Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin

Shun Ichiro Kawabata, Takashi Morita, Sadaaki Iwanaga, Hideo Igarashi

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52 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1603-1614
Number of pages12
JournalJournal of biochemistry
Volume98
Issue number6
DOIs
Publication statusPublished - Dec 1985

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Coagulase
Prothrombin
Thrombin
Isoflurophate
Catalytic Domain
Photofluorography
Molecular Weight
Molecular weight
Affinity chromatography
Column chromatography
Substrate Specificity
Electrophoresis
Titration
Affinity Chromatography
Sodium Dodecyl Sulfate
Sepharose
Purification
Staphylococcus aureus
Gels
Chemical activation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin. / Kawabata, Shun Ichiro; Morita, Takashi; Iwanaga, Sadaaki; Igarashi, Hideo.

In: Journal of biochemistry, Vol. 98, No. 6, 12.1985, p. 1603-1614.

Research output: Contribution to journalArticle

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AB - 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.

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