified by measuring the binding property to rab3A p25 by the cross-linking method. That is, radioiodinated GTPγS
The bound rab3A p25 was reacted with the fractionated fractions from each column, and the reaction mixture was subjected to SDS-PAGE (10% polyacrylamide gel), and the presence or absence of a band with a molecular weight of about 110,000 was measured to detect rabphilin-3A. Can be identified and confirmed. Escherichia coli-derived rab3A p25 can be prepared from Escherichia coli overexpressing rab3A p25 by the method of Araki et al. (Araki, S. et al., Mol. Cell. Biol.,
11, 1438-1447, 1991).

(3) Structural analysis of rabphilin-3A (i) Partial peptide amino sequence rabphilin-3A obtained by the above method was achromobac
It is possible to determine the partial peptide amino acid sequence by degrading with ter pretease I, subjecting the treated solution to C ₈ reverse phase column chromatography, collecting the peaks of the appearing peptides, and analyzing the amino acid sequence with an amino acid sequencer. B) Cloning and structural analysis of rabphilin-3A cDNA Based on the partial amino acid sequence of rabphilin-3A, select an appropriate one and synthesize an oligonucleotide corresponding to it and use it as a probe. The rabphilin-3A cDNA can be determined by selecting clones that hybridize with these probes from the bovine cerebral cDNA library and performing base structure analysis. The method for cloning is the plaque hybridization method (Sambrook, J. et al., Molecul.
ar cloning, a laboratory manual. Cold Spring Harbo
r Laboratory, Cold Spring Harbor, NY 1989), and the selected clones are subcloned into a plasmid or a phage vector as required. The structure of the nucleotide sequence is Maxam, Gilbert method (Maxam,
AM and Gilbert, W., Proc. Natl. Acad. Sci. USA,
74, 560, 1977) or dideoxy method (Messing, J. et.
al., Nucl. Acids Res., 9, 309, 1981). Finally, the rabphilin-3A cDNA was determined as SEQ ID NO: 1 and its amino acid sequence was deduced as described in SEQ ID NO: 1.

(4) Preparation of anti-rabphilin-3A antibody As the antigen, rabphilin-3A or a peptide having a partial structure based on the amino acid sequence thereof is used. For the preparation of a complex of an antigen and a carrier protein, a conventional condensing agent can be used, and a commonly used protein such as bovine serum albumin, thyroglobulin and hemocyanin can be used. Examples of animals to be immunized include mice, rats, rabbits, and guinea pigs. The method of inoculation is subcutaneous, intramuscular or intraperitoneal. Upon administration, complete Freund's adjuvant and incomplete Freund's adjuvant may be mixed, and the administration is usually performed once every 2 to 5 weeks. The polyclonal antibody can be collected from blood or serum from a fully immunized animal. The antibody can be purified by a known method and can be easily achieved by means such as ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, use of anion exchanger, and affinity chromatography. The monoclonal antibody can be prepared by a known method. For example, antibody-producing cells obtained from the spleen or lymph node of an immunized animal are fused with myeloma cells and isolated as a hybridoma. Myeloma cells include mice, rats,
Those derived from humans and the like are used, and those derived from the same species as the antibody-producing cells are preferable, but it may be possible between different species.

The operation of cell fusion is known, for example, the method of Kohler and Milstein (Nature, 256, 495, 1975).
It can be carried out according to. Examples of the fusion promoter include polyethylene glycol and Sendai virus. Usually, polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of about 20 to 50% is used at 20 to 40 ° C, preferably 30 to
Cell fusion can be carried out by reacting at a temperature of 37 ° C. for a ratio of the number of antibody-producing cells to the number of myeloma cells of usually about 1: 1 to 10: 1 for about 1 to 10 minutes. Anti-rab3A
Various immunochemical methods can be used for screening p25 antibody-producing hybridomas. For example, an ELISA (enzyme-based assay) using a microplate coated with rab3A p25.
Linked immunosorbent assay) method, EIA (Enzyme) using microplate coated with anti-immunoglobulin antibody
immunoassay) method and the like. Wells producing the desired antibody are obtained by these immunochemical techniques. From such wells, cloning is further performed by, for example, the limiting dilution method to obtain a clone. Hybridomas are usually selected and bred by adding HAT (hypoxanthine, aminopterin, thymidine), and using an animal cell medium containing 10 to 20% fetal bovine serum (eg, RPMI 1640).

The clones thus obtained were transplanted into the abdominal cavity of BALB / C mice to which pristane had been administered in advance, and 10 to 14 days later, ascites containing a high concentration of monoclonal antibody was collected and used as a raw material for antibody purification. can do. Alternatively, the clone can be cultured and the culture can be used as a raw material for antibody purification. For the recovery of the monoclonal antibody, the above method may be used as the immunoglobulin purification method.
Immunoassays using these antibodies enable the identification and quantification of human prohibitin in human biological samples, and are expected to be applied to cancer diagnostic reagents. The immunological measurement method of human prohibitin may be based on a known method, for example, a fluorescent antibody method, a passive agglutination reaction method, an enzyme antibody method and the like. The monoclonal antibody of the present invention is Ig
G, IgA, IgM may be of any class, and the Fc 'or Fab' or Fab in which the Fc region is removed from the antibody
Fractions or polymers thereof may be used. Further, it may be a chimeric antibody thereof.

(5) As a result of structural analysis of rabphilin-3A of the present invention, it was found to have hydrophobic moieties at two positions near amino acid numbers 105 and 590. Also the rabphilin-3A amino acid number
The sequences at positions 396-530 and 548-669 are protein k
It was found to have a sequence with a homology to the C ₂ region present in inase C and synaptotagmin (Perin, MS
et al., Nature, 345, 260-263, 1990). synaptotagm
In is a substance that is present in synaptic vesicles and is considered to be involved in the release of neurotransmitters, and has two C ₂ regions on the C-terminal side. rabphilin-3A is C
It is a target protein of rab3A p25, a low molecular weight G protein that has two C ₂ regions on the end side and is considered to be involved in secretory reaction, and is a neurotransmitter because it is specifically distributed in brain tissue. It is expected to be involved in the release of erythrocyte and is closely related to various brain diseases.

[0015]

The present invention will be described in detail and specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Identification of rabphilin-3A (Cross-linking Assay) Radioiodinated GTPγS-binding rab3A p25 (1
pmol, 2.2 −2.6 × 10 ⁵ cpm) 45 μl of sample solution
(22.2mM HEPES / NaOH pH 7.4, 5.56mM MgCl 2, 1.11 μM
GTPγS, 222mM NaCl, 0.056% Nacholate) and 30 ℃ 5
After reaction for 20 minutes, 20 mM disuccinimidyl suberate / 20% DMSO
Add 5 μl. Continue the reaction at 30 ° C for 30 minutes to obtain 25 μl.
Solution (200 mM Tris / HCl pH 6.7, 9% SDS, 6% 2-
The reaction was stopped by adding mercaptoethanol and 15% glycerol). SDS-PAGE of 60 μl of the reaction mixture
After applying (10% polyacrylamide gel), Fujix BA
Radioactivity was measured by S 2000 Bio-imaging analyzer (standard is radioiodinated rab3A p25). rab3A p25
Can be identified by being detected at the position of molecular weight 25,000, and when it is bound to rabphilin-3A, it is detected at the position of molecular weight 110,000. rab3A p25 is prepared from Escherichia coli, and radioactive iodine can be labeled using Bolton-Hunter reagent (Shirataki, H. et al., J. Biol. Chem.,
267, 10946-10949, 1992). In the following purification process of rabphilin-3A, each purified fraction was treated by this method.
The in-3A was identified.

Example 2 Purification of rabphilin-3A Using bovine brain as a raw material, the method of Kikuchi et al. (Kikuchi, A. et.
al., J. Biol. Chem., 263. 2897-2904, 1988) to prepare a crude membrane fraction. This crude membrane fraction (800ml, 9.6g
(protein) was centrifuged at 20,000 × g for 1 hour to obtain a pellet. 320 ml of 2% sodium cholate Buffer A (20 mM HEPES / NaON pH7.4, 5 mM MgCl ₂ ,
1 mM dithiothreitol) 100,000 × g, 1
Centrifuge for 2 hours to remove insoluble matter and remove the supernatant (320 ml, 2.9
g protein) was subjected to the following purification. 80% of supernatant is 1%
Hil equilibrated with Buffer A containing sodium cholate
Load on an oad 26/10 Q-Sepharose column (2.6 x 10 cm), wash with 148 ml of the same buffer, and then perform gradient elution of a 0-1.0 M sodium chloride gradient using the same buffer (600 ml). 37-39 were obtained. The remaining 240 ml of the supernatant was pooled by performing the same operation. After concentration of this active fraction, 0.6
A solution (144 ml, 72 mg protein) obtained by adding Buffer A containing% CHAPS and removing sodium cholate was loaded on a Mono Q HR 10/10 column (1 × 10 cm) equilibrated with Buffer A containing 0.6% CHAPS. After washing with 56 ml of the same buffer,
The same buffer (240 ml) was used for elution with a 0-0.5 M sodium chloride concentration gradient, and 2 ml each was collected to obtain an active fraction 128-141. Next, this fraction (28 ml, 1.4 mg protein) was loaded onto a hydroxyapatite column (0.8 × 20 cm) equilibrated with Buffer A containing 0.6% CHAPS and 50 mM KH ₂ PO ₄ , and 0.6%
Buffer A containing CHAPS and 80 mM KH ₂ PO ₄ After washing with 42 ml of 0.6% CHAPS and 120 mM KH ₂ PO ₄ Buffer A
Was eluted with 1.5 ml of each fraction to obtain active fractions 49-52.
This active fraction (6 ml, 10 μg protein) was concentrated to 1.2 ml and subjected to 5-20% sucrose concentration gradient ultracentrifugation (tube volume).
4.8ml) 238,000 × g, perform for 19 hours, from the bottom of the tube
As a result of assaying by collecting 0.25 ml each, fractions 7-10 (6 ml, 5
μg protein) was obtained as high-purity rabphilin-3A. The above purification procedure was repeated 20 times to obtain about 100 μg of rabphilin-3A.
Got Finally, these high-purity products were separated by SDS-PAGE and transferred to a PVDF membrane, then stained with Ponceau S and rabphilin
A band corresponding to −3A was cut out to isolate rabphilin-3A. Rabphilin-3A and rab3A thus obtained
The formation of a complex with p25 was confirmed by the method of Example 1. Rabphilin-3A in the presence of GDP-bound rab3A p25
Appeared at a molecular weight of about 85,000. GTPγS-bound r
In the presence of ab3A p25, rabphilin-3A has a molecular weight of approximately 110,000.
, And part of rab3A p25 moved to the position of molecular weight 110,000. Rabphilin-3A isolated from this
Was confirmed to form a complex only with GTPγS-bound rab3A p25.

Example 3 Analysis of partial amino acid sequence of rabphilin-3A The rabphilin-3A obtained in Example 2 was subjected to Achromopacter pr
After enzymatic degradation (molar ratio 1:50 = API: rabphilin-3A) in 0.2M Tris / HCl pH 9.0, 8% acetonitrile solution with otease I (API) for 24 hours at 37 ° C, C ₈ reverse phase column chromatography. Then, about 30 peptide peaks were collected. Of these 11 peaks, the amino acid sequence was analyzed using an automatic gas phase sequencer (Applied Biosystems, Model 470A). The results are shown in Table 1.

[0019]

[Table 1]

Example 4 Cloning of rabphilin-3A cDNA and analysis of nucleotide sequence Based on the results of the amino acid sequence of the partial peptide of rabphilin-3A of Example 3, the peptide LysMetGlu contained in peak 10 was identified.
GluMetGluGlnGlu and peptide G contained in peak 11
Oligonucleotides encoding luPheAsnGluGluPhePheTyr were converted to DNA synthetic groups (Applied Biosystems, Model 380A).
Was used as a probe. cDN using λgt10
Bovine brain cDNA using A cloning kit (Amersham)
Twenty-four clones hybridizing with both probes were obtained from about 600,000 phage plaques in the library. The plaque hybridization was performed by a known method according to the method of Maniatis et al. The obtained clone is Plas
Recloning was performed using mid pUC 19 and the respective nucleotide sequences were analyzed. A clone containing one of them, a cDNA of about 2.7 kb, was obtained and the nucleotide sequence was determined by the dideoxy method. The base sequence and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 1. This cDNA has an open reading frame consisting of 704 amino acids and is shown in Table 1.
The partial peptide amino acid sequence of rabphilin-3A shown in (3) was included. It also had a Kozak sequence before the start codon ATG. Derived amino acid sequence from rabphil
The molecular weight of in-3A is 77,976, and that of purified rabphilin-3A
The molecular weight obtained from SDS-PAGE and sucrose density gradient centrifugation is close to about 85,000. This suggests that rabphilin-3A consists of a single-chain peptide that does not have a subunit structure. Of the 24 clones isolated, 4 clones hybridized with 2 kinds of probes, one clone had an N-terminal translation region, and the other had a C-terminal translation region. Was. CDNA isolated in this way
In order to examine whether or not rabphilin-3A is encoded, homogenates of cells expressing Cb7 cells transformed with an expression vector containing the cDNA and expressing rabphilin-3A were treated with SDS by the same method as described in Example 5 below. -PAGE after anti-rabph
It was immunoblotted with ilin-3A antibody. The transformation method was to first ligate BamHI and KpnI restriction enzyme sites to the ends of the start and stop codons of the rabphilin-3A cDNA.
A 2.1 kb fragment was generated by PCR. That PCR
The product is cleaved with BamHI and the expression of the foreign gene is SRα promo
It was inserted into the BamHI site of ter-controlled pCEV4 and transfected into COS7 cells by the DEAE dextran method. The cells were collected by culturing for 72 hours after the transfection, and the expression of rabphilin-3A was examined by immunoblotting with an anti-rabphilin-3A polyclonal antibody. As a result, a band was observed at a position of a molecular weight of about 85,000 as in bovine cerebrum rabphilin-3A, and the isolated cDNA was rabphilin-
It was confirmed that it coded 3A.

Example 5 Preparation of anti-rabphilin-3A antibody and tissue distribution of rabphilin-3A From the 18 amino acids excluding the N-terminal His of the peptide of peak 2 from the rabphilin-3A partial amino acid sequence shown in Table 1. Was synthesized and used as an antigen. This synthetic peptide was used to prepare a complex using carrier protein KLH and a condensing agent maleimide active ester, and then mixed with complete Freund's adjuvant, and rabbits were subcutaneously immunized 5 times every 2 weeks to prepare an antibody. Confirmation of antibody production was carried out by immobilizing each antigenic peptide on a microplate, adding blood serum, and then performing an ELISA method using an anti-rabbit immunoglobulin antibody labeled with horseradish peroxidase. The obtained antiserum was purified by applying it to an affinity column against the same antigen to prepare an anti-rabphilin-3A polyclonal antibody. Rabphilin− using this antibody
The tissue distribution of 3A was examined. Various organs of rat (about 0.1
g) with 10 ml of 20 mM Tris / HCl pH 7.5, 0.25 M sucrose, 0.
017% sodium deoxycholate, 10 μM p-amidino
After homogenizing with phenyl-methanesulfonyl fluoride solution, add trichloroacetic acid to a final concentration of 6%. After centrifugation, the precipitate was dissolved in Laemmli's Huffer, heat-treated, and then subjected to SDS-PAGE (12% polyacrylamide gel). After electrophoresis, the protein was transferred to a nitrocellulose membrane and reacted with an anti-rabphilin-3A polyclonal antibody and then with a peroxydis labeled sheep anti-rabbit immunoglobulin antibody to test for the presence of a positive band. As a result, an immunoreactive positive band was found only in the brain, and the pancreas, liver, spleen,
Not found in kidney or lung.

[0022]

INDUSTRIAL APPLICABILITY The present invention provides an amino acid sequence of a novel target protein rabphilin-3A of low molecular weight G protein rab3A p25, a cDNA encoding the same and an anti-rabphilin-3A antibody. Rabphilin-3A is specifically distributed in brain tissues and is involved in the release of neurotransmitters, and its cDNA, including its cDNA, is useful as a research reagent for elucidating the secretory mechanism. In addition, rabphilin of biological samples was examined by immunological techniques using the antibody.
-3A can be identified and quantified, and its application in the field of diagnostic agents is expected by studying the relationship with various cranial nerve diseases.

[0023]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2135 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Bovine Sequence characteristics Characteristic symbol: Peptide Location :twenty one. . 2132 Character that determined the feature: S sequence TGGAGTGCTG GGCTTCAGCC ATG ACT GAC ACC GTG TTC AGC AGC AGC TCA AGC 53 Met Thr Asp Thr Val Phe Ser Ser Ser Ser Ser Ser 1 5 10 CGT TGG ATG TGT CCC AGT GAC CGA CCC CTG CAG TCA AAT GAT AAA GAG 101 Arg Trp Met Cys Pro Ser Asp Arg Pro Leu Gln Ser Asn Asp Lys Glu 15 20 25 CAG CTC CAG ACA GGA TGG TCT GTC CAC CCC AGC GGT CAG CCG GAC AGA 149 Gln Leu Gln Thr Gly Trp Ser Val His Pro Ser Gly Gln Pro Asp Arg 30 35 40 CAG AGG AAA CAG GAA GAG CTG ACA GAT GAG GAG AAG GAA ATC ATC AAC 197 Gln Arg Lys Gln Glu Glu Leu Thr Asp Glu Glu Lys Glu Ile Ile Asn 45 50 55 AGG GTG ATC GCT CGC GCT GAG AAG ATG GAA GAG ATG GAA CAG GAG CGA 245 Arg Val Ile Ala Arg Ala Glu Lys Met Glu Glu Met Glu Gln Glu Arg 60 65 70 75 ATC GGG CGC CTG GTG GAC CGC CTA GAG AAC ATG AGG AAG AAC GTG GCC 293 Ile Gly Arg Leu Val Asp Arg Leu Glu Asn Met Arg Lys Asn Val Ala 80 85 90 GGG GAC GGG GTG AAC CGC TGC ATC CTG TGT GGA GAA CAG CTG GGG ATG 341 Gly Asp Gly Val Asn Arg Cys Ile Leu Cys Gly Glu Gln Leu Gly Met 95 100 105 CTG GGC TCT GCC T GT GTG GTG TGT GAG GAC TGT AAG AAG AAT GTC TGC 389 Leu Gly Ser Ala Cys Val Val Cys Glu Asp Cys Lys Lys Asn Val Cys 110 115 120 ACC AAG TGT GGG GTG GAG ACC TCC AAC AAC CGC CCA CAC CCT GTG TGG 437 Thr Lys Cys Gly Val Glu Thr Ser Asn Asn Arg Pro His Pro Val Trp 125 130 135 CTC TGC AAA ATC TGC ATC GAG CAG AGA GAG GTG TGG AAG CGC TCT GGA 485 Leu Cys Lys Ile Cys Ile Glu Gln Arg Glu Val Trp Lys Arg Ser Gly 140 145 150 155 GCA TGG TTC TTC AAG GGC TTC CCC AAA CAG GTC CTC CCA CAG CCT ATG 533 Ala Trp Phe Phe Lys Gly Phe Pro Lys Gln Val Leu Pro Gln Pro Met 160 165 170 CCC ATA AAG AAG AAC AAG CCC CAG CAG CCA GTC AGT GAG CCT GTG CCC 581 Pro Ile Lys Lys Asn Lys Pro Gln Gln Pro Val Ser Glu Pro Val Pro 175 180 185 GCT GCC CCT GAG CCA GCC ACT CCT GAG CCC AAG CAC CCT GCC CGG GCT 629 Ala Ala Pro Glu Pro Ala Thr Pro Glu Pro Lys His Pro Ala Arg Ala 190 195 200 CCA ACT CGA GGT GAC ACT GAA GAC AGG AGG GGC CCA GGT CAG AAG ACA 677 Pro Thr Arg Gly Asp Thr Glu Asp Arg Arg Gly Pro Gly Gln Lys Thr 205 210 215 GGC C CT GAC ATG ACT TCT GCT CCT GGG CGA GGA AGC TAC GGG CCT CCT 725 Gly Pro Asp Met Thr Ser Ala Pro Gly Arg Gly Ser Tyr Gly Pro Pro 220 225 230 235 GTG CGC AGG GCC TCT GAG GCA CGA ATG AGC TCC TCT GGC CGG GAC TCA 773 Val Arg Arg Ala Ser Glu Ala Arg Met Ser Ser Ser Gly Arg Asp Ser 240 245 250 GAC AGC TGG GAC CAG GGC CAC GGC ATG GCT GCT GGG GAC CCC AGC CAG 821 Asp Ser Trp Asp Gln Gly His Gly Met Ala Ala Gly Asp Pro Ser Gln 255 260 265 AGC CCA GCA GGT TTG AGA CGG GCC AAC TCA GTC CAG GCT TCT AGA CCT 869 Ser Pro Ala Gly Leu Arg Arg Ala Asn Ser Val Gln Ala Ser Arg Pro 270 275 280 GCC CCA GCC TCA ATG CAG AGC CCG GCA CCT CCC CAG CCG GGG CAA CCA 917 Ala Pro Ala Ser Met Gln Ser Pro Ala Pro Pro Gln Pro Gly Gln Pro 285 290 295 GGA CCC CCA GGA GGC AGC AGA CCC AGT CCG GGG CCG ACG GGA CGC TTT 965 Gly Pro Pro Gly Gly Ser Arg Pro Ser Pro Gly Pro Thr Gly Arg Phe 300 305 310 315 CCA GAT CAG AGA CCA GAG GTG GCT CCC AGT GAC CCC GAC TAT ACA GGG 1013 Pro Asp Gln Arg Pro Glu Val Ala Pro Ser Glu Pro Glu Tyr Thr Gly 320 325 330 GCC GCC GCC CAA CCC CGG GAG GAG AGA ACC GGG GGA ATC GGA GGC TAC 1061 Ala Ala Ala Gln Pro Arg Glu Glu Arg Thr Gly Gly Ile Gly Gly Tyr 335 340 345 TCG GCA GCT GGA ACC AGG GAG GAC CGA GCG GGC CAC CCC CCG GGC TCC 1109 Ser Ala Ala Gly Thr Arg Glu Asp Arg Ala Gly His Pro Pro Gly Ser 350 355 360 TAT ACC CAA GCC TCT GCG GCT GCT CCC CAG CCG GTG GTG GCC TCG GCC 1157 Tyr Thr Gln Ala Ser Ala Ala Ala Pro Gln Pro Val Val Ala Ser Ala 365 370 375 CGT CAG CCC CCA CCC CCT GAG GAG GAC GAG GAG GAA GCT AAC AGC TAC 1205 Arg Gln Pro Pro Pro Pro Glu Glu Asp Glu Glu Glu Ala Asn Ser Tyr 380 385 390 395 GAT TCG GAT GAA GCA ACC ACC CTG GGT GCC CTG GAG TTC AGC CTT CTC 1253 Asp Ser Asp Glu Ala Thr Thr Leu Gly Ala Leu Glu Phe Ser Leu Leu 400 405 410 TAT GAC CAG GAC AAC AGC TCC CTG CAC TGC ACC ATC ATA AAG GCC AAG 1301 Tyr Asp Gln Asp Asn Ser Ser Leu His Cys Thr Ile Ile Lys Ala Lys 415 420 425 GGA CTG AAG CCC ATG GAT TCG AAT GGC TTG GCC GAT CCC TAC GTT AAG 1349 Gly Leu Lys Pro Met Asp Ser Asn Gly Leu Ala Asp Pro Tyr Val Lys 430 435 440 CTG CAC CTC CTG CCG GGA GCC AGC AAG TCC AAC AAG CTT CGT ACA AAA 1397 leu His Leu Leu Pro Gly Ala Ser Lys Ser Asn Lys Leu Arg Thr Lys 445 450 455 ACC CTG CGG AAC ACC AGA AAC CCC ATC TGG AAC GAA ACT CTG GTC TAC 1445 Thr Leu Arg Asn Thr Arg Asn Pro Ile Trp Asn Glu Thr Leu Val Tyr 460 465 470 475 CAT GGC ATC ACA GAC GAG GAC ATG CAA AGG AAG ACC CTC AGA ATC TCT 1493 His Gly Ile Thr Asp Glu Asp Met Gln Arg Lys Thr Leu Arg Ile Ser 480 485 490 GTC TGT GAT GAG GAC AAA TTC GGC CAT AAT GAG TTT ATT GGT GAG ACC 1541 Val Cys Asp Glu Asp Lys Phe Gly His Asn Glu Phe Ile Gly Glu Thru 495 500 505 AGA TTC TCC CTC AAG AAA CTG AAG CCC AAT CAG AGG AAG AAC TTC AAC 1589 Arg Phe Ser Leu Lys Lys Leu Lys Pro Asn Gln Arg Lys Asn Phe Asn 510 515 520 ATC TGT CTG GAG CGG GTG ATC CCG ATG AAG CGT GCC GGA ACC ACT GGG 1637 Ile Cys Leu Glu Arg Val Ile Pro Met Lys Arg Ala Gly Thr Thr Gly 525 530 535 TCA GCC CGA GGC ATG GCC CTC TAT GAG GAG GAG CAG GTG GAA CGT ATT 1685 Ser Ala Arg Gly Met Ala L eu Tyr Glu Glu Glu Gln Val Glu Arg Ile 540 545 550 555 GGT GAC ATA GAG GAA CGC GGC AAG ATC CTG GTG TCC CTC ATG TAC AGC 1733 Gly Asp Ile Glu Glu Arg Gly Lys Ile Leu Val Ser Leu Met Tyr Ser 560 565 570 ACA CAG CAG GGA GGC CTC ATC GTG GGC ATC ATA CGC TGC GTG CAC CTG 1781 Thr Gln Gln Gly Gly Leu Ile Val Gly Ile Ile Arg Cys Val His Leu 575 580 585 GCG GCC ATG GAT GCC AAT GGC TAC TCA GAT CCA TTT GTC AAG CTC TGG 1829 Ala Ala Met Asp Ala Asn Gly Tyr Ser Asp Pro Phe Val Lys Leu Trp 590 595 600 CTG AAA CCA GAC ATG GGA AAG AAA GCC AAA CAC AAG ACT CAA ATT AAG 1877 Leu Lys Pro Asp Met Gly Lys Lys Ala Lys His Lys Thr Gln Ile Lys 605 610 615 AAG AAA ACC TTG AAT CCT GAA TTT AAT GAG GAA TTT TTC TAT GAC ATC 1925 Lys Lys Thr Leu Asn Pro Glu Phe Asn Glu Glu Phe Phe Tyr Asp Ile 620 625 630 635 AAA CAC AGT GAC CTG GCG AAG AAG TCA CTG GAC ATC TCA GTC TGG GAC 1973 Lys His Ser Asp Leu Ala Lys Lys Ser Leu Asp Ile Ser Val Trp Asp 640 645 650 TAT GAC ATC GGC AAG TCC AAT GAT TAC ATT GGA GGC TGC CAG CTG GGG 2021 T yr Asp Ile Gly Lys Ser Asn Asp Tyr Ile Gly Gly Cys Gln Leu Gly 655 660 665 ATC TCG GCC AAG GGA GAG CGC TTA AAA CAC TGG TAC GAG TGT CTG AAA 2069 Ile Ser Ala Lys Gly Glu Arg Leu Lys His Trp Tyr Glu Cys Leu Lys 670 675 680 AAC AAG GAC AAG AAG ATC GAA CGC TGG CAC CAG CTA CAG AAC GAG AAC 2117 Asn Lys Asp Lys Lys Ile Glu Arg Trp His Gln Leu Gln Asn Glu Asn 685 690 695 CAC GTG TCG AGC GAT TAG 2135 His Val Ser Ser Asp 700

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/50 T 7055-2J // C12Q 1/68 Z 7823-4B G01N 33/58 A 7055- 2J

Claims (3)

[Claims] 1. A target protein of a low molecular weight G protein rab3A p25 having the amino acid sequence of SEQ ID NO: 1. 2. The low molecular weight G protein rab3A according to claim 1.
DNA of SEQ ID NO: 1 encoding the p25 target protein
. 3. An antibody whose antigen is the target protein or its partial peptide according to claim 1.