title: A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma.
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 Km = 80 microM and Vmax = 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.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195.
authors.forename: S
authors.initials: S
authors.lastname: Kawabata
authors.forename: E W
authors.initials: EW
authors.lastname: Davie
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D001798:Blood Proteins; D002413:Cations, Divalent; D010618:Phenanthrolines; D011498:Protein Precursors; D002731:4-Chloromercuribenzenesulfonate; D004492:Edetic Acid; D010450:Endopeptidases; D008628:Mercury; C025205:1,10-phenanthroline