Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase.
Enkephalin convertase, an enkephalin-synthesizing carboxypeptidase present in adrenal medulla chromaffin granules, has also been detected in brain and pituitary. To determine whether these three carboxypeptidase activities represent the same enzyme, we purified and characterized enkephalin convertase from adrenal medulla, whole brain, and whole pituitary. Enzyme from all three tissues co-purifies on DEAE-cellulose, gel filtration, concanavalin A, and L-arginine affinity columns, resulting in a 135,000-fold, 110,000-fold, and 2,800-fold purification for bovine adrenal medulla, brain, and pituitary, respectively. Purified enkephalin convertase appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showing a single band with an apparent molecular weight of 50,000 for enzyme isolated from all three tissues. Adrenal, brain, and pituitary enkephalin convertase are similarly inhibited by hexapeptide enkephalin precursors and active site-directed inhibitors. Both [Met]-and [Leu]enkephalin-Arg6 inhibit enkephalin convertase with Ki values between 50 and 80 microM, while [Met]-and [Leu]enkephalin-Lys6 are 3-fold less potent. Two active site-directed inhibitors, guanidinopropylsuccinic acid and guanidinoethylmercaptosuccinic acid, are potent inhibitors of all three enzymes with Ki values of 8-9 nM. A series of dansylated di-, tri-, and tetrapeptide substrates are hydrolyzed by enkephalin convertase with similar kinetic properties (Km, Vmax, and Kcat/Km) for the three enzymes. This evidence suggests that enkephalin convertase activity represents the same enzyme in adrenal medulla, brain, and pituitary. Enkephalin convertase may be involved in the production of other peptide neurotransmitters and hormones besides enkephalin.
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D001921:Brain / Q000201:enzymology; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D007700:Kinetics; D008970:Molecular Weight; D010902:Pituitary Gland / Q000201:enzymology
D002268:Carboxypeptidases; D043423:Carboxypeptidase H