The Active Site of Bovine Plasminogen Activator

Summaria, Louis; Ling, Chung‐Mei; Groskopf, William R.; Robbins, Kenneth C.

doi:10.1016/s0021-9258(18)99335-x

Cited by 36 publications

(8 citation statements)

References 13 publications

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“…In the streptokinase-human plasmin activator complex, many investigators have proposed that the active site resided in the plasmin moiety. This is based on the fact that reagents such as diisopropyl fluorophosphate were incorporated into the plasmin moiety of the complex at rates which paralleled the rate of loss of plasminogen activator activity of the complex (De Renzo et a!., 1967b;Summaria et al, 1968). Further, in the streptokinase-human plasminogen activator complex, it is thought that binding of streptokinase to human plasminogen induces a conformational alteration in the plasminogen moiety leading to formation of the activator active site (Werkheiser and Markus, 1964;Hummel et al, 1966;McClintock and Bell, 1971;Reddy and Markus, 1972).…”

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confidence: 99%

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

Brockway¹,

Castellino²

1974

Biochemistry

101

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The properties of native streptokinase and streptokinase purified from the human plasminogen activator complex (altered streptokinase) have been compared. Native streptokinase possesses a molecular weight of 44,000 and a s°o,w value of 3.03 S whereas the values for altered streptokinase are 36,000 and 2.82 S, respectively. Native streptokinase possesses an amino-terminal isoleucine and a carboxylterminal lysine residue whereas altered streptokinase possesses an amino-terminal serine and a carboxyl-terminal lysine residue. The amino-terminal amino acid sequence of native streptokinase is NH2-Ile-Ala-Gly-Pro-Glu-Trp-Leu-Leu-Asp-Arg-Pro-Ser-and the amino-terminal amino acid sequence for altered streptokinase is NH2-Ser-Lys-Pro-Phe-Ala-X-Asp-T A he mechanism of the activation of the single-chain proenzyme, plasminogen to the two-chain enzyme, plasmin by streptokinase has been a widely studied subject. Any study of this mechanism must include the fact that at least one bond in the plasminogen molecule must be cleaved in the activation process (Robbins et al., 1967;Summaria et al., 1967). Although synthetic ester substrates have been found for all other plasminogen activators, none has been found to be a substrate for streptokinase (De Renzo et al., 1967a). This finding appears to rule out direct activation of plasminogen by streptokinase; however, such a mechanism has been nonetheless proposed (Summaria et al., 1969). On the other hand, most investigators have suggested an indirect activation mechanism via an activator complex. It was found that streptokinase could form a 1:1 complex with human plasmin and this complex could in fact directly activate bovine plasminogen (Zybler et al., 1969;Blatt et al., 1964;Ling et al., 1965Ling et al., , 1967). This same activator complex was formed whether streptokinase and human plasmin or streptokinase and human plasminogen were used as the starting materials (Ling et al., 1967). More recently, these considerations have been incorporated into a unified mechanism for the streptokinase induced activation of human plasminogen (Reddy and Markus, 1972) and for the streptokinase-induced activation of rabbit plasminogen (Schick and Castellino, 1973). These studies demonstrate that the principal activator of human plasminogen is a complex of streptokinase and human plasminogen, containing an active site.

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confidence: 99%

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

Brockway¹,

Castellino²

1974

Biochemistry

101

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“…A-a-Cbz-L-lysine-pnitrophenyl ester was purchased from Calbiochem. Protein concentrations were determined spectrophotometrically at 280 nm (Summaria et al, 1968). Spectrophotometric assays were performed in a Cary 118B spectrophotometer with the cell compartment maintained at 30°.…”

Section: Methodsmentioning

confidence: 99%

Kinetic mechanism of the activation of human plasminogen by streptokinase

Kosow¹

1975

Biochemistry

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A method of determining the initial rate of plasminogen activation has been developed. The method has been used to investigate the mechanism of activation of human plasminogen by streptokinase. Plasmin formation follows saturation kinetics. Inhibition of plasmin formation by epsilon-aminocaproic acid is uncompetitive with a Ki of 0.6 mM. A model consistent with the data is that streptokinase induces a conformational change in the plasminogen molecule, producing an active center which cleaves an internal peptide bond to produce plasmin. Thus, streptokinase functions as a catalytic allosteric effector.

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“…The only contaminant was the parent Lys-Plg, about 31%, which was completely activatable. The amidase parameters of this Lys-Plga W^en human plasminogen and streptokinase react to form a stoichiometric complex, either plasminogen-streptokinase (Plg-SK)1 or plasmin-streptokinase (Pln-SK) can be identified, depending upon the conditions of the reaction (De Renzo et al, 1963;Hummel et al, 1966;Buck et al, 1968;Summaria et al, 1968). In the equimolar human Plg-SK (with either Glu-Plg or Lys-Plg) complex, an active site develops in the Pig moiety in minutes prior to the formation of plasmin (Glu-Pln or Lys-Pln) (McClintock & Bell, 1971;Reddy & Markus, 1972, 1973.…”

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confidence: 99%

“…This enzyme has been shown to react with serine protease inhibitors, such as DFP, NPGB, and bovine pancreatic trypsin inhibitor or Trasylol. However, the enzyme apparently does not react with the naturally occurring plasma protease inhibitors, such as a2-plasmin inhibitor, a2-macroglobulin, arantitrypsin, or antithrombin III (Summaria et al, 1968;McClintock & Bell, 1971;Reddy & Markus, 1972Summaria et al, 1977;Wohl et al, 1979;Cederholm-Williams et al, 1979). Kinetic parameters have been determined for both the Plg-SK and Pln-SK enzyme complexes (Buck & Boggiano, 1971; Reddy & Markus, 1974;Wohl et al, 1977Wohl et al, -1980Morris et al, 1981).…”

mentioning

confidence: 99%

“…Kinetic parameters have been determined for both the Plg-SK and Pln-SK enzyme complexes (Buck & Boggiano, 1971; Reddy & Markus, 1974;Wohl et al, 1977Wohl et al, -1980Morris et al, 1981). Various methods have been used to dissociate the Plg-SK enzyme complex in an effort to study the individual moieties (De Renzo et al, 1967;Buck et al, 1968;Summaria et al, 1968Summaria et al, , 1971Brockway & Castellino, 1974), but a pure, structurally intact Pig moiety, with or without an active site, had not been pre-preparation were determined on two synthetic chromogenic substrates. With H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide, a Km of 417 /tM and a A:cat of 8.44 s_1 were obtained; with Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide, a Km of 307 juM and a t of 12.83 s_1 were obtained.…”

mentioning

confidence: 99%

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