Covalent cross-linking of porcine small-intestine microvillar aminopeptidase: subunit structure of the membrane-bound and the solubilized enzyme [peptide mapping]

The porcine intestinal microvillar aminopeptidase (EC 3.4.11.2) consists of three types of subunits, alpha, beta and gamma, of molecular weights determined to 168,000, 118,000 and 54,000, respectively. The isolated detergent-form of the enzyme was cross-linked in dilute solution in the presence of Triton X-100 by diimidates varying in chain length from five to twelve carbon atoms. The tendency of the subunits to participate in inter-protomer cross-linking depended on the chain length of the reagent as shown by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Diimidates of chain lengths from seven to twelve carbon atoms caused formation of products of molecular weights up to about 660,000, whereas the six carbon atom diimidate generated complexes lower in molecular weight and the five carbon atom diimidate introduced very few inter-protomer cross-linkages. Cross-linking of brush border membrane vesicles, with dimethylsuberimidate, followed by Triton X-100 solubilization and isolation of the microvillar aminopeptidase by immunoadsorbent chromatography on anti-aminopeptidase M-Sepharose, indicated a maximum molecular weight of the membrane-bound enzyme of 330,000. The beta- and gamma-polypeptide chains were previously demonstrated to be formed by limited protcolysis of the alpha-polypeptide chain. Peptide mapping excluded the possibility of the gamma-chain being generated from the beta-chain. The subunit structures of the membrane-bound enzyme were suggested as alpha(,2), alpha beta gamma, and beta(,2)gamma(,2). Small amounts of a larger polypeptide chain of molecular weight 330,000 are present in preparations of microvillar aminopeptidase. Peptide mapping indicated structural homology between this component, termed 2alpha, and alpha, beta and gamma. It is suggested that the large polypeptide is a precursor of the intestinal aminopeptidase.