CHARACTERIZATION OF AMINOPEPTIDASE-N FROM TORPEDO-MARMORATA KIDNEY

A major antigen of the brush border membrane of Torpedo marmorata kidn ey was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very sim ilar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed amino peptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa aft er SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefor e heavily glycosylated. Depending on solubilization conditions, both t he antigen and peptidase activity were recovered either as a broad pea k with a sedimentation coefficient of 18S (2% CHAPS) or as a single pe ak of 7.8S (1% CHAPS plus 0.2% C(12)E(9)), showing that Torpedo aminop eptidase N behaves as an oligomer stabilized by hydrophobic interactio ns, easily converted into a 160 kDa monomer. The antigen is highly con centrated in the apical membrane of proximal tubule epithelial cells ( 600 gold particles/mu m(2) of brush border membrane) whereas no labeli ng could be detected in other cell types or in other membranes of the same cells (basolateral membranes, vacuoles or vesicles). Monoclonal a ntibodies prepared here will be useful tools for further functional an d structural studies of Torpedo kidney aminopeptidase N.