A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma

S. I. Kawabata, E. W. Davie

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Abstract

The microsomal fraction of rabbit liver contains an endopeptidase that cleaves synthetic peptides that mimic the amino acid sequences of the processing sites of many proproteins, including the vitamin K-dependent proteins. The endopeptidase (M(r) 69,000) was extracted from liver microsomes with 1% Lubrol and purified about 2,700-fold. The substrate employed for isolation and characterization of the enzyme was the decapeptide acetyl-Ala- Arg-Val-Arg-Arg-Ala-Asn-Ser-Phe-Leu (prothrombin peptide), in which hydrolysis occurred on the carboxyl side of the paired Arg-Arg residues. The purified enzyme, whose activity was enhanced 1.8-fold by 0.1 mM CoCl2, has a K(m) = 80 μM and V(max) = 21,000 nmol · min-1 · mg-1 and a pH optimum of 8.7. Proteolytic cleavage of decapeptide substrates was dependent on an arginine residue at positions P1 and P4. The enzyme was completely inhibited by EDTA and 1,10-phenanthroline as well as by p-chloromercuriphenylsulfonic acid and Hg2+. Inhibitors of serine proteases and cysteine proteases had no effect. Based on the substrate preference, the endopeptidase appears to be a good candidate for the enzyme responsible for the precursor processing of the vitamin K-dependent proteins and a number of other proproteins that are synthesized via the secretory pathway in liver and other tissues.

Original languageEnglish
Pages (from-to)10331-10336
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number15
Publication statusPublished - 1992
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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