Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M.
Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.
Bausback
H H
HH
Ward
P E
PE
D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents; D018826:CD13 Antigens; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004723:Endorphins / Q000378:metabolism*; D004744:Enkephalin, Methionine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides; D011691:Puromycin / Q000494:pharmacology; D013552:Swine
D000900:Anti-Bacterial Agents; D004723:Endorphins; D009842:Oligopeptides; D010455:Peptides; D011691:Puromycin; D004744:Enkephalin, Methionine; C016977:amastatin; D000626:Aminopeptidases; D018826:CD13 Antigens; D007930:Leucine; C012211:ubenimex