Characterization of aminopeptidase N from Torpedo marmorata kidney.
A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian amino-peptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2% C12E9), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/microns2 of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolateral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.
O'Callaghan
B
B
Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, 91198 Gif-sur-Yvette, France.
Synguelakis
M
M
Le Gal la Salle
G
G
Morel
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D018826:CD13 Antigens / Q000276:immunology / Q000302:isolation & purification*; D005455:Fluorescent Antibody Technique; D007684:Kidney Tubules / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D014101:Torpedo / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018826:CD13 Antigens