Purification and characterization of a membrane-bound enkephalin-forming carboxypeptidase, "enkephalin convertase".
Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001921:Brain / Q000201:enzymology; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D002846:Chromatography, Affinity; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010902:Pituitary Gland / Q000201:enzymology; D051381:Rats; D012995:Solubility; D014018:Tissue Distribution
D002268:Carboxypeptidases; D043423:Carboxypeptidase H