Hydrolysis of opioid hexapeptides by carboxypeptidase N. Presence of carboxypeptidase in cell membranes.
Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys6- or Arg6-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the Km and kcat: Arg6-Met5-enkephalin, 49 microM, 1024 min-1; Arg6-Leu5-enkephalin, 57 microM, 375 min-1; Lys6-Met5-enkephalin, 216 microM, 6204 min-1; bradykinin, 19 microM, 58 min-1. Thus, while bradykinin had the lowest Km, the specificity constants (kcat/Km) for all the enkephalin hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl2 increased both the kcat and Km of bradykinin and Arg6-Met5-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental microvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg6- or Lys6-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.
D000311:Adrenal Glands / Q000201:enzymology; D000653:Amniotic Fluid / Q000201:enzymology; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002268:Carboxypeptidases / Q000032:analysis*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D004745:Enkephalins / Q000378:metabolism*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D001121:Lysine Carboxypeptidase / Q000032:analysis* / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy
D004745:Enkephalins; D009842:Oligopeptides; D002268:Carboxypeptidases; D001121:Lysine Carboxypeptidase; D001920:Bradykinin