A succinyl-trialanine p-nitroanilide hydrolase in hog kidney cytosol: Its identification as proline endopeptidase.

Publisher Website
Google Scholar
Abstract
A succinyl-trialanine p-nitroanilide [Suc-(Ala)3-pNA] hydrolase which is able to hydrolyze an artificial elastase substrate, Suc-(Ala)3-pNA, but unable to hydrolyze a naturally occurring substrate, elastin, was highly purified from hog kidney cytosol. The apparent MW of the enzyme was estimated to 65,000 by gel filtration on Sephadex G-150 and 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point of the enyzme was 5.0. The enyzme is an endopeptidase which catalyzes the hydrolysis of peptides with the general structure Y-Ala (or Pro)-X (Y = peptide or N-protected amino acid; X = amino acid moiety, peptide or amide) at the carboxyl side of alanine and proline residues. The enzyme was markedly inhibited by diisopropyl fluorophosphate and p-chloromercuribenzoate. EDTA and 1,10-phenanthroline, however, were not inhibitors of the enzyme. The enzyme activity was retained on an affinity column having a proline endopeptidase [EC 3.4.21.26] inhibitor, Z-Gly-Pro, as ligand and could be eluted at 0.125 M NaCl (mean value). Suc-(Ala)3-pNA-hydrolytic activity coincided with the peak of proline endopeptidase activity as determined with a sensitive fluorogenic substrate, succinyl/glycyl-L-proline 4-methylcoumaryl-7-amide (Suc-Gly-Pro-MCA). The optimum pH and Kcat/Km values (mM-1 .cntdot. s-1) were pH 7.5 and 5.4 for Suc-(Ala)3-pNA and pH 6.8 and 24.8 for Suc-Gly-Pro-MCA, and the enyzme activity was competitively inhibited by Z-Ala-Ala and Z-Gly-Pro, as is the case with proline endopeptidase. Suc-(Ala)3-pNA hydrolase in hog kidney cytosol may be identical with proline endopeptidase which was first found in human uterus as an oxytocin degrading enzyme.

This publication has 1 reference indexed in Scilit: