Molecular conversions of recombinant staphylokinase during plasminogen activation in purified systems and in human plasma.
Recombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-delta 6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-delta 10 with NH2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma. In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-delta 10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with Km = 3.6 microM and k2 = 0.38 s-1. The specific clot lysis activities of HMW-STAR (122,000 +/- 8,000 units/mg) and LMW-STAR (129,000 +/- 8,000 units/mg) were indistinguishable. In an in vitro system consisting of a 60 microliters plasma clot submerged in 250 microliters plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR.(ABSTRACT TRUNCATED AT 250 WORDS)
Ueshima
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S
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Silence
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Collen
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Lijnen
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HR
D000595:Amino Acid Sequence; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000097:blood / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000097:blood / Q000737:chemistry*
D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus