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Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

ABSTRACT: A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.

SUBMITTER: Wang TT

PROVIDER: S-EPMC1172639 | biostudies-other | 1976 Mar

REPOSITORIES: biostudies-other

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Project description:Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

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Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

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