Characterization of native streptokinase and altered streptokinase isolated from a human plasminogen activator complex

Brockway, William J.; Castellino, Francis J.

doi:10.1021/bi00707a010

Cited by 101 publications

(59 citation statements)

References 33 publications

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“…This experiment demonstrated extensive degradation of the streptokinase to fragments of molecular weights comparable to those reported for streptokinase in complex with rabbit plasmin (24). Degradation was considerably more extensive than previously observed when streptokinase was reacted with human plasminogen (8). However, in these previous studies, the incubation time was only 2-5 min as contrasted to 30 min.…”

Section: Resultsmentioning

confidence: 62%

“…Human plasminogen complexed to streptokinase resulted in much less degradation under these conditions (8). However, when studied at 30 min, we found that streptokinase was extensively degraded when in complex with human plasminogen.…”

Section: Discussionmentioning

confidence: 63%

“…The a2M-plasmin complex is cleared from the circulation via specific a2M-protease receptors in the liver (7). Streptokinase released from the complex also clears in the liver and may exist in a degraded form that clears more rapidly than native streptokinase (8).…”

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confidence: 99%

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A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.

Rajagopalan¹,

Gonias²,

Pizzo³

1985

J. Clin. Invest.

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Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG4-streptokinase were slightly more active. Streptokinpse incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by >95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with a2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 1251-plasmin;however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.Address reprint requests to Dr. Pizzo.

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Section: Resultsmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 63%

See 1 more Smart Citation

A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.

Rajagopalan¹,

Gonias²,

Pizzo³

1985

J. Clin. Invest.

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“…Clearly, at incubation times of up to 30 min, during which the time-dependent activity changes of importance to this study are occurring, the only components present are SK and HPg. At longer times, the HPg remains intact while a slight degradation of SK occurs (16). Therefore, all events that take place on incubation of the two proteins, at least up to times of 90 min, that lead to the ionic-strength-related expression of amidolytic activity occur prior to the conversion of HPg to HPm within the complex.…”

Section: Resultsmentioning

confidence: 99%

Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.

Chibber¹,

Radek²,

Morris³

1986

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the time-dependent appearance of amidolytic activity in equimolar complexes of streptokinase (SK) and human [Glul]plasminogen (HPg) under various conditions. When stoichiometric levels of the two proteins are incubated and assayed in hypotonic buffers at 4°C, amidolytic activity toward the chromogenic substrate D-ValLeu-Lys-p-nitroanilide (S-2251), within the resulting complex, appears with an observed first-order rate constant of 1.03 ± 0.06/min. On the other hand, when the assay for amidolytic activity is conducted at a Cl-concentration of 0.15 M, this same activity develops with an observed first-order rate constant of 0.13 ± 0.01/min. Under all conditions of assay of importance to the mechanism proposed, the only molecular components present are SK and H]Pg. The rate of appearance of an enzyme species displaying amidolytic activity is dependent on the anion in its assay; a much slower rate constant is obtained with Cl than with AcO-. These observations are consistent with the formation, within the complex, of an early anion-sensitive active site (SK-HPg*) that is converted to a form (SK-HPg') that is much less sensitive to the presence of anions. During the time period of this process, no conversion of plasminogen to plasmin occurs within the complex. Steadystate kinetic properties of SK-HPg* and SK-HPg' have been measured toward the substrate S-2251. Consistent with the mechanism suggested above, the amidolytic activity of SK-HPg* is inhibited by Cl to a much greater extent than is that of SK-HPg'.Fibrinolytic activity in mammalian plasma is primarily expressed through the action of plasmin, a serine protease generated consequent to activation of its plasma protein precursor, plasminogen. This activation occurs as a result of cleavage of a single peptide bond, ArgSWq-Vall6l, in the zymogen (1) and is mediated by a variety of proteins, such as urokinase (2), streptokinase (SK) (3), and tissue activators (4). Of these, the bacterial protein SK is unique in that it does not appear to be a protease, since a synthetic substrate, or a protein substrate, for SK, other than plasminogen, has not as yet been identified.Current understanding of the mechanism whereby SK activates human plasminogen (HPg) is based on studies that correlate appearance of enzymic activity with the molecular species of proteins present in equimolar complexes of HPg and SK (for a comprehensive review of this area, see ref. 5). From such work, it has been concluded that the major activators of HPg are equimolar complexes of SK and HPg (6), in which an active site has developed in the plasminogen moiety of the complex (7), and a similar complex of SK and human plasmin (HPm), which uses the active site present in HPm for its activator activity (8). This latter complex forms by intramolecular (9, 10) peptide bond cleavage of HPg, within the SK-HPg complex, and/or by stoichiometric interaction of SK with previously formed HPm.Recent work from our laboratory has shown that at very early reaction times, at least two types o...

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“…Several fragments and modified forms of SK have been isolated previously (Brockway & Castellino, 1974;Siefring & Castellino, 1976;Jackson et al, 1986;Malke et al, 1987;Misselwitz et al, 1992;Rodriguez et al, 1994Rodriguez et al, , 1995Shi et al, 1994;Reed et al, 1995;Young et al, 1995). A number of SK fragments remain folded when isolated in solution and a minority maintain an ability to cause Plg activation, although, in most cases, this occurs at substantially reduced rates when compared with native SK.…”

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confidence: 99%

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

et al. 1996

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Streptococcus equisirnilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al ., 1996, Protein Sci 5693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46°C and its stability is largely independent of the presence of the other domains. The hightemperatwe transition in intact SK (at approximately 63 "C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (AI and A2) adopt unstructured conformations when separated. AI binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

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Characterization of native streptokinase and altered streptokinase isolated from a human plasminogen activator complex

Cited by 101 publications

References 33 publications

A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.

A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.

Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

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