HK1136844B - Enhancement of vanadium-containing phosphatase inhibitors - Google Patents

Description

Enhancement of vanadium-containing phosphatase inhibitors

The present invention relates to the biochemistry of phosphatases. In particular, the invention relates to compounds useful as additives which can coexist with vanadium-containing inhibitors of different phosphatases. The compounds used as additives in the present invention enhance the inhibitory effect. Thus, lower concentrations of vanadium-containing compounds can adequately inhibit phosphatases.

Background

Phosphatases are enzymes that hydrolyze phosphate monoesters to phosphate ions and molecules containing free hydroxyl groups. This effect is in contrast to the effect of phosphorylases and kinases which can add phosphate groups to their substrates via chemical energy storing molecules such as ATP.

Phosphatases can be divided into two main categories: metalloenzymes (which require the presence of two or more metal ions at their active site to produce activity) and metalloenzymes. These classes can be further divided into subclasses. Metalloenzymes have hitherto included most phosphatases and include enzymes such as alkaline phosphatase (containing three metal ions, only two of which are catalytically active), serine threonine phosphatase and myoinositol monophosphatase. The best known non-metalloenzymes are protein tyrosine phosphatases, which hydrolyze phospho-tyrosine residues.

The presence or absence of phosphate groups on proteins is known to play a regulatory role in a variety of biochemical pathways, particularly signaling pathways. Tyrosine residues can be linked to phosphate groups (phosphorylated) by protein kinases. In the phosphorylated state, the tyrosine residue is called phosphotyrosine (phosphotyrosine). Tyrosine phosphorylation is considered to be one of the key steps in signal transduction and regulation of enzymatic activity. Phosphotyrosine can be detected by specific antibodies. Together, specific kinases and phosphatases regulate the activity of enzymes, receptors and other components of signal transduction pathways, transcription factors, and other functional proteins.

Thus, there are a variety of biochemical methods for detecting phosphorylated proteins in biological samples. For example, sample cellular material is lysed, protein fractions are separated in one dimension (e.g., SDS-PAGE) or two dimensions (e.g., isoelectric focusing in the first dimension and SDS-PAGE in the second dimension), and phosphorylated protein is detected in each band or spot by phosphoserine or phosphotyrosine specific antibodies. Other specific detection methods use antibodies that specifically bind to a particular phosphorylated protein. Such antibodies are commonly used for histochemical detection of phosphorylated target proteins in formalin-fixed paraffin-embedded tissue sections from biopsy material.

Regardless of the detection method used, it is desirable that the result of the analysis of phosphorylated proteins reflect the time point at which the experiment is initiated, i.e., the phosphorylation state upon cell lysis or tissue fixation and sectioning. More generally, it is desirable to maintain the phosphorylated state of a protein at a particular point in time. Retention can be by inhibiting hydrolysis of phosphate esters from their target proteins. For this purpose, the art provides a variety of substances that inhibit phosphatase activity. The inhibitors are generally useful in assays for detecting phosphorylated proteins, as well as in processes for the purification of phosphorylated proteins in large quantities.

Among the vanadium-containing inhibitors of phosphatase activity, peroxovanadate and vanadate are the most widely known substances. Vanadate is a phosphate analog that mimics the transition state of phosphate hydrolysis and is therefore considered a universal phosphatase inhibitor. However, it is recognized that vanadates are particularly suitable for inhibiting tyrosine phosphatases (Huyer, G., et al., J.biol. chem.272(1997)843-851) and alkaline phosphatases (e.g., Stankiewicz, P.J., et al., in Met. Ions biol. Syst.31(1995) 287-324). However, inhibition of other phosphatases, such as ATPases, glucose-6-phosphatase, acid phosphatase or fructose-2, 6-bisphosphatase, has also been reported.

However, it is disadvantageous that the concentration of vanadate must be in the micromolar or even millimolar range in order to provide an effective amount. In this regard, an "effective amount" is understood to mean that the concentration of inhibitor in aqueous solution is between 1x (1 fold) and 50x (50 fold), and an effective amount in a 1x concentration can reduce phosphatase activity by a factor of 20. Other inhibitors of phosphatase activity are known to be effective at nanomolar concentrations relative to vanadate. Such as cantharidin.

However, it has been reported that the inhibition can be enhanced by forming stable vanadium-containing complexes. For example, potassium diperoxy (bipyrimidine) oxovanadate (V) and potassium diperoxy (1, 10 phenanthroline) oxovanadate (V) are both more effective than orthovanadate. However, complexation by vanadate is not a prerequisite for increased effectiveness. For example, hydroxylamine or dimethylhydroxylamine can spontaneously form complexes with vanadates. The effectiveness of these complexes remains at the original level or is somewhat reduced (Cuncic, C., et al., biochem. Pharmacol.58(1999) 1859-1867). In cellular level assays, both compounds have been found to enhance vanadate uptake in the lumen of cells (Nxumulo, F., et al., J.biol.Inorg.chem.3(1998) 534-542; Cuncic, C., et al., biochem.Pharmacol.58(1999) 1859-1867).

It is further known that the inhibition of phosphatase by vanadate can be reduced by a factor of about 1,000 in the presence of specific complex-forming agents, such as EDTA (Huyer, G., et al., J.biol.chem.272(1997) 843-851). This is highly disadvantageous because EDTA is commonly used in biochemistry as a stabilizer, and as an inhibitor of metalloenzymes such as phosphatases and proteases. Another important stabilizer for the preparation of cell lysates is Dithiothreitol (DTT). The inventors have found that DTT can also significantly reduce the inhibitory effect of vanadate on phosphatases.

In view of the disadvantages of the prior art, it is an object of the present invention to provide alternative compounds which enhance the inhibitory effect of vanadium-containing compounds on enzymes having phosphatase activity. It is another object of the present invention to provide compounds that counteract the negative effects of EDTA and DTT on vanadium-containing compounds.

The inventors have surprisingly found that the inhibitory effect of vanadate on phosphatases, in particular phosphotyrosine-specific phosphatases, is enhanced in the presence of a polyol. This effect can be observed even in very simple polyols, such as glycerol, and sugar-containing alcohols, such as mannitol. Even more surprising was the discovery that the negative effects of EDTA and DTT on vanadate were significantly reduced, or even completely eliminated.

Summary of The Invention

A first embodiment of the present invention is the use of a composition comprising (i) an ionic compound selected from vanadyl (vanadyl) (II), vanadate (IV), vanadate (V), oligomeric vanadate (V), and mixtures thereof, and (II) a polyol, for inhibiting an enzyme having phosphatase activity. A second embodiment of the invention is a composition comprising (i) an ionic compound selected from vanadyl (II), vanadate (IV), vanadate (V), oligomeric vanadate (V), and mixtures thereof, and (II) a polyol, characterized in that the molar ratio of polyol to vanadium-containing compound is equal to or greater than 1: 1. A third embodiment of the present invention is a method for inhibiting an enzyme having phosphatase activity comprising the steps of (a) dissolving in an aqueous solution a composition comprising (i) an ionic compound selected from the group consisting of vanadyl (II), vanadate (IV), vanadate (V), oligomeric vanadate (V), and mixtures thereof, and (II) a polyol, and (b) contacting the enzyme having phosphatase activity with the solution of step (a). A fourth embodiment of the invention is a kit comprising a packaging material and a component comprising a combination of (i) an ionic compound selected from the group consisting of vanadyl (II), vanadate (IV), vanadate (V), oligomeric vanadate (V), and mixtures thereof, and (II) a polyol.

Detailed description of the invention

Certain terms have certain meanings, or are defined for the first time, in the description of the invention. For the purposes of understanding the present invention, the terms used to describe the present invention are defined in an art-accepted sense unless those definitions conflict or partially conflict with the definitions set forth below. In case of conflict in the definitions, the meaning of a term is defined first by the definitions set out below.

The term "comprising" as used in the description and claims of the present invention means "including, but not limited to". The indefinite article "a" when used with a term indicating a chemical compound, such as an inhibitor or an additive, is intended to mean "one or more". A "phosphatase inhibitor" is a substance that effectively inhibits the hydrolysis of a phosphate by phosphatase activity, thereby releasing phosphate ions from the target molecule. An "aqueous" solution is a solution wherein the liquid solvent is at least 80% [ v/v ] water, more preferably 95% [ v/v ], more preferably 99% [ v/v ], more preferably 100% [ v/v ]. It will be understood by those skilled in the art that the solution may further contain one or more compounds such as salts, buffers, inhibitors, additives and biomolecules, wherein the one or more compounds are soluble in the liquid solvent.

The composition according to the invention contains an ionic compound selected from vanadyl (II), vanadate (IV), vanadate (V), oligomeric vanadate (V) and mixtures thereof. More preferably, the vanadium ion containing compound is orthovanadate (V) and oligomers thereof. Preferred oligomers are selected from di-, tri-and tetra-poly-vanadate ions. Another particularly preferred ionic compound in the composition according to the invention is a peroxovanadate ion. Most preferred, however, is orthovanadate (VO)₄ ^3-)。

The "polyhydroxy compound" in the compositions according to the present invention is a water-soluble organic compound in which two or more hydroxyl groups are covalently linked to a carbon atom. Preferably, the two hydroxyl groups of the polyhydroxy compounds according to the invention are bound to two adjacent carbon atoms. That is, the preferred polyol contains two adjacent hydroxyl groups. However, polyols having more than two adjacent hydroxyl groups are preferred. Well known and particularly preferred polyhydroxy compounds having adjacent hydroxyl groups are sugar alcohols. "sugar alcohols" are hydrogenated forms of carbohydrates whose carbonyl groups (aldehydes or ketones) are reduced to primary or secondary hydroxyl groups. The non-hydrogenated form of the carbohydrate is also referred to as the "reduced" sugar.

The composition according to the invention preferably contains a sugar alcohol having 4 to 100 carbon atoms. In this case, non-reducing mono-, di-, tri-and tetrasaccharides are very preferred.

A highly preferred sugar alcohol is a non-reducing monosaccharide. Such sugar alcohol is a C4 sugar alcohol, preferably selected from threitol and erythritol. Also preferably, the sugar alcohol is a C5 sugar alcohol, preferably selected from ribitol, arabitol, xylitol and lyxitol. Also preferably, the sugar alcohol is a C5 deoxy sugar alcohol, preferably selected from the group consisting of deoxyribose alcohol and deoxyarabitol. Also preferably, the sugar alcohol is a C6 sugar alcohol, preferably selected from allitol, altritol, mannitol, sorbitol, gulitol, iditol, galactitol, and talitol.

According to the present invention, a deoxy sugar alcohol is included in the term sugar alcohol, as long as the deoxy sugar provides four or more pairs of adjacent hydroxyl groups. The preferred sugar alcohol is a C6 deoxy sugar alcohol, preferably selected from the group consisting of deoxy sorbitol and deoxy mannitol. Also preferred sugar alcohols are those with other substituent groups at the C2 position, a preferred example being N-acetyl-sorbitan-2, and others with other substituent groups at the C2 position, such as N-acetyl-sorbitan-2.

Both disaccharides and monosaccharides may form sugar alcohols; however, sugar alcohols derived from disaccharides (e.g. mannitol and lactitol) are not fully hydrogenated, since only one aldehyde group can be reduced due to glycosidic bonds. The same is true for trisaccharides and higher sugars. Thus, non-reducing disaccharides or trisaccharides having up to 100C atoms or higher oligosaccharides having non-reducing properties are very advantageous in the practice of the present invention. Sucrose is a highly preferred sugar alcohol. Notably, sucrose, unlike most polysaccharides, glycosidic linkages are formed at the reducing ends of glucose and fructose, rather than at the reducing end of one and the non-reducing end of the other. This effect inhibits further bonding with other saccharide units. Sucrose is a non-reducing sugar because it does not contain a free anomeric carbon atom.

According to the invention, the molar ratio of the preferred polyhydroxy compound to the vanadium-containing ionic compound is higher than 1: 1. Particularly preferably, the molar ratio is between 100: 1 and 1: 1. More preferably, the molar ratio is between 70: 1 and 5: 1. More preferably, the molar ratio is between 50: 1 and 10: 1.

With reference to phosphatase activity in a sample in the absence of a vanadium-containing inhibitor (100%), the phosphatase activity is substantially reduced when a combination of an ionic compound selected from vanadyl (II), vanadate (IV), vanadate (V), oligomeric vanadate (V) and a polyol according to the invention is added to the sample. Preferably, the activity reduction is of a value of 1.5% to 40%, more preferably of a value of 1.5% to 30%, more preferably of a value of 1.5% to 20%, more preferably of a value of 1.5% to 10%.

In one aspect, the polyol enhances the inhibitory effect of the vanadium ion-containing compound on phosphatase activity. On the other hand, the polyhydroxy compound may reduce the negative effects of the complex forming agent. That is, inhibition of phosphatase activity is not adversely affected by the presence of a chelator for divalent or trivalent positively charged metal ions. Thus, formation of a complex of divalent ions can, for example, inhibit undesirable activities of various proteases without adverse effects. Therefore, the composition of the present invention additionally contains a chelating agent for a divalent or trivalent positively charged metal ion. Preferred chelating agents are selected from EDTA, citrate, EGTA and 1, 10-phenanthroline. The preferred concentration of chelating agent in the composition according to the invention is 0.1mM to 50mM, more preferably 0.2mM to 10mM, most preferably about 1 mM.

In addition, the polyol in the composition can reduce the negative impact of the reducing agent on the vanadium-containing ionic compound. In particular, DTT reduces the inhibitory effect of orthovanadate on phosphatase activity. However, in the presence of polyols, the reduction is reversed. Preferred reducing agents are selected from the group consisting of DTT (dithiothreitol), β -mercaptoethanol, glutathione and thioredoxin. The preferred concentration of reducing agent in the composition according to the invention is 0.1mM to 30mM, more preferably 0.2mM to 10mM, most preferably about 1 mM.

In another embodiment of the present invention, the composition is provided to the end user in a convenient form. Another example is therefore a package containing a measured amount of the composition. The packaging material is preferably protected from water contact with water vapor. In addition, one or more packages may be stored in the presence of a dry material such as silica gel or other suitable substance. The composition may be in the form of free-flowing granules. More preferably, the composition is in the form of a tablet. In this case, the composition may additionally contain other materials which facilitate tablet formation.

In addition, the compositions of the present invention may contain one or more other phosphatase inhibitors in addition to the vanadium-containing compound. Preferably, the one or more other phosphatase inhibitors are ionic inhibitors such as NaF. Also preferably, the inhibitor may be a low molecular weight compound selected from cantharidin, naphthalene phosphate (napthyl phosphate) and microcystin poison. Also preferably, the inhibitor may also be a peptidic compound selected from calcineurin self-inhibitory peptides and protein phosphatase inhibitor 2.

Due to the surprising effect of the presence of a polyol in addition to a vanadium containing compound, the composition according to the invention is suitable for inhibiting enzymes with phosphatase activity. The composition is particularly suitable for inhibiting a phosphatase selected from the group consisting of acid phosphatase, alkaline phosphatase, phosphotyrosine phosphatase, ATPase, phosphoserine/threonine phosphatases, and binary phosphatases. Most preferably, the composition is useful for inhibiting protein tyrosine phosphatases, i.e., it hydrolyzes the phosphate ester of a phosphotyrosine residue that is part of a phosphorylated polypeptide. Examples of polypeptides that contain phosphotyrosine residues and serve as substrates for phosphotyrosine-specific protein phosphatases are the phosphorylated receptors for erythropoietin, pErk1, pErk2 and pJak 2.

Thus, another embodiment of the invention is a method of inhibiting an enzyme having phosphatase activity comprising the steps of (a) dissolving in an aqueous solution a composition comprising (i) an ionic compound selected from the group consisting of vanadyl (II), vanadate (IV), vanadate (V), oligomeric vanadate (V), and mixtures thereof, and (II) a polyol, and (b) contacting the enzyme having phosphatase activity with the solution of step (a).

Thus, a further embodiment of the present invention is a liquid composition comprising a composition according to the present invention and an aqueous solution. Preferably, the pH of the liquid composition is the pH at which the phosphatase (one or more target phosphatases) is active. The pH is preferably pH5.5 to pH8.5, more preferably pH6.7 to pH 8.0.

The following examples, figures and sequence listing are provided for the understanding of the present invention, the scope of which is indicated in the appended claims. It is to be understood that appropriate modifications can be made without departing from the spirit of the invention.

Drawings

FIG. 1 inhibition of phosphotyrosine specific protein phosphatases by orthovanadate in the presence and absence of polyhydroxy compounds. The experimental principle is shown in example 1. The ordinate represents the residual phosphatase activity. The line represents the results obtained with the following composition: (1) no inhibitor was added (control, 100% residual activity); (2)1mM orthovanadate; (3)2mM orthovanadate; (4)1mM orthovanadate, 27mM mannitol; (5)1mM orthovanadate, 54mM glycerol.

FIG. 2 inhibition of phosphotyrosine specific protein phosphatases by orthovanadate in the presence and absence of polyols and in the presence of EDTA and DTT. The experimental principle is shown in example 2. The ordinate represents the residual phosphatase activity. The line represents the results obtained with the following composition: (1) no inhibitor was added (control, 100% residual activity); (2)1mM orthovanadate; (3)2mM orthovanadate; (4)1mM orthovanadate, 27mM mannitol; (5)1mM orthovanadate, 54mM glycerol.

FIG. 3 inhibition of phosphotyrosine specific protein phosphatases by orthovanadate in the presence and absence of polyols and in the presence of EDTA, with or without DTT. The experimental principle is shown in example 3. The ordinate represents the residual phosphatase activity. The line represents the results obtained with the following composition: (1)1mM orthovanadate, Hepes buffer containing EDTA; (2)1mM orthovanadate, Hepes buffer containing EDTA and DTT; (3)1mM orthovanadate, Hepes buffer with EDTA, in the presence of 27mM mannitol; (4)1mM orthovanadate, Hepes buffer containing EDTA and DTT, in the presence of 27mM mannitol; (5)1mM orthovanadate, Hepes buffer with EDTA, in the presence of 54mM glycerol; (6)1mM orthovanadate, Hepes buffer with EDTA and DTT, in the presence of 54mM glycerol.

FIG. 4 inhibition of phosphatase activity by orthovanadate in insect cell lysates in the presence and absence of mannitol. The experimental principle is shown in example 5. The ordinate represents the residual phosphatase activity. The line represents the results obtained with the following composition: (1) no inhibitor was added (control, 100% residual activity); (2)1mM orthovanadate; (3)1mM orthovanadate, 27mM mannitol.

FIG. 5 inhibition of phosphatase activity by orthovanadate in COS7 cell lysates in the presence and absence of mannitol. The experimental principle is shown in example 6. The ordinate represents the residual phosphatase activity. The line represents the results obtained with the following composition: (1) no inhibitor was added (control, 100% residual activity); (2)1mM orthovanadate; (3)1mM orthovanadate, 27mM mannitol.

Example 1

Inhibition of phosphotyrosine-specific protein phosphatases

Recombinantly produced phosphotyrosine-specific protein phosphatases (human T cells; Calbiochem, No. Cat 539732) were stored in a medium containing 0.1mM CaCl₂50mM Tris pH7.0 in 1: 200. The following mixtures (a-e) were prepared in separate reaction tubes: mu.l of the diluted enzyme solution was mixed with 4. mu.l of (a) water (as 100% control), or (b)10mM orthovanadate; (c)20mM orthovanadate (d)10mM orthovanadate, 270mM mannitol; (e)10mM orthovanadate, 540mM glycerol. Each mixture was incubated at Room Temperature (RT) for 15 minutes. Then, a 10. mu.l aliquot was transferred from each mixture to a microplate. Mu.l of a test peptide containing phosphotyrosine residues (peptide sequence R-R-L-I-E-D-A-E-pY-A-A-R-G; 1mM in water) solution and 10. mu.l of a solution containing 0.1mM CaCl were added to each well₂50mM Tris pH7.0, incubated at 37 ℃ for 30 minutes. By adding 100. mu.l of a solution additionally containing0.034％[w/v]Malachite green, 10mM sodium molybdate and 3.4% [ v/v ]]Ethanol in 1M HCI to carry out the detection reaction on the free phosphate obtained by enzymatic hydrolysis of the phosphate bond. The detection reaction was carried out at room temperature for 10 minutes while the microplate was continuously shaken. The relative concentration of phosphoric acid was determined by measuring the extinction (620nm) of each reaction well.

Example 2

Inhibition of phosphotyrosine specific protein phosphatases in the Presence of EDTA and DTT

The recombinantly produced phosphotyrosine-specific protein phosphatase (human T cells; Calbiochem, No. Cat 539732) was diluted 1: 200 in a buffer containing 25mM Hepes, 50mM NaCl, 2.5mM EDTA, 5mM DTT, pH 7.2. The following mixtures (a-e) were prepared in separate reaction tubes: 36 μ l of diluted enzyme solution with 4 μ l of (a) water (as 100% control), or (b)10mM orthovanadate; (c)20mM orthovanadate (d)10mM orthovanadate, 270mM mannitol; (e)10mM orthovanadate, 540mM glycerol. The mixture was incubated at Room Temperature (RT) for 15 minutes. Then, a 10. mu.l aliquot was transferred from each mixture to a microplate. Mu.l of a test peptide containing phosphotyrosine residues (peptide sequence R-R-L-I-E-D-A-E-pY-A-A-R-G; 1mM in water) solution and 10. mu.l of a buffer containing 25mM Hepes, 50mM NaCl, 2.5mM EDTA, 5mM DTT, pH7.2 were added to each well and incubated at 37 ℃ for 30 minutes. The detection reaction of the free phosphate obtained by enzymatic hydrolysis of the phosphate bond was carried out by adding 100. mu.l of a 1M HCl solution additionally containing 0.034% [ w/v ] malachite green, 10mM sodium molybdate and 3.4% [ v/v ] ethanol. The detection reaction was carried out at room temperature for 10 minutes while the microplate was continuously shaken. The relative concentration of phosphoric acid was determined by measuring the extinction (620nm) of each reaction well.

Example 3

Inhibition of phosphotyrosine-specific protein phosphatases in the Presence of EDTA, with or without DTT

The recombinantly produced phosphotyrosine-specific protein phosphatase (human T cells; Calbiochem, No. Cat 539732) was diluted 1: 200 in a buffer containing 25mM Hepes, 50mM NaCl, 2.5mM EDTA, 5mM DTT, pH7.2 (enzyme solution (i)) or in a buffer with the same components but without DTT (enzyme solution (ii)). The following mixtures (a-f) were prepared in separate reaction tubes: 36. mu.l of the diluted enzyme solution (i) or (ii) and 4. mu.l of the following certain solution (a, d)10mM orthovanadate; (b, e)10mM orthovanadate, 270mM mannitol; (c, f)10mM orthovanadate, 540mM glycerol. The mixture was incubated at Room Temperature (RT) for 15 minutes. Then, 10. mu.l of each mixture was transferred to a microplate. Mu.l of a test peptide containing phosphotyrosine residues (peptide sequence R-R-L-I-E-D-A-E-pY-A-A-R-G; 1mM, dissolved in water) solution and 10. mu.l of a buffer containing 25mM Hepes, 50mM NaCl, 2.5mM EDTA, 5mM DTT, pH7.2 (reacted with the enzyme solution (I)) or 10. mu.l of a buffer containing 25mM Hepes, 50mM NaCl, 2.5mM EDTA, pH7.2 (reacted with the enzyme solution (ii)) were added to each well and incubated at 37 ℃ for 30 minutes. The detection reaction of the free phosphate obtained by enzymatic hydrolysis of the phosphate bond was carried out by adding 100. mu.l of a 1M HCl solution additionally containing 0.034% [ w/v ] malachite green, 10mM sodium molybdate and 3.4% [ v/v ] ethanol. The detection reaction was carried out at room temperature for 10 minutes while the microplate was continuously shaken. The relative concentration of phosphoric acid was determined by measuring the extinction (620nm) of each reaction well.

Example 4

Inhibition of phosphotyrosine specific protein phosphatases by orthovanadate

TABLE 1

The residual phosphatase activity in the composition containing orthovanadate and other compounds in an aqueous buffer is shown below. The data in the tables are illustrated with reference to figures 1-3.

	Buffer solution	Activity of
			FIG. 1:
1) non-inhibitor	50mM Tris；0,1mM CaCl2pH7,0	100％
			2)Na3VO41mM	50mM Tris；0,1mM CaCl2pH7,0	16,6％
3)Na3VO42mM	50mM Tris；0,1mM CaCl2pH7,0	15,7％
			4)Na3VO41mM + mannitol 27mM	50mM Tris；0,1mM CaCl2pH7,0	8,5％
5)Na3VO41mM + glycerol 54mM	50mM Tris；0,1mM CaCl2pH7,0	9,7％
			FIG. 2:
1) non-inhibitor	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	100％
			2)Na3VO41mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	87,7％
3)Na3VO42mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	58,0％
			4)Na3VO41mM + mannitol 27mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	12,4％
5)Na3VO41mM + glycerol 54mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	9,3％
			FIG. 3:
1)Na3VO41mM	25mM Hepes；50mM NaCl；2,5mM EDTA；pH7,2	29,5％
			2)Na3VO41mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	46,3％
3)Na3VO41mM + mannitol 27mM	25mM Hepes；50mM NaCl；2,5mM EDTA；pH7,2	14,1％
			4)Na3VO41mM + mannitol 27mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	5,8％
5)Na3VO41mM + glycerol 54mM	25mM Hepes；50mM NaCl；2,5mM EDTA；pH7,2	14,9％
			6)Na3VO41mM + glycerol 54mM	25mM Hepes；50mM NaCl；2,5mM EDTA；5mM DTT pH7,2	7,3％

Example 5

Inhibition of phosphatases in insect cell lysates

270mg of insect cells (SF9) were collected, washed 3 times with 50mM Hepes, 50mM NaCl pH7.0, and lysed with 2.5ml M-Per (Pierce) for 10 minutes. After centrifugation, the lysate supernatant was collected.

Detection of phosphotyrosine Activity: mu.l of water or orthovanadate (10mM) or orthovanadate/mannitol (10mM/270mM) was added to 45. mu.l of the lysate and incubated for 10 minutes at room temperature.

Then, 10. mu.l of each mixture was transferred to a microplate. Mu.l of the peptide to be tested (R-R-L-I-E-D-A-E-pY-A-A-R-G; 1mM in water) and 10. mu.l of CaCl containing 0.1mM were added to each well₂50mM Tris pH7.0, incubated at 37 ℃ for 30 minutes. By adding 100. mu.l of a solution additionally containing 0.034% [ w/v ]]Malachite green, 10mM sodium molybdate and 3.4% [ v/v ]]Ethanol in 1M HCl to carry out the detection reaction on the free phosphate obtained by enzymatic hydrolysis. The detection reaction was carried out at room temperature for 10 minutes while the microplate was continuously shaken. The relative concentration of phosphoric acid was determined by measuring the extinction (620nm) of each reaction well.

TABLE 2

The residual phosphatase activity in the composition containing orthovanadate and other compounds in an aqueous buffer is shown below. The data in the table refer to the diagram given in fig. 4.

	Phosphatase Activity
		No inhibitor (Water control)	100％
1mM orthovanadate	14％
		1mM orthovanadate/27 mM mannitol	2％

Example 6

Inhibition of phosphatase in COS7 cell lysate

COS7 cells were collected, washed 3 times with 50mM Hepes, 50mM NaCl pH7.0, and lysed with 5ml M-Per (Pierce) for 10 minutes. After centrifugation, the lysate supernatant was collected.

Detection of phosphotyrosine Activity: mu.l of water or orthovanadate (10mM) or orthovanadate/mannitol (10mM/270mM) was added to 45. mu.l of lysate (1+4, containing M-Per (Pierce)), and incubated for 10 minutes at room temperature.

Then, 10. mu.l of each mixture was transferred to a microplate. Mu.l of the peptide to be tested (R-R-L-I-E-D-A-E-pY-A-A-R-G; 1mM in water) and 10. mu.l of CaCl containing 0.1mM were added to each well₂50mM Tris pH7.0, incubated at 37 ℃ for 30 minutes. By adding 100. mu.l of a solution additionally containing 0.034% [ w/v ]]Malachite green, 10mM sodium molybdate and 3.4% [ v/v ]]Ethanol in 1M HCl to carry out the detection reaction on the free phosphate obtained by enzymatic hydrolysis. The detection reaction was carried out at room temperature for 10 minutes while the microplate was continuously shaken. The relative concentration of phosphoric acid was determined by measuring the extinction (620nm) of each reaction well.

TABLE 3

The residual phosphatase activity in the composition containing orthovanadate and other compounds in an aqueous buffer is shown below. The data in the table is illustrated with reference to fig. 5.

	Phosphatase Activity
		No inhibitor (Water control)	100％
1mM orthovanadate	52％
		1mM orthovanadate/27 mM mannitol	36％

Sequence listing

<110>Roche Diagnostics GmbH

F.Hoffmann-La Roche AG

<120> enhancement of vanadium-containing phosphatase inhibitor

<130>24107

<150>EP 07001593.8

<151>2007-01-25

<160>1

<170>PatentIn version 3.2

<210>1

<211>14

<212>PRT

<213> Artificial

<220>

<223> protein phosphatase substrate

<220>

<221>MISC FEATURE

<222>(10)..(10)

<223> in the substrate peptide, tyrosine residue is phosphorylated

<400>1

Arg Arg Leu Ile Glu Asp Ala Glu Pro Tyr Ala Ala Arg Gly

1 5 10

Claims (13)

1. Liquid composition comprising an aqueous solvent, an enzyme having phosphatase activity, orthovanadate, mannitol and dithiothreitol, characterized in that the concentration of mannitol is between 1mM and 100mM and the molar ratio between mannitol and orthovanadate is equal to or greater than 1: 1.

2. A composition according to claim 1, characterized in that the concentration of orthovanadate is between 0.1mM and 10 mM.

3. A composition according to claim 2, characterized in that the concentration of orthovanadate is between 0.5mM and 5 mM.

4. A composition according to any of claims 1 to 3, wherein the composition further comprises a chelating agent for a divalent or trivalent positively charged metal ion.

5. In vitro use of mannitol or glycerol for enhancing the inhibitory effect of an ionic compound selected from the group consisting of vanadate, oligomeric vanadate, and mixtures thereof, wherein vanadium of said vanadate is pentavalent and vanadium of said oligomeric vanadate is pentavalent, the enzyme being hydrolytically soluble in an enzyme having a neutral pH and containing Ca in a concentration of 0.7mM to 1.2mM²⁺An aqueous buffered ionic solution having the sequence of SEQ ID NO: 1, the phosphoester bond of a phosphotyrosine residue in the peptide of 1.

6. Use according to claim 5, characterized in that the phosphatase activity is reduced by a value between 1.5% and 40%.

7. A method for enhancing in vitro the inhibitory effect of an ionic compound selected from the group consisting of vanadate, oligomeric vanadate, and mixtures thereof, wherein the vanadium of said vanadate is pentavalent and the vanadium of said oligomeric vanadate is pentavalent, said enzyme being hydrolytically soluble in an enzyme having a neutral pH and containing Ca in a concentration of 0.7mM to 1.2mM²⁺An aqueous buffered ionic solution having the sequence shown in SEQ ID NO: 1, the method comprising the steps of:

(a) dissolving in an aqueous solvent a composition comprising (i) an ionic compound and (ii) mannitol or glycerol, wherein the molar ratio of mannitol or glycerol to the ionic compound is equal to or greater than 1: 1, and

(b) contacting an enzyme having phosphatase activity with the solution of step (a).

8. The method of claim 7, wherein the concentration of mannitol or glycerol in the solution is between 1mM and 100 mM.

9. A method according to claim 7 or 8, characterized in that the vanadate is orthovanadate and the concentration of orthovanadate in solution is between 0.1mM and 10 mM.

10. A method according to any one of claims 7 to 8, characterised in that the ionic compound and mannitol or glycerol are added to the aqueous sample containing the enzyme having phosphatase activity in dry form.

11. A kit comprising a packaging material and a composition comprising (i) an ionic compound selected from the group consisting of vanadates, wherein vanadium is pentavalent, oligomeric vanadates, wherein vanadium is pentavalent, and mixtures thereof, and (ii) mannitol or glycerol, wherein the molar ratio of mannitol or glycerol to the ionic compound is equal to or greater than 1: 1.

12. A kit according to claim 11, wherein the composition further comprises a reducing agent and/or a chelating agent for a divalent or trivalent positively charged metal ion.

13. A kit according to any one of claims 11 and 12, characterized in that the composition is provided as a dry substance in the form of a tablet.