Cystinosis results from a defect in the efflux of cystine from the lysosome to the cytosol in cystinotic cells. However, many questions concerning this mechanism remain unanswered. Our major objective is to understand this disease thoroughly at the molecular level. An important aspect of this will be to understand the molecular differences leading to the different forms of cystinosis. We will study the kinetics of lysosomal cystine transport, and at the same time will attempt to isolate and characterize the cystine transport protein itself. Our kinetic studies will include the use of cell types from normal individuals and from patients with different types of cystinosis. The cell types studied will include cultured lymphoblasts, fibroblasts, renal tubular cells and endothelial cells. We plan to establish a system to measure counter transport (trans-stimulation) of cystine across the lysosomal membrane and to study the effect of inhibitors and pH on both cystine efflux and counter transport. We will prepare membrane vesicles from highly purified rat liver lysosomes in an attempt to reconstitute the cystine efflux (and counter transport) system. In addition to using this system for kinetic studies of cystine efflux, we will utilize methods of sequential solubilization of membrane proteins before resealing the membranes of these vesicles. This will provide a more purified starting material for our attempts to make an antibody to the cystine transport protein and for our attempts to isolate this protein. These results will be applied to studies of the human transport protein in both normal and cystinotic tissues.
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