Funds are requested for the continuation of a program of projects on the biochemistry of glycosphingolipids and some of the enzymes involved in their metabolism. Research on the regulation of ganglioside biosynthesis and metabolism will be focused on the sialyltransferases of GM3 and GD3 biosynthesis, which catalyze the transfer of sialic acid from CMP-sialic acid to form Alpha2-3 and Alpha2-8 glycosidic bonds, respectively. These enzymes will be purified from rat liver Golgi vesicles by affinity chromatography and their kinetic characteristics determined. The regulation of their activities will be examined by an immunochemical approach, with an emphasis on an evaluation of transcriptional control and post-translational covalent modifications. The proposal by Hakomori and his co-workers that plasma membrane GM3 ganglioside modulates growth of cultured cells by inhibition of the mitogenic response to growth factors will be studied in depth. Using doubly labeled GM3 for turnover studies and analyses of sialic acid and sialidase in the medium, a new model of GM3 synthesis and metabolism during the cell cycle and in sparse and confluent cultures will be experimentally tested. The effects of agents that increase the secretion of extracellular proteases will be determined, and an extracellular inhibitor of growth from conditioned medium of dense cultures will be used to test the hypothesis that turnover of GM3 in plasma membrane to lactosylceramide will become inhibited in dense cultures. Characterization of human liver lysosomal Alpha-galactosidase A and Alpha-N-acetylgalactosaminidase (Alpha-Gal B) will be continued. Monospecific, polyclonal antibodies against Alpha-Gal B will be affinity purified and used to screen a human hepatoma cDNA expression library in Lambdagt11. Knowledge of the primary sequences of Alpha-Gal B will permit the design of experiments to determine the locations of asparagine-linked carbohydrate chains on these enzymes and to assess the enzymatic activities of translation products in microbial and viral/mammalian expression systems. An interesting newly described Alpha-galactosidase from watermelon will be studied in some detail because it exists in a monomeric form and has catalytic activity in the hydrolysis of the glycosphingolipid that accumulates in Fabry's disease. A method will be developed for the analysis of human serum glycosphingolipids and used to assess the presence and structural nature of shed glycosphingolipids in various neoplasias.
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