Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin.
Staphylocoagulase with a molecular weight of 64,000 and subspecies ranging in molecular weight from 36,000 to 64,000 were purified by affinity column chromatography on bovine prothrombin-Sepharose 4B from the culture filtrates of the Staphylococcus aureus strains, st-213 and 104. The samples containing all molecular species from both strains had the same NH2-terminal sequence, Ile-Val-Thr-Lys-Asp-Tyr-Ser-Lys-Glu-, implying that the molecular heterogeneity was due to proteolytic degradation to some extent of the COOH-terminal portion during cultivation or purification. Staphylocoagulase (Mr = 64,000) from strain st-213 formed an active complex, "staphylothrombin," with human prothrombin in a molar ratio of 1 to 1.1. Staphylothrombin was unstable at 37 degrees C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmentation of prothrombin into prethrombin 1 and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent fluorography for the products of prothrombin activation by staphylocoagulase in the presence of [3H]diisopropylphosphofluoridate (DFP) demonstrated the formation of a DFP-sensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of alpha-thrombin was found. The enzymatic properties including the pH-dependency of the activity, substrate specificity and behavior towards thrombin inhibitors of staphylothrombin differed from those of alpha-thrombin, although the active site titration of staphylothrombin with p-nitrophenyl-p'-guanidinobenzoate showed 0.95 +/- 0.2 mol of active site/mol of enzyme.
D000581:Amidohydrolases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003030:Coagulase / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000378:metabolism; D008970:Molecular Weight; D055503:Protein Multimerization; D011516:Prothrombin / Q000378:metabolism; D013211:Staphylococcus aureus / Q000032:analysis; D013917:Thrombin / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D003030:Coagulase; D004792:Enzyme Precursors; D007531:Isoflurophate; D011516:Prothrombin; D005340:Fibrinogen; C012007:prethrombins; D013917:Thrombin; C022654:staphylothrombin; D000581:Amidohydrolases; C019665:amidase