Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme.
A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.
D000818:Animals; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification; D010902:Pituitary Gland / Q000201:enzymology*; D011480:Protease Inhibitors; D013379:Substrate Specificity
D002412:Cations; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D010450:Endopeptidases