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°C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmen tation of prothrombin into prethrombin 1 and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylarnide 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 DFPsensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of α-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 α-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.
|Number of pages||12|
|Journal||Journal of biochemistry|
|Publication status||Published - Dec 1985|
All Science Journal Classification (ASJC) codes
- Molecular Biology