Influence on Me2SO and incubation time on papain activity studied using fluorogenic substrates

• Autorzy: Szabelski M. , Stachowiak K. , Wiczk W.
• Języki publikacji: EN

Abstrakty

EN

Papain activity in a buffer containing Me2SO was studied using fluorogenic sub­strates. It was found that the number of active sites of papain decreases with increas­ing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain in­cubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an in­crease of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michae- lis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of kcat/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis kcat.

Słowa kluczowe

EN

proteolytic enzyme; fluorogenic substrate; activity; protease; papain activity; enzyme activity; papain; amino acid

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2001
• Tom: 48
• Numer: 4
• Opis fizyczny: p.995-1002,fig.

Twórcy

autor

Szabelski M.

• University of Gdansk, Gdansk, Poland

autor

Stachowiak K.

autor

Wiczk W.

Bibliografia

• 1.Gershkovich,A.A. & Kholodovych, V.V. (1996) Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J. Biochem. Biophys. Methods 33, 135-162.
• 2.Klibanov,A.M. (1997) Why are enzyme less active in organic solvents than in water? Trends Biotechnol. 15, 97-101.
• 3.Sarath, G., De La Motte, R.S. & Wagner, F.W. (1989) Protease assay methods; in Proteolytic Enzymes: a Practical Approach (Beynon, R.J. & Bond, J.S., eds.) pp. 25-55, IRL Press at Oxford University Press, Oxford, New York, Tokyo.
• 4.Barrett,A.J. & Kirschke, H. (1981) Cathepsin B, cathepsin H and cathepsin L. Methods Enzymol. 80, 535-561.
• 5.Zaks,A. & Klibanov, A.M. (1988) Enzymatic catalysis in nonaqueous solvent. J. Biol. Chem. 263(7), 3194-3201.
• 6.Sirotkin, V.A., Zinatulin, A.N., Solomonov, B.N., Fazillin, D.A. & Fedotov, V.D. (2001) Colorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents. Biochim. Biophys. Acta 1547, 359-369.
• 7.Grard, E. & Legoy, M.-D. (1999) Activity and stability of dextransucrase from Leuconostoc mesenteroides NRRL B-512F in the presence of organic solvents. Enzyme Microb. Technol. 24, 425-432.
• 8.Potier,P., Bouchu, A., Descotes, G. & Queneau, Y. (2000) Proteinase N-catalyzed transestrification in Me2SO-water and DMF- water; preparation of sucrose monomethacrylate. Tetrahedron Lett. 41, 3597-3600.
• 9.Simon, L.M., Laszlo, K., Vertesi, A., Bagi, K. & Szajani, B. (1998) Stability of hydrolytic enzymes in water-organic solvent systems. J. Mol. Catal. B: Enzymatic 4, 41-45.
• 10.Okazaki, S., Goto, M. & Furusaki, S. (2000) Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb. Technol. 26, 159-164.
• 11.Schmid, H., Koop, M., Utermann, S., Lambacher, L., Mayer, P. & Schaefer, L. (1997) Specific catalytic activity of cathepsin S in comparison to cathepsins B and L along the rat nephron. Biol. Chem. 378, 61-69.
• 12.Kirschke,H., Clausen, T., Gohring, B., Gunther, D., Heucke, E., Laube, F., Lowe, E., Neef, H., Papesch, H., Peinze, S., Plehn, G., Rebmann, U., Rinne, A., Rudrich, R. & Weber, E. (1997) Concentrations of lysosomal cystein proteases are decreased in renal cell carcinoma compared with normal kidney. J. Cancer Res. Clin. Oncol. 123, 402-406.
• 13.Dwojakowska,D., Dabrowska, A., <@163>ankiewicz, L., Wiczk, W. & Stachowiak, K. (1999) Fluorogenic substrates of papain with structural resemblance to the inhibitory center of family 2 cystatins; in Peptides 1998 (Bajusz, S. & Hudecz, F., eds.) p. 632, Proc. 25th Europ. Peptide Symp., Akademiai Kiado, Budapest.
• 14.Garcia-Echeverria,C. & Rich, D.H. (1992) New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain. FEBSLett. 297, 100-102.
• 15.Blumberg,S., Schechter, I. & Berger, A. (1970) The purification of papain by affinity chromatography. Eur. J. Biochem. 15, 97-102.
• 16.Bodanszky,M. & Bodanszky, A. (1984) The Practice of Peptide Chemistry. Springer-Verlag, New York.
• 17.Jou,G., Gonzalez, I., Albericio, F., Lloyd-Williams, P. & Giralt, E. (1997) Total synthesis of dehydrodidemnin B. Use of uranium and phosphonium salt coupling reagents in peptide synthesis in solution. J. Org. Chem. 62, 354-366.
• 18.Barrett,A.J., Kembahavi, A.A., Brown, M.A., Kirschke, H., Knight, C.G., Tamai, M. & Hanada, K. (1982) L- trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem. J. 201, 189-198
• 19.Lakowicz,J.R. (1999) Principles of Fluorescence Spectroscopy, 2 edn., chap. 6, pp. 185- 210, Kluwer Academic/Plenum Publisher, New York, Boston, Dordrecht, London, Moscow.
• 20.Matsumoto,K., Murata, M., Sumiya, S., Kitamura, K. & Ishida, T. (1994) Clarification of substrate specificity of papain by crystal analyses of complexes with covalent-type inhibitors. Biochim. Biophys. Acta 1208, 268-276.
• 21.Matsumoto,K., Murata, M., Sumiya, S., Mizoue, K., Kitamura, K. & Ishida, T. (1998) X-Ray crystal structure of papain complexed with cathepsin B-specific covalent-type inhibitor: Substrate specificity and inhibitory activity. Biochim. Biophys. Acta 1383, 93-100.
• 22.Guo,Z., Ramirez, J., Li, J. & Wang, P.G. (1998) Peptidyl N-nitrosoanilines: A novel class of cysteine protease. J. Am. Chem. Soc. 120, 3726-3734.
• 23.Iguchi, S., Kawasaki, K., Okamoto, H., Umezawa, C. & Okada, Y. (1999) Synthesis of some pseudo-peptide analogs of tiol proteinase inhibitors. Chem. Pharm. Bull. 47, 423-427.
• 24.Maurel, P. (1978) Relevance of dielectric constant and solvent hydrophobicity to the organic solvent effect in enzymology. J. Biol. Chem. 253, 1677-1683.
• 25.Turk, B., Dolenc, I., Lenarcic, B., Krizaj, I., Turk, V., Bieth, J.G. & Bjork, I. (1999) Acidic pH as a physiological regulator of human cathepsin L activity. Eur. J. Biochem. 259, 926-932.