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Titolo:
PURIFICATION AND PROPERTIES OF MEMBRANE-BOUND AMINOPEPTIDASE-P FROM RAT LUNG
Autore:
ORAWSKI AT; SIMMONS WH;
Indirizzi:
LOYOLA UNIV,STRITCH SCH MED,DEPT MOLEC & CELLULAR BIOCHEM MAYWOOD IL 60153 LOYOLA UNIV,STRITCH SCH MED,DEPT MOLEC & CELLULAR BIOCHEM MAYWOOD IL 60153
Titolo Testata:
Biochemistry
fascicolo: 35, volume: 34, anno: 1995,
pagine: 11227 - 11236
SICI:
0006-2960(1995)34:35<11227:PAPOMA>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
DIPEPTIDYL PEPTIDASE-IV; BRADYKININ METABOLISM; RENAL DIPEPTIDASE; KININ METABOLISM; BOVINE LUNG; ENZYME; BRAIN; IDENTIFICATION; DEGRADATION; EXPRESSION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
38
Recensione:
Indirizzi per estratti:
Citazione:
A.T. Orawski e W.H. Simmons, "PURIFICATION AND PROPERTIES OF MEMBRANE-BOUND AMINOPEPTIDASE-P FROM RAT LUNG", Biochemistry, 34(35), 1995, pp. 11227-11236

Abstract

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified 670-fold to apparent homogeneityfrom rat lung microsomes. The enzyme was solubilized from the membranes using a phosphatidylinositol-specific phospholipase C. The purification scheme also resulted in homogeneous preparations of dipeptidylpeptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19). Aminopeptidase P had a subunit molecular weight of 90 000, which included at least 17% N-linked carbohydrate. The molecular weight by gel permeation chromatography varied from 220 000 to 340 000, depending on the conditions used. The amino acid composition was determined and the N-terminal sequence was found to be er(5)-Leu(6)-Gly(7)-Arg(8)-Glu(9)-Asp(10)-Val(11)- 19)-Arg(20)-Leu(21)-X(22)-Val(23)-Thr(24)-Ala(25). Aminopeptidase P cleaved the Arg(1)-Pro(2) bond of bradykinin with a k(cat)/K-m of 5.7 x 10(5) s(-1) M(-1). N-Terminal fragments of bradykinin including Arg-Pro-Pr, but not Arg-Pro, were also cleaved. The enzyme was shown to have four binding subsites (S-1, S-1', S-2', S-3'), the first three of which must be occupied for hydrolysis to occur. Neuropeptide Y and allatostatin I were hydrolyzed at the Tyr(1)-pro(2) bond and Ala(1)-Pro(2) bond, respectively. The pH optimum for Arg-Pro-Pro cleavagewas 6.8-7.5 in most buffers. The enzyme was most stable in the range of pH 7.0-10.5 in the presence of poly(ethylene glycol). NaCl inhibited activity completely at 2 M. Mn2+ had variable effects on activity, depending on its concentration and the substrate used. Various peptideshaving an N-terminal Pro-Pro sequence were inhibitory. The enzyme wasalso inhibited by EDTA, o-phenanthroline, 2-mercaptoethanol, dithiothreitol, p-(chloromercuri)benzenesulfonic acid, apstatin, and captopril. The carboxyalkyl angiotensin-converting enzyme inhibitors, ramiprilat and enalaprilat, inhibited activity in the micromolar range only in the presence of Mn2+.

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Documento generato il 30/11/20 alle ore 00:21:32