Purification and characterization of an enkephalin-degrading dipeptidyl-aminopeptidase from porcine brain.
A porcine brain dipeptidyl-aminopeptidase (DAP) has been purified more than 2400-fold from a crude mitochondrial fraction containing synaptosomes. This enzyme catalyzes the release of free Tyr-Gly from Leu-enkephalin (Km = 2.5 microM) with an optimal activity between pH 6.0 and pH 8.0. The enzyme appears homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis devoid of detectable contaminating aminopeptidase activities. The native enzyme is a monomeric protein with a molecular weight of 51,000 +/- 1,000 and an isoelectric point of 4.6 +/- 0.1. This enzyme cosediments with synaptosomes on a Ficoll-sucrose gradient and is partially associated with synaptic plasma membranes. Its activity is inhibited by the metal-chelating agents ethylenediaminetetraacetate and o-phenanthroline. It is not inhibited by the OH-reactive agent phenylmethanesulfonyl fluoride and SH-reactive agents such as p-(chloromercuri)benzoate and N-ethylmaleimide. Among the various biologically active peptides tested, the purified enzyme releases efficiently the N-terminal dipeptide moiety from enkephalins, Trp-Met-Asp-Phe-NH2 (CCK4), and Gly-Trp-Met-Asp-Phe-NH2 (CCK5). At variance, the native peptides CCK8, substance P, neurotensin, and angiotensin II are not cleaved by the DAP. This enzyme is different from other unspecific DAPs, as well as from enkephalin-degrading DAPs previously reported, by its molecular weight and substrate specificity.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000302:isolation & purification* / Q000378:metabolism; D004743:Enkephalin, Leucine / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013574:Synaptosomes / Q000201:enzymology
D011480:Protease Inhibitors; D004743:Enkephalin, Leucine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases