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) and 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.