Effect of staphylokinase concentration of plasminogen activation.
Activation of Glu- and Lys-plasminogen by various concentrations of recombinant staphylokinase (SAK) were studied by the generation of amidolytic activity from the chromogenic substrate S-2251(H-D-Val-Leu-Lys-pNA) and by SDS-PAGE analysis. Surprisingly, excess SAK decreased and fixed the rate of S-2251 hydrolysis in a mixture of Lys-plasminogen and SAK. Since the effect of SAK on S-2251 hydrolysis by plasma was similar, the hydrolysis kinetics by free plasmin and plasmin-SAK complex were studied. Hydrolysis by either enzyme form followed Michaelis-Menten kinetics with a Km of 0.38 mM for plasma and 3.74 mM for SAK-plasmin complex. The catalytic rate constant was 22.7 s-1 for plasmin and 21.0 s-1 for the SAK-plasmin complex. With excess SAK and vigorous removal of plasmin activity from plasminogen, the pre-activation lag period differed greatly between Glu- and Lys-plasminogen. Based on the different substrate specificity of plasmin and plasmin-SAK complex, we analyzed the Glu-plasminogen activation with either catalytic or excess SAK. With excess SAK, almost no Lys-plasminogen was detectable and whole Glu-plasminogen was converted directly to Glu-plasmin, then gradually to Lys-plasmin. In contrast, Lys-plaminogen appeared rapidly with catalytic amount of SAK. These results suggest that inhibition of Glu-plasminogen to Lys-plasminogen to Lys-plasminogen conversion in the plasminogen-SAK complex in the presence of excess SAK prolonged the initial lag phase of activation.
Arai
K
K
Division of Thrombosis and Hemostasis, Institute of Hematolog, Jichi Medical School, Tochigi-ken, Japan.
Mimuro
J
J
Madoiwa
S
S
Matsuda
M
M
Sako
T
T
Sakata
Y
Y
D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism / Q000494:pharmacology*; D010958:Plasminogen / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000494:pharmacology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010958:Plasminogen; D005341:Fibrinolysin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus