Kinetic characterization of affinity chromatography purified clostripain.
The cysteine protease clostripain, purified by affinity chromatography on a large scale, shows very high activity against BAEE using the titrimetric standard assay. Furthermore, titration of the active site with the irreversible inhibitor tosyl-lysyl-chloromethane resulted in a more than two times higher specific activity compared with literature data (Porter et al. (1971) J. Biol. Chem. 246, 7675-7682). Based on the molar enzyme concentration determined, the hydrolysis kinetics of the standard substrate BAEE were compared with those for the N alpha-protected dipeptide ester Mal-Tyr-Arg-OEt. It was demonstrated from the kinetic data that the highly purified clostripain is the most active enzyme preparation available up to now. In contrast to the standard substrate, Mal-Tyr-Arg-OEt shows a threefold lower specificity constant.
Ullmann
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Department of Biochemistry, University of Leipzig, Germany.
Jakubke
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HD
D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
D004151:Dipeptides; C087424:maleyltyrosylarginine ethyl ester; D001120:Arginine; C009727:benzoylarginine ethyl ester; D003546:Cysteine Endopeptidases; C019913:clostripain; D002122:Calcium Chloride