UDP-galactosyltransferase (GT) is primarily a Golgi membrane- bound enzyme that participates in the coordinate biosynthesis of the carbohydrate moieties of glycoproteins and glycolipids. In mammary tissue, GT can interact with the hormonally regulated protein-lactalbumin. This complex (lactose synthetase) is responsible for the biosynthesis of the unique mammalian disaccharide, lactose. Galactosyltransferase has also been localized to the plasma membrane of a diverse variety of cells and tissues by immunohistochemical procedures. This cell surface distribution supports the hypothesis that, in addition to its biosynthetic role, this transferase also has a functional role in intercellular recognition/adhesion. Our long range goals are to determine the structure, function and organization of GT in both intracellular and plasma membranes and to understand the mechanism responsible for compartmentalization of this membrane bound enzyme. The proposed experiments outlined in this proposal lay the foundation for these goals. This proposal takes advantage of: (1) a partial cDNA clone isolated from a Lambdal gt11 expression library that encodes bovine GT and (2) a series of monoclonal antibodies that identify four unique structural/functional domains within the bovine GT polypeptide including the alpha-lactalbumin binding site. Additionally, several of these antibodies are able to selectively recognize either the cell surface GT or the intracellular Golgi-associated GT as analyzed by immunohistochemical staining. These observations suggest a structural difference between the intracellular and cell surface form of GT. Using these highly specific and complementary probes for GT, we plan: (1) to isolate a full- length cDNA clone for bovine GT. (2) To characterize the mRNA structure including any alternatively spliced intermediates. (3) To construct an expression vector containing the complete protein coding sequence, in order to determine after transfection, if GT is synthesized and inserted into both the intracellular Golgi membrane and the plasma membrane. (4) To localize the four structure/function domains within the GT polypeptide as identified by our monoclonal antibodies by utilization the Lambdal gt11 expression system in conjunction with Western blot analysis.
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