title: Purification and characterization of prolyl endopeptidase from rat skin.
abstract: An enzyme with the specificity of a prolyl endopeptidase was purified approximately 329-fold from rat skin. The enzyme has a molecular weight of 70,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pH optimum of 5.8 as checked with 7-(Succinyl-Gly-Pro)-4- methylcoumarinamide (Suc-Gly-Pro-MCA) as the substrate. The optimal temperature for the enzyme activity was 40 degrees C. The Km and Vmax values for Suc-Gly-Pro-MCA were 0.7 mM and 68 nmol/min per mg protein, respectively. The enzyme activity was markedly inhibited by diisopropyl fluorophosphate, p-chloromercuribenzoic acid, N-ethylmaleimide, Zn2+ and Cu2+, while it was partially inhibited by phenylmethanesulphonyl fluoride. The purified enzyme was shown to release the N-terminal tetrapeptide, Arg-Pro-Lys-Pro, from substance P producing the C-terminal heptapeptide, Gln-Gln-Phe-Phe-Gly-Met- CONH2. In the skin, this enzyme might be related to the inactivation of substance P.
authors.affiliation: Department of Dermatology, Kurume University School of Medicine, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kusuhara
authors.forename: H
authors.initials: H
authors.lastname: Hachisuka
authors.forename: S
authors.initials: S
authors.lastname: Nakano
authors.forename: Y
authors.initials: Y
authors.lastname: Sasai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D012867:Skin / Q000201:enzymology*; D013373:Substance P / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D013373:Substance P; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases