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Purification and properties of membrane-bound aminopeptidase P from rat lung
1Department of Molecular and Cellular Biochemistry, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
Abstract:
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+.
Insights
This study purified membrane-bound aminopeptidase P from rat lung microsomes, revealing its molecular weight, N-terminal sequence, and substrate-binding properties. The enzyme cleaves specific peptide bonds, with optimal activity and stability under defined conditions, and is inhibited by various compounds.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Membrane-bound aminopeptidase P (aminoacylprolyl-peptide hydrolase) is an enzyme involved in peptide metabolism.
- Understanding its properties is crucial for various biological processes and potential therapeutic applications.
Purpose of the Study:
- To purify and characterize the membrane-bound form of aminopeptidase P from rat lung microsomes.
- To determine its molecular properties, N-terminal sequence, and substrate specificity.
- To investigate its catalytic mechanism and inhibition profile.
Main Methods:
- Purification of aminopeptidase P from rat lung microsomes using phosphatidylinositol-specific phospholipase C.
- Determination of subunit molecular weight, N-terminal sequence, and amino acid composition.
- Enzyme kinetics studies using bradykinin, neuropeptide Y, and allatostatin I as substrates.
- Analysis of enzyme inhibition by various peptides and chemical agents.
Main Results:
- Aminopeptidase P was purified 670-fold with a subunit molecular weight of 90,000 (17% N-linked carbohydrate) and a variable oligomeric state (220,000–340,000).
- The N-terminal sequence was determined, and the enzyme efficiently cleaved the Arg1-Pro2 bond of bradykinin (kcat/Km = 5.7 x 10^5 s^-1 M^-1).
- The enzyme possesses four binding subsites (S1, S1', S2', S3'), requiring the first three for hydrolysis, and showed activity against neuropeptide Y and allatostatin I.
Conclusions:
- The study successfully purified and characterized membrane-bound aminopeptidase P, providing insights into its structure and function.
- The enzyme's substrate specificity and catalytic mechanism, involving specific binding subsites, were elucidated.
- The identification of inhibitors, including angiotensin-converting enzyme inhibitors, suggests potential roles in regulating peptide signaling pathways.