The goal of the project is to characterize the biochemical and biological properties of cerebroside sulfate activator (CS-Act). CS-Act is a small, heat stable protein involved in the catabolism of cerebroside sulfate and several other glycosphingolipids. It has been referred to by a number of names including sphingolipid activator protein 1 (SAP-1), saposin Band nonspecific activator protein. Genetic defects in human CS-Act lead to arylsulfatase A-positive forms of metachromatic leukodystrophy, a progressive neuro-degenerative disease. The gene for the precursor to CS- Act, prosaposin, has recently been cloned and several disease related mutations identified. The function of CS-Act is believed to involve the binding and solubilization of certain lipids making them available to soluble hydrolytic enzymes. It is now felt that this protein might also participate in additional enzymatic processes and facilitate the transport of lipids between membranes. Recent discoveries suggest that CS-Act and its precursor may represent a widely distributed class of proteins involved in various aspects of complex lipid metabolism.
The specific aims of the present project focus on the detailed characterization of CS- Act from pig kidney, so that its structure and biological roles can be better understood. This in turn should enhance our ability to detect and eventually treat defects in this protein. The detailed structure of pig kidney CS-Act will be determined, micro- heterogeneity evaluated, disulfide bonding patterns established and subunit association examined. The entire pig CS-Act and putative subdomain sequences will be synthesized and their properties compared to the native material. The lipid binding characteristics will be examined in detail. Naturally occurring lipids of various types and structural analogs of cerebroside sulfate will be tested individually and in membrane-like mixtures. Structure-activity correlations will be characterized for the protein in a number of environments. The possible range of physiological functions for CS-Act will be better defined. The protein will be tested for its ability to modulate a wider spectrum of enzyme reactions involving lipoidal substrates. A role for CS- Act in membrane assembly and the transport of lipids between membranes will be investigated. The use of CS-Act for selectively modifying membrane composition and properties in vitro will also be evaluated.
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