Purification and characterization of a soluble endopeptidase from rat bone.

Abstract

A soluble endopeptidase was purified from rat bone using ammonium sulphate precipitation followed by Fast Protein Liquid Chromatography on gel, anion exchange, and chromatofocusing columns. The enzyme was silver stain-pure (SDS-PAGE) and its apparent molecular weight was 60,000. In general, the enzyme favored the hydrolysis of the X-Y bond in substrates with the sequence of -Pro-X-Y-Pro-, where either X or Y (or both) were hydrophobic residues. The presence of imino acid residues near the scissile bond favored hydrolysis. The enzyme was strongly inactivated by metal chelating agents and p-hydroxymercuribenzoic acid, indicating that SH-groups may be necessary for full activity of the enzyme and that the enzyme may be a metallopeptidase.