The proposed studies on the distribution of specific glycosphingolipids (GSLs) in subcellular membranes and on processes which regulate their biosynthesis and cellular levels will relate to a number of basic normal and pathological phenomena in which these complex carbohydrates participate. We now recognize that two independent processes can dramatically increase the levels of mouse kidney glycolipids, i.e. the stimulation of their biosynthesis by testosterone and the exocytosis of the multi-lamellar lysosomal bodies. To help understand these processes we propose to test the following hypotheses: that the metabolism and intracellular sorting of GSLs is determined by their ceramide as well as their carbohydrate structure; that testosterone induces the biosynthesis of specific molecular species that are unique precursors of GSL components that characterize """"""""lysosomal"""""""" membranes; that enzymes which are rate limiting in the biosynthesis of lysosomal GSLs are induced by testosterone and that mutations which block """"""""lysosomal"""""""" exocytosis include defects that affect the synthesis or expression of lysosomal or plasma membrane glycoproteins and/or glycolipids.
The specific aims designed to study aspects of these hypotheses are: 1) To characterize the GSL molecular species present in subcellular fractions from mouse kidney. Preparations from developing male and female mice will be analyzed by reversed-phase HPLC and liquid chromatography-mass spectrometric techniques. 2) To measure the in vitro incorporation of radiolabelled precursors into individual GSLs of the subcellular fractions. Pulse-chase techniques will be utilized to follow the synthesis of the GSLs and their flux through subcellular compartments. 3) To measure the activities of ceramide: UDP-Glc glucosyltransferase and four GSL galactosyltransferases with endogenous and exogenous substrate in kidney homogenates and subcellular fractions. 4) To analyze kidney GSLs and lectin binding proteins in subcellular fractions from pigmentation mutants that have blocks in the exocytosis of lysosomes.
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