The long-range objectives of the proposed research are to elucidate the biosynthesis, structure, action, membrane topology, and regulation of Golgi enzymes, with emphasis on the Alpha-D-mannosidases. Relevant studies on brain Alpha-D-mannosidases and on lysosomal Alpha-D-mannosidase will also be performed.
Specific aims are the following. 1) Determine the structure, biosynthetic processing, and membrane topology of Golgi mannosidase II. This enzyme is a glycoprotein which in 3T3 cells incorporates sulfate, phosphate, and palmitate. 2) Purify, characterize, and investigate the biosynthesis and membrane localization of Golgi mannosidases IA and IB. 3) Determine the biosynthetic pathway and membrane localization of Golgi Beta-N-acetylglucosaminyl transferases I and II. These enzymes alternate with the Golgi mannosidases in the synthesis of asparagine-linked glyco-proteins containing complex oligosaccharides. 4) Characterize brain Alpha-D-mannosidases. 5) Determine the biosynthetic pathway of lysosomal Alpha-D-mannosidase. Biosynthetic studies will be carried out in cultured cells. Membrane localization studies will utilize immunocytochemical procedures. The research on brain enzymes will involve the characterization and purification of the various Alpha-D-mannosidases and the effects of the locoweed toxin, swainsonine, which produces in animals a neurological condition resembling the hereditary lysosomal storage disease, Alpha-mannosidosis. Swainsonine blocks the synthesis of glycoproteins with asparagine-linked oligosaccharides by inhibiting Golgi mannosidase II. Since lysosomal enzymes, membrane constituents, hormones, brain receptors and other important biological substances have such structures, and since the Golgi apparatus plays a central role in their biosynthesis and routing within the cell, the studies proposed should provide results of broad applicability.
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