Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries.
A metalloendopeptidase optimally active at a neutral pH was purified 10000-fold from particulate fractions of bovine pituitaries. The solubilized enzyme has an apparent molecular weight of about 90 000, as determined by gel filtration on Sephadex G-200 and G-100 columns. The enzyme is not sensitive to inhibition by SH-blocking agents, diisopropyl fluorophosphate, leupeptin, pepstatin, antipain, and chymostatin. Thiols and metal chelators such as ethylenediaminetetraacetic acid (EDTA) o-phenanthroline are inhibitory. An EDTA-treated enzyme can be reactivated by several divalent metal ions, with zinc giving reactivation at the lowest concentrations. The specificity and kinetic parameters of the enzyme were studied with a series of synthetic peptide naphthylamides. The enzyme cleaves bonds in which the amino group is provided by a hydrophobic amino acid residue (position P1'). Replacement of this residue by small neutral amino acids decreases or virtually eliminates activity. The nature of substituents in positions P1, P2, P3, and P4 greatly influences specificity. Relatively high kcat and kcat/Km ratios were obtained with substrates containing arginine residues in positions P1 and P2. In such cases the impression of a "trypsin-like" activity was created. High reaction rates were also observed with substrates containing small neutral amino acids in positions P1 and P2, provided that position P3 was occupied by the acidic (polar) glutaryl residue. Replacement of this residue with hydrophobic substituents greatly decreased the rate of reaction. When positions P1 and P2, however, were occupied by arginine residues, the unfavorable effect of hydrophobic substituents in position P3 or P4 on catalysis was eliminated.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis*; D010902:Pituitary Gland / Q000201:enzymology*; D013379:Substrate Specificity
D009842:Oligopeptides; D010450:Endopeptidases; D008666:Metalloendopeptidases