STRUCTURAL DOMAINS OF STREPTOKINASE INVOLVED IN THE INTERACTION WITH PLASMINOGEN

Two fragments of recombinant streptokinase, comprising amino acids Val 143-Lys293 (17-kDa rSK) or Val143-Lys386 (26-kDa rSK), were cloned and expressed in Escherichia coli, purified to homogeneity and their inte ractions with plasmin(ogen) were evaluated. Both 17-kDa rSK and 26-kDa rSK bound to plasminogen with a 1:1 stoichiometry and with affinity c onstants of 3.0X10(8) M(-1) and 12X10(8) M-1 , respectively, as compar ed to 6.3X10(8) M(-1) for the binding of intact recombinant streptokin ase to plasminogen. Binding of 17-kDa rSK to plasminogen-Sepharose was displaced by addition of increasing concentrations of recombinant str eptokinase, whereas bound recombinant streptokinase was not displayed by 17-kDa rSK. In equimolar mixtures of plasminogen and 26-kDa rSK, th e appearance of amidolytic activity as monitored with a chromogenic su bstrate, was significantly delayed compared to the equimolar mixture w ith recombinant streptokinase (60% of the maximal activity after 30 mi n, compared to maximum activity within less than or equal to 2 min). I n contrast, no amidolytic activity was generated in equimolar mixtures of plasminogen and 17-kDa rSK. Plasminogen was rapidly activated by c atalytic amounts (1:100 molar ratio) of recombinant streptokinase (60- 70% within 10-15 min), whereas only 4% of the plasminogen was activate d within 60 min with 26-kDa rSK, and no plasmin was generated with 17- kDa rSK. Complexes of plasmin with 17-kDa rSK or with 26-kDa rSK were very rapidly inhibited by alpha(2)-antiplasmin (apparent second-order inhibition rate constant of approximately 2X10(7) M(-1) s(-1)), wherea s the complex with recombinant streptokinase was resistant to inhibiti on. With 26-kDa rSK, inhibition by alpha(2)-antiplasmin resulted in di ssociation of the complexes and recycling of functionally active 26-kD a rSK to other plasminogen molecules; 17-kDa rSK, in contrast, remaine d associated with the plasmin-alpha(2)-antiplasmin complex. These find ings suggest that different regions of the streptokinase molecule are involved in binding to plasminogen, in active-site exposure, and in im pairment of the inhibition of plasmin by alpha(2)-antiplasmin. Thus, t he 17-kDa region spanning Val143-Lys293 in streptokinase mediates its binding to plasminogen but does not induce activation, Furthermore, th is region does not interfere with the inhibition of the complex with p lasmin by alpha(2)-antiplasmin.