Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex.
Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.
D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis*; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000032:analysis*; D015260:Neprilysin; D009842:Oligopeptides / Q000378:metabolism; D010902:Pituitary Gland / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology
D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; C037284:benzyloxycarbonylleucyl-leucyl-glutamine-2-naphthylamide; C037286:benzyloxycarbonylglycyl-glycyl-leucine-4-nitroanilide; D011189:Potassium Chloride; C037285:benzyloxycarbonylalanyl-leucyl-arginine-2-naphthylamide; D010450:Endopeptidases; D015260:Neprilysin