Purification and properties of membrane-bound aminopeptidase P from rat lung.
The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified 670-fold to apparent homogeneity from 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 X1-Gly2-Pro3-Glu4-Ser5-Leu6-Gly7-Arg8-Glu9-As p10-Val11-Arg12-Asp13-X14-Ser15- Thr16-Asn17-Pro18-Pro19-Arg20-Leu21- X22-Val23-Thr24-Ala25-. Aminopeptidase P cleaved the Arg1-Pro2 bond of bradykinin with a kcat/Km of 5.7 x 10(5) s-1 M-1. N-Terminal fragments of bradykinin including Arg-Pro-Pro, but not Arg-Pro, were also cleaved. The enzyme was shown to have four binding subsites (S1, S1', S2'. S3'), the first three of which must be occupied for hydrolysis to occur. Neuropeptide Y and allatostatin I were hydrolyzed at the Tyr1-Pro2 bond and Ala1-Pro2 bond, respectively. The pH optimum for Arg-Pro-Pro cleavage was 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 peptides having an N-terminal Pro-Pro sequence were inhibitory. The enzyme was also 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+.
Orawski
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Department of Molecular and Cellular Biochemistry, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
Simmons
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000378:metabolism; D004150:Dipeptidases / Q000302:isolation & purification; D018819:Dipeptidyl Peptidase 4 / Q000302:isolation & purification; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D008168:Lung / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D010455:Peptides; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D004150:Dipeptidases; C038937:dipeptidase; D018819:Dipeptidyl Peptidase 4; D001920:Bradykinin