Activation of human Glu-plasminogen to Glu-plasmin by urokinase in presence of plasmin inhibitors. Streptomyces leupeptin and human plasma alpha1-antitrypsin and antithrombin IIi (plus heparin)

Summaria, L.; Boreisha, I.G.; Arzadon, L.; Robbins, K.C.

Journal of Biological Chemistry 252(11): 3945-3951

1977


ISSN/ISBN: 0021-9258
PMID: 140876
Accession: 068521856

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Abstract
Human Glu-plasminogen can be activated to Glu-plasmin by urokinase in the presence of molar excesses of either leupeptin, .alpha.1-antitrypsin or antithrombin III (plus heparin) in a 25% glycerol buffer, at 25.degree. C pH 8.0. Glu-plasmin was identified by its derived Glu-heavy(A) chain in an acrylamide gel/dodecyl sulfate electrophoretic system using reduced and carboxymethylated activation mixtures. Glu-plasminogen was not converted to Lys-plasminogen in any of the activation mixtures. Since leupeptin apparently was an inhibitor of urokinase, smaller molar ratios of Glu-plasminogen to urokinase were required to obtain nearly complete conversion to Glu-plasmin in 5 h. In the .alpha.1-antitrypsin system, 72% of the zymogen was converted to Glu-plasmin in 20 h. In the antithrombin III (plus heparin) system, 68% of the zymogen was converted to Glu-plasmin in 5 h, but in 20 h this enzyme form was completely converted to Lys-plasmin. In the 2 activation systems containing .alpha.1-antitrypsin and antithrombin III (plus heparin), undissociable enzyme.cntdot.inhibitor complexes were identified in the acrylamide gel/dodecyl sulfate electrophoretic system. After complete reduction and carboxymethylation of the activation mixtures, the Cm-light(B) chains were not found, showing that Cm-light(B)chain.cntdot.inhibitor complexes were formed and present in these mixtures. In the enzyme/.alpha.1-antitrypsin activation mixture, the Cm-light(B) chain.cntdot.inhibitor complex was not identified in the presence of the Cm-Glu-heavy(A) chain but identified in the presence of the Cm-Lys-heavy(A) chain. To prove that the Cm-light(B) chain.cntdot.inhibitor complex co-migrates with the Cm-Glu-heavy(A) chain, this complex was isolated from a partially reduced and carboxymethylated Lys-plasmin.cntdot.inhibitor complex by an affinity chromatography method using an L-lysine-substituted Sepharose column. After complete reduction and carboxymethylation, the isolated Cm-light(B) chain.cntdot.alpha.1-antitrypsin complex had a similar electrophoretic mobility to the Cm-Glu-heavy(A) chain in the acrylamide gel/dodecyl sulfate system, showing that these 2 proteins co-migrated. After removal of excess inhibitor from a Glu-plasmin.cntdot.alpha.1-antitrypsin complex by absorption and elution from an L-lysine-substituted Sepharose column, it converted to the Lys-plasmin.cntdot.inhibitor complex, indicating an enzymatically active complex. Diisopropyl phosphorofluoridate-plasmin did not form a complex with .alpha.1-antitrypsin.