Purification and characterization of an enkephalin-degrading dipeptidyl-aminopeptidase from porcine brain

Abstract

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.

Synthesis of Hydroxamic Esters via Alkoxyaminocarbonylation of β-Dicarbonyl Compounds

“…The products are closely related to well-known hydroxamic acid derivatives of malonates, which are inhibitors of MMPs. Previous syntheses of these compounds by other methods were complicated by side reactions, such as hydroxylamine reacting with the coupling reagent and N−O bond cleavage during deprotection 4a. These problems are completely avoided with the 2-pyridylsulfonyloxy reagent.…”

“…As shown by elegant X-ray crystallographic studies, these compounds are known to inhibit matrix metalloproteases (MMPs), a class of enzymes implicated in several diseases, by virtue of their ability to form a bidentate chelate with an active site zinc atom . The more potent inhibitors for some MMPs, such as thermolysin, collagenase, elastase, neutral endopeptidase, and endothelin converting enzyme, are hydroxamic acid analogues of malonic esters …”

Cloning and functional expression of rat kidney dipeptidyl peptidase II

“…In 1992, based on the results of a cytochemical study, Smid et al [13] proposed that DPP II may have a role in programmed cell death in rat incisor tooth ameloblasts. There are several reports on mammalian enzymes having DPP activities [14][15][16][17][18][19], but they have not been classified by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology.…”