PREPARATION OF ANTISERA TO RECOMBINANT, SOLUBLE N-ACETYLGLUCOSAMINYLTRANSFERASE-V AND ITS VISUALIZATION IN-SITU

N-Acetylglucosaminyltransferase V (GlcNAc-T V) is a glycosyltransferas e which transfers N-acetylglucosamine in beta(1,6) linkage to the alph a(1,6)-linked mannose residue of Asn-linked oligosaccharides. This enz yme is characterized by several unusual properties: GlcNAc-T V is the largest lumenal Golgi glycosyltransferase described thus far, and its multiple mRNA transcripts range from 4.5 to about 9.5 kb; GlcNAc-T V m RNA and activity are regulated by the src tyrosine kinase signalling p athway; in brain tissue, large levels of GlcNAc-T V mRNA are present, but only relatively low levels of catalytic activity can be detected; a lectin-resistant cell line, Lec4A, expresses active GlcNAc-T V which is mislocalized intracellularly. In addition, the cell surface oligos accharide products of this enzyme have been hypothesized to regulate i ntercellular adhesion. In order to devise specific inhibitors of this enzyme it is necessary to understand its physical structure and how st ructural changes can influence its activity and localization. We have expressed milligram amounts of a soluble form of recombinant rat GlcNA c-T V, purified it from CHO cell-conditioned media, and used it to pre pare specific antisera. This antisera binds selectively to GlcNAc-T V and has been used to visualize B-16 mouse melanoma cell GlcNAc-T V on immunoblots after SDS-PAGE. When the antisera was used in immunofluore scence microscopy experiments or permeabilized B-16 and baby hamster k idney cells, intense, specific staining was observed in intracellular structures which appear to correspond to the Golgi apparatus.