The activation of type 1 and type 2 plasminogen by type I and type II tissue plasminogen activator.
Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.
Department of Biochemistry, University of Oxford, United Kingdom.
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Mori
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Dwek
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Downing
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Opdenakker
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Rudd
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003545:Cysteine; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005340:Fibrinogen / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008545:Melanoma; D008958:Models, Molecular; D008970:Molecular Weight; D010446:Peptide Fragments / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D017433:Protein Structure, Secondary; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004220:Disulfides; D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D005340:Fibrinogen; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D003545:Cysteine