Characterization of a protein C activator from the venom of Agkistrodon contortrix contortrix.
An enzyme capable of activating protein C has been purified 60-fold from the venom of the Southern copperhead snake (Agkistrodon contortrix) by ion-exchange and gel filtration chromatography. The purified enzyme consists of a single polypeptide with an apparent molecular weight of 37,000. The isoelectric point of the protein C activator was determined to be 6.3 when measured by chromatofocusing. The enzyme was inhibited by p-nitrophenyl p-guanidinobenzoate, phenylmethanesulfonyl fluoride, and D-Phe-Pro-Arg-CH2Cl but was not affected by cysteine-directed reagents or by metal chelators. These results suggest that the enzyme is a serine protease. Protein C activator was capable of hydrolyzing the thrombin substrate tosyl-Gly-Pro-Arg-p-nitroanilide (TGPRpNA), and steady-state kinetic studies determined that the Km for amidolysis of this substrate was 1.1 mM while the Vmax was 66 s-1. The activator demonstrated considerable substrate specificity since the amidolysis of D-Phe-Pip-Arg-pNA, D-Ile-Pro-Arg-pNA, Bz-Ile-Glu-Gly-Arg-pNA, D-Val-Leu-Arg-pNA, and pyrGlu-Pro-Arg-pNA was less than 10% of that of TGPRpNA when measured under identical conditions using 1.0 mM substrate concentrations. The enzyme appears to be thrombin-like in its preference for arginyl as compared to lysyl chloromethyl ketones as well as by its inhibition by benzamidine and p-aminobenzamidine. However, the substrate specificity of the activator is distinguished from alpha-thrombin in that it does not clot fibrinogen and does not react with antithrombin III or hirudin.(ABSTRACT TRUNCATED AT 250 WORDS)
Orthner
C L
CL
American Red Cross, Biomedical Research and Development Division, Rockville, Maryland 20855.
Bhattacharya
P
P
Strickland
D K
DK
D003435:Crotalid Venoms / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009842:Oligopeptides / Q000302:isolation & purification / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D009842:Oligopeptides; D011486:Protein C; C047135:protein C activator peptide