Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

M. J. Gething, B. Adler, J. A. Boose, R. D. Gerard, E. L. Madison, D. McGookey, R. S. Meidell, L. M. Roman, J. Sambrook

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76 Scopus citations

Abstract

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.

Original languageEnglish (US)
Pages (from-to)2731-2740
Number of pages10
JournalThe EMBO journal
Volume7
Issue number9
StatePublished - Sep 1988

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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    Gething, M. J., Adler, B., Boose, J. A., Gerard, R. D., Madison, E. L., McGookey, D., Meidell, R. S., Roman, L. M., & Sambrook, J. (1988). Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain. The EMBO journal, 7(9), 2731-2740.