Abstract
A chymotrypsin-like enzyme was purified from rat plasma, involving ammonium sulfate fractionation and chromatographgy on CM-sephadex and red sepharose. The purified enzyme effectively hydrolysed the ester substrates for chymotrypsin (N-acetyl L-tyrosine ethyl ester and N-acetyl L-tryptophan ethyl ester). The Km values for the two substrates were 2.2×10−3M and 9.0×10−3M respectively. The hydrolytic activity of the enzyme was inhibited by phenylmethyl sulfonyl fluoride and tosylphenylalanine chloromethylketone, suggesting the presence of serine and histidine at the active centre. The enzyme exhibited anionic nature and possessed a high molecular weight (MW 71,000) as observed by gel exclusion chromatography on Sephadex G-200. The enzyme was stable upon exposure to pH 7.0–9.0, but was inactivated upon heat treatment at 60°C for 5 min.
Similar content being viewed by others
References
Woodbury, R.G., Katunuma, N., Kobayashi, K., Titani, K. and Neurath, H. (1978) Covalent structures of group specific protease from rat small intestine. Biochemistry 17, 811–819.
Kido, H., Fukusen, N. and Katunuma, N. (1984) A simple method for purification of chymase from rat peritoneal cells. Anal. Biochem. 137, 449–453.
Kawaik, J., Vensel, W.H., Komender, J. and Bernard, E.A. (1971) A chymotrypsin-like protease from rat mast cells. Biochim. Biophys. Acta. 235, 172–187.
Vensel, W.H., Komender, J. and Barnard, E.A. (1971) Purification and criteria for homogeneity of elastase and cathepsin G. Biochem. J. 144, 255–263.
Starkey, P.M. and Barrett, A.J. (1976) Purification and criteria for homogeneity of elastase and cathepsin G. Biochem. J. 155, 255–263.
Schechter, N.M., Fraki, J.E., Geesin, J.C. and Lazarus, G.S. (1983) Human skin chymotryptic proteinase—isolation and relation to cathepsin-G and rat mast cell proteinase I. J. Biol. Chem. 258, 2973–2978.
Woodbury, R.G. and Neurath, H. (1980) Structure, specificity and localization of the serine protease of connective tissue. F.E.B.S. Lett. 114, 189–196.
Powers, J.C., Tanaka, T., Harper, J.W., Minematsu, Y., Barker, L., Lincoln, D., Crumley, K.V., Fraki, J.E., Schechter, N.M., Lazarus, G.G., Nakajima, K., Nakashino, K., Neurath, H. and Woodbury, G. (1985) Mammalian chymotrypsin-like enzyme. Comparative reactivities of rat mast cell proteases, human and dog skin chymases and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethylketone and sulfonyl fluoride inhibitors. Biochemistry 24, 2048–2058.
Syner, F.N. and Moghissi, K.M. (1972) Purification and properties of human seminal proteinase. Biochem. J. 126, 1135–1140.
Pattabiraman, T.N. and Senthil Kumar, R. (1994) Protease-like activities in plasma of different animals. Biochem. Archives. 10, 51–57.
Prabhu, K.S. and Pattabiraman, T.N. (1977) A colorimetric method for the estimation of esterolytic activity of chymotrypsin. Ind. J. Biochem. Biophys. 14, 96–98.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, B.J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265–275.
Prabhu, K.S. and Pattabiraman, T.N. (1980) Isolation and characterization of trypsin/chymotrypsin inhibitor from Indian red wood (Adenanthera pavonia) seed. J. Sci. Food. Agric. 31, 967–980.
Nayak, N., Rajaram, S.V. and Pattabiraman, T.N. (1976) Studies on human trypsin and chymotrypsins. Ind. J. Biochem. Biophys. 13, 355–338.
Sanada, Y., Yasagowa, N. and Katunuma, N. (1978) Crystallization and amino acid composition of a serine protease from rat skeletal muscle. Biochem. Biophys. Res. Commun. 82, 108–11.
Starkey, P.M. and Barret, A.J. (1976) Human Cathepsin G. Catalytic and immunological properties. Biochem. J. 155, 273–278.
Yamguchi, T., Fukase, M., Nishikawa, M., Fujimi, T. and Fujita, T. (1989) Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the clonal osteogenic UMR-106 cell. Biochim. Biophys. Acta. 1010, 177–183.
Post, L.L., Schuel, R. and Schuel, H. (1988) Evidence that hatching enzyme of the sea-urchin ‘Strongylocentratus purpuratus’ is a chymotrypsin-like portease. Biochem. Cell. Biol. 66, 1200–1209.
Wintroub, B.U., Kaempfer, C.E., Schechter, N.M. and Proud, D. (1986) A human mast cell chymotrypsin-like enzyme. Identification and partial characterization. J. Clin. Invest. 77, 196–201.
Gerber, A.C.H., Carson, J.H. and Hadorn, B. (1974) Partial purification and characterization of a chymotrypsin-like enzyme from human neutrophil leucocytes. Biochim. Biophys. Acta. 364, 103–112.
Goldstein, S.M., Leong, J., Schawartz, L.B. and Cooke, D. (1992) Protease composition of exocytosed human skin mast cell protease proteoglycan complexes-Tryptase residues in a complex distinct from chymase and carboxypeptidase. J. Immunol. 148, 2475–2482.
Pejler, G. and Karlstorm, A. (1993) Thrombin is inactivated by mast cell secretory granule chymase. J. Biol. Chem. 268, 11817–11822.
McNeil, H.P., Austen, K.F., Somerville, L.L., Gurish, M.F. and Stevens, R.L. (1991) Molecular cloning of the mouse mast cell protease-5 gene. J. Biol. Chem. 266, 20316–20322.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kumar, R.S., Pattabiraman, T.N. Purification and characterization of chymotrypsin-like enzyme from rat plasma. Indian J Clin Biochem 11, 152–157 (1996). https://doi.org/10.1007/BF02896434
Issue Date:
DOI: https://doi.org/10.1007/BF02896434