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ATP-regulated activity of the plasmin-streptokinase complex: a novel mechanism involving phosphorylation of streptokinase.

ABSTRACT: Streptokinase, an extracellular protein produced by Streptococci, is capable of activating the human fibrinolytic zymogen plasminogen. The rate of amidolytic activity of the plasminogen-streptokinase complex is greatly diminished by micromolar concentrations of ATP and heparin oligosaccharides. In addition, the plasminogen activator activity of the plasminogen-streptokinase complex is also inhibited by these effectors. ATP and heparin oligosaccharides show structural similarity, suggesting that the inhibition is caused by binding of these molecules to a common newly formed binding pocket in streptokinase, which appears after interaction with plasminogen. Addition of the bivalent cations Ca2+ and Mg2+ reverses the inhibition caused by ATP and heparin. In the presence of ATP and bivalent cations, the complex between plasminogen and streptokinase develops an autophosphorylating activity whose target is the sequence LTSRPAHG in the 4.5 kDa streptokinase N-terminal peptide, which is an early autolysis peptide. This streptokinase N-terminal peptide, which is essential for streptokinase activating activity, may serve, once phosphorylated, in mechanisms related to the pathogenicity of Streptococci. These studies suggest a critical role for plasminogen in regulating the activity of the streptokinase molecule.

SUBMITTER: Serrano RL

PROVIDER: S-EPMC1216879 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

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Project description:Streptokinase-human plasmin complex (Sk-hPm) reacted rapidly with purified mouse alpha 2-macroglobulin (m alpha 2M) in vitro at 37 degrees C. Approx. 98% of the plasmin in Sk-hPm bound covalently to at least one m alpha 2M subunit. Most of the streptokinase dissociated (95%). The rate of Sk-hPm inactivation clearly depended on the m alpha 2M concentration. With 1.2 microM-m alpha 2M, 50% of the Sk-hPm (0.02 microM) reacted in less than 50 s. A double-reciprocal plot comparing pseudo-first-order rate constants (kapp.) and m alpha 2M concentration yielded a second-order rate constant of 2.3 x 10(4) M-1.s-1 (r = 0.97). This value is an approximation, since Sk-hPm preparations are heterogeneous. Sk-hPm reacted with human alpha 2M (h alpha 2M), forming alpha 2M-plasmin complex (98% covalent). More than 99% of the streptokinase dissociated. The rate of reaction of Sk-hPm with h alpha 2M did not clearly depend on inhibitor concentration. The kapp. values determined with 0.6-1.2 microM-h alpha 2M were decreased 10-20-fold compared with m alpha 2M. In order to study the effect of Sk-hPm heterogeneity on the reaction with alpha 2M, the proteinase was incubated for various amounts of time at 37 degrees C before addition of inhibitor. The enzyme amidase activity was maximal within 5 min; however, reaction of Sk-hPm with m alpha 2M or h alpha 2M was most extensive after 20 min and 2 h respectively. After incubation for more than 1 h, Sk-hPm acquired fibrinogenolytic activity, suggesting plasmin dissociation. Therefore the enhanced reaction of h alpha 2M with 'older' Sk-hPm preparations may have resulted in part from dissociated plasmin or 'plasmin-like' species. By contrast, the reaction of Sk-hPm with m alpha 2M was most rapid when the proteinase preparation was free of plasmin, indicating direct reaction of Sk-hPm with m alpha 2M as the only major mechanism. Finally, streptokinase-cat plasminogen complex reacted more extensively with m alpha 2M than with h alpha 2M, suggesting that m alpha 2M may be a superior inhibitor with this class of plasminogen activators in general.

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ATP-regulated activity of the plasmin-streptokinase complex: a novel mechanism involving phosphorylation of streptokinase.

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