Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides.
An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.
D002118:Calcium / Q000494:pharmacology; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011480:Protease Inhibitors; D013379:Substrate Specificity
D010455:Peptides; D011480:Protease Inhibitors; D010447:Peptide Hydrolases; C023092:succinyltrialanine p-nitroanilide hydrolase; D002118:Calcium