Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain

Histatin 5 is a low molecular weight salivary protein which is known to exh ibit inhibitory activity against several proteinases, including the cystein e proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine pr oteinase clostripain derived from the pathogen Clostridium histolyticum. Us ing a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to th at of leupeptin, a known competitive inhibitor of clostripain, It was found that the concentration of histatin 5 required to inhibit 50% of clostripai n activity was 23.6 +/- 1.6 nM. Kinetic analysis revealed that histatin 5 i s a competitive inhibitor of clostripain with an inhibition constant (K-i) of 10 nM. The K-i for the inhibition of clostripain activity against nitroa nilide substrate by leupeptin was found to be 60 nM, significantly higher t han that of histatin 5, Thus, histatin 5 inhibits clostripain more effectiv ely than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incu bated at physiologic concentrations with clostripain, The potent inhibition of clostripain by histatin 5 points towards the possibility that this prot ein may prevent establishment of clostridial infections and therefore may h ave significant potential for the treatment of diseases associated with thi s enzyme. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.