Purification and characterization of two aminopeptidases from guinea-pig small-intestinal mucosa. Cavian intestinal tripeptide hydrolases

Abstract

Two electrophoretically distinct cytosolic peptide hydrolases from guinea-pig small-intestinal mucosa have been highly purified by a six-step procedure comprising extraction from mucosal homogenate, ammonium sulphate fractionation, DEAE-cellulose chromatography, chromatofocusing, calcium phosphate chromatography and Sephadex G-100 gel filtration. They have similar apparent molecular masses as determined by gel filtration (Mr = 68 000) or by sodium dodecyl sulphate gel electrophoresis (Mr = 72 000). Both are aminopeptidases with optimum activity at pH 7.6. They are strongly inhibited by p-hydroxymercuribenzoate, o-phenanthroline and bestatin. Although both hydrolyse some dipeptides they have a distinctive kinetic preference for tripeptides composed of aromatic or non-polar residues. Their affinities for some tripeptides are particularly high and also the hydrolysis of some substrates exhibits biphasic kinetics. These two aminotripeptidases are similar but they can be differentiated from each other and from a number of other aminopeptidases.