1. Several hypotheses have been advanced to explain the activating function of streptokinase. The predominant hypothesis suggests a stable equimolar streptokinase-plasmin(ogen) complex, activating free plasminogen by an active centre, which is located in the plasmin(ogen) part of the complex. 2. This hypothesis cannot explain a number of phenomena and certain accumulated experimental data, for example: rabbit and bovine plasminogen activation by streptokinase, not forming stable complexes with these plasminogens; possible activation with pH less than or equal to 2, in the presence of urea, during modification of streptokinase tyrosine residues, i.e. when these two proteins cannot form a stable complex. 3. On the basis of acquired experimental data the following concept is suggested: the activating function of streptokinase is oxygen-dependent and is realised with the help of superoxide radical due to the O(2-.)-generating ability of plasminogen and the O(2-.)-converting ability of streptokinase.
Human, rabbit and bovine plasminogens, having different sensitivity to streptokinase-activating action, differ, according to spectrophotometric titration, tryptophan fluorescence and circular dichroism spectroscopy, in the state of tyrosine and tryptophan residues, and in secondary and tertiary structures. Human plasminogen-streptokinase equimolar complex formation (according to gel chromatography) is accompanied by a differential ultraviolet spectrum. Difference spectroscopy is a convenient and adequate means of studying the formation of the said complexes. Streptokinase-human plasminogen complex formation is not hindered by partial substitution of water (20%) with ethanol or dimethylsulphoxide or by addition of 0.001 M sodium dodecylsulphate. The complex is not formed in 6 M urea, in solution, at pH less than 2.0 or approximately 12.0-13.0, or with bovine plasminogen. Circular dichroism and tryptophan fluorescence spectral pattern changes during streptokinase-plasminogen complex formation enable us to conclude that streptokinase secondary and tertiary structures undergo certain rearrangements in the framework of the complex, while tryptophan-containing sites of the molecule are not drastically changed. The data obtained enable us to presuppose formation of streptokinase-rabbit plasminogen complexes which differ from human plasminogen complexes with streptokinase.
We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001-0.06 M) enhances by 50-250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2-12.0 times enhances protein lysis by trypsin, alpha-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40-50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10(-8)-10(-1) M enhanced the cleavage of a number of proteins by serine proteases and, at concentrations of 10(-5) -10(-3) M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of > 10(-3) and >10(-4) M inhibited pepsin- and metalloprotease-induced lysis of virtually all proteins. ATP increased casein lysis by serine proteases, metalloprotease, and pepsin by 20-60% at concentration of 10(-3) M and by 30-260% at 10(-2) M concentration. At concentrations of 10-2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloprotease by 20-100%, and, at concentrations of 10(-3) M, lysis of albumin with pepsin and other proteins (except for fibrinogen) by metalloprotease. A GTP concentration of 10(-7)-10(-2) M increased protein degradation by serine proteases, papain, and gelatin lysis by pepsin by 20-90%, whereas albumin lysis was inhibited by 40-70%. The presence of 10(-6)-10(-5) M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloprotease, while 10(-3) M GTP induced a drop in the activity of the metalloprotease by 20-50%. ADP could enhance gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloprotease activity by 20-100% (at 10(-3) M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.
SUMMARYThe formation of stable equimolar complexes of streptokinase or plasminogen with muscle lactate dehydrogenase or pyruvate kinase, heart mitochondrial malate dehydrogenase and hepatic catalase at pH 7.4, 3.0 and 10.0 was first detected by differential spectroscopy methods. All complexes, except those of plasminogen with dehydrogenases, were resistant to 6 M urea. Judging from circular dichroism spectra, tertiary and secondary structures were considerably changed in the complexes. These changes were significantly dependent upon the nature of interacting proteins; in some cases their structures were more ordered. NAD (but not NADH) hampered the formation of streptokinase complexes with dehydrogenases. The plasminogen--activating function of streptokinase and the ability of plasminogen to be activated by streptokinase in the complexes with oxidoreductases were essentially unchanged. Pyruvate kinase induced a moderate (by 35%) increase in the streptokinase activating function. It is assumed that the formation of complexes of streptokinase or plasminogen with enzymes may serve as a link in metabolic regulation and/or intercellular interactions.
Scavengers of different active oxygen species affect fibrin plate lysis, catalysed by various proteinases, only at relatively high concentrations (> 10(-2) M). Singlet oxygen scavengers change proteinase activity insignificantly except for strong inhibition of pepsin and papain by sodium azide, but pepsin-by histidine, and fibrinolytic urokinase activity-by all used O2 delta 1 scavengers. Of all used scavengers of OH-radical only ethanol caused significant changes in the proteinases under study, except for alpha-chymotrypsin. The most strong inhibitory effect on proteinase activity was demonstrated by scavengers of superoxide radical. Thus, nitrotetrazolium blue strongly inhibited the activity of plasmin, urokinase (fibrinolytic activity), papain and pepsin. Catalase changed proteinase activity insignificantly, though it leads to total inhibition of pepsin activity at final 4.5 x 10(-4) M concentration. These facts and our previous findings on generating of active oxygen species by proteinases give us grounds to suppose that minor active oxygen species, endogenous for the "proteinase-substrate" system, can participate in the catalytic function of some proteinases.
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