title: Post-proline cleaving enzyme from lamb brain.
abstract: A post-proline cleaving enzyme [post-proline endopeptidase: EC 3.4.21.26] was purified from lamb brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and Sephadex G-150. The purified enzyme appeared homogeneous on disc gel and sodium dodecyl sulfate (SDS) gel electrophoreses. The enzyme was most active at pH 7.0 with carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap) as a substrate and catalyzed the hydrolysis of oxytocin, vasopressin, thyrotropin releasing hormone (TRH), substance P, luteinizing hormone releasing hormone (LH-RH), and angiotensin at the carboxyl side of their proline residues, except for the Pro2-Lys3 bond in substance P. From the results of subsite mapping using synthetic peptides, five subsites, S3 to S2', for substrate interaction with the enzyme were deduced to be present, and high stereospecificity was observed at S2, S1, and S1'. The isoelectric point of the enzyme was at pH 4.9, and the molecular weights estimated by gel filtration and SDS gel electrophoresis were 74,000 and 77,000, respectively. The enzyme was markedly inhibited by diisopropylphosphoro fluoridate (DFP), carbobenzoxy-Gly-Pro-chloromethyl ketone (Z-Gly-Pro-CH2Cl), p-chloromercuribenzoate (PCMB), Hg2+, and Cu2+ ions. These enzymatic and protein chemical properties of post-proline cleaving enzyme from lamb brain closely resemble those of the lamb kidney enzyme, except for the molecular weight. In the present work, however, we decided that the molecular weight of the enzyme from lamb kidney was also 74,000, which is different from that reported previously (J. Biol. Chem. 251, 7593 (1976) but is in accord with the value of post-proline cleaving enzyme from lamb brain.
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D004355:Drug Stability; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D012756:Sheep; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010455:Peptides; D011480:Protease Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases