A zinc metalloendopeptidase associated with dog pancreatic membranes.

Abstract

Assay of solubilized dog pancreas microsomes revealed the presence of an endopeptidase which hydrolyzed the fluorogenic peptide substrate Suc-Ala-Ala-Phe-7-amino-4-methylcoumarin (AMC) between the alanine and phenylalanine positions. This activity was inhibited by phosphoramidon, 1,10-phenanthroline, and a number of synthetic inhibitors of thermolysin indicating that the enzyme is a zinc metallopeptidase. Endopeptidase activity was not inhibited by the serine protease inhibitors elastatinal, antipain, leupeptin, N-carbobenzyloxy-L-phenylethyl chloromethyl ketone, L-tosylamido-2-lysyethyl chloromethyl ketone, L-tosylamido-2-phenylethyl chloromethyl ketone, phenyl-methanesulfonyl fluoride, or low levels of chymostatin. The endopeptidase had a pH optimum between 7.0 and 7.5. The enzyme also hydrolyzed Suc-Ala-Ala-Ala-AMC and Suc-Ala-Gly-Ala-AMC in an analogous way to yield Ala-AMC. Thermolysis hydrolyzed Suc-Ala-Ala-Phe-AMC in an analogous way to the endopeptidase. However, thermolysin did not hydrolyze Suc-Ala-Ala-Ala-AMC or Suc-Ala-Gly-Ala-AMC, demonstrating that its substrate specificity differs from the endopeptidase.