Activation of human plasminogen by urokinase. Partial characterization of a pre activation peptide

P. J. Walther, H. M. Steinman, R. L. Hill, P. A. McKee

Research output: Contribution to journalArticle

66 Scopus citations

Abstract

The activation of human plasminogen by urokinase has been examined by sodium dodecyl sulfate gel electrophoresis and by amino acid sequence analysis. Plasminogen, purified from human plasma by affinity chromatography, was found to have a molecular weight of 87,000 and an NH2 terminal sequence of Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly. Neither lysine nor valine was detected at the NH2 terminus. Activation in the presence of 1 chloro 3 tosyl amido 7 amino 2 heptanone resulted in the formation of a peptide with a molecular weight of 8,200 and an intermediate form of plasminogen which possessed a molecular weight of 80,000 and lysine at the NH2 terminus. This intermediate form was then converted to plasmin, composed of a heavy chain (molecular weight 55,000) and a light chain (molecular weight 26,000). The peptide cleaved prior to plasmin formation, henceforth called the preactivation peptide, is not linked by disulfide bonds to the rest of the molecule; amino acid sequence analysis indicates that it originates from the NH2 terminus of plasminogen with NH2 terminal glutamic acid. The sequence of the first 8 NH2 terminal residues of the heavy chain of plasmin was found to be Lys Val Tyr Leu Ser Glu Phe Lys; a minor amount of the des lysyl NH2 terminal sequence was also found. Activation of plasminogen in the absence of 1 chloro 3 tosylamido 7 amino 2 heptanone indicated that the preactivation peptide was not only rapidly released but also rapidly degraded. In addition, loss of plasmin activity occurred concomitantly with proteolysis of the light chain, which resulted in formation of 2 fragments with molecular weights of 18,000 and 6,500. Degradation of the heavy chain was not observed. The light chain fragments are joined by disulfide bonds, and are clearly different from the preactivation peptide by several criteria. In 25% glycerol, the loss of plasmin activity was also correlated with light chain degradation; after prolonged incubation, heavy chain was also degraded to a lower molecular weight species. It is concluded that the activation of human plasminogen by urokinase occurs by the cleavage of at least 2 peptide bonds in a sequential manner, involving the initial release of an NH2 terminal preactivation peptide to form an intermediate plasminogen which is then converted to the active enzyme, plasmin.

Original languageEnglish (US)
Pages (from-to)1173-1181
Number of pages9
JournalJournal of Biological Chemistry
Volume249
Issue number4
StatePublished - Jan 1 1974
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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