Identification of the chick neural retina cell surface N-acetylgalactosaminyltransferase using monoclonal antibodies.
Intact embryonic chick neural retina cells have at their surface an N-acetylgalactosaminyltransferase which catalyzes the incorporation of N-acetylgalactosamine from UDP-N-acetylgalactosamine into endogenous macromolecular acceptors. The enzyme along with its endogenous acceptors can be isolated as a particulate complex following treatment of membrane-enriched fractions with Triton X-100. In this paper we report on two separate fusions generating monoclonal antibodies: one using as immunogen the particulate complex and the second using as immunogen a soluble N-acetylgalactosaminyltransferase found in tissue-culture-conditioned medium which lacks endogenous acceptor activity. Antibodies from both fusions recognize an antigen which is tightly associated with the particulate transferase/acceptor complex and a soluble antigen having N-acetylgalactosaminyltransferase activity toward exogenously added acceptors. The antibodies recognize a component of ca Mr 220,000, which shows N-acetylgalactosaminyltransferase activity after SDS-gel electrophoresis and transfer to nitrocellulose. This component comigrates on two-dimensional gel electrophoresis with an iodinatable cell surface component whose presence at the cell surface correlates with endogenous transferase activity. We conclude that the antibodies recognize the transferase enzyme itself. Immunohistochemical analysis shows that the enzyme is initially localized throughout the embryonic neural retina in a pattern indicative of a cell surface disposition but becomes restricted to the outer plexiform layer and to outer segments in the adult.
D000818:Animals; D000911:Antibodies, Monoclonal; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D046508:Culture Techniques; D005455:Fluorescent Antibody Technique; D005700:Galactosyltransferases / Q000276:immunology*; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D017350:N-Acetylgalactosaminyltransferases; D012160:Retina / Q000201:enzymology*; D012995:Solubility
D000911:Antibodies, Monoclonal; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase