The proposed studies are designed to test the hypotheses that uricase is a membrane-associated protein that transports urate across renal plasma membranes, and that at least two transporters, an anion exchanger and the putative transporter, uricase, are present in brush border and/or basolateral membranes of the renal proximal tubule cells of several mammalian species. The mechanism(s) of urate transport will be assessed in unstimulated and copper stimulated brush border and basolateral membrane vesicles of rat, dog, and rabbit renal cortex prepared with several techniques: free-flow electrophoresis, magnesium aggregation, calcium precipitation, and percoll gradients. Transport will be examined in the presence and absence of inwardly directed electrolyte gradients, and in the presence and absence of pH gradients. Kinetic studies will assess saturation, inhibition and temperature dependence of transport. The kinetic constants of transport in vesicles will be compared to those obtained for urate oxidation by membranes. To further characterize the mechanism(s) of urate transport, the phenomena of homeo and heteroexchange diffusion will be evaluated. Cytochemical studies will be performed on renal and hepatic tissue slices and renal membrane vesicles to localize intramembranous and intracellular sites of uricase. This technique is based on a coupled oxidation-peroxidation reaction between hydrogen peroxide, produced by enzymatic (uricase) oxidation of urate, and cerium chloride. The product of this reaction, cerium perhydroxide, forms fine electron dense deposits at the site of the reaction, thereby permitting electron microscopic mapping of the distribution of uricase. In species and renal membranes in which uricase is detected in transport and cytochemical studies, uricase will be isolated and purified. Solubilized membranes will be subjected to affinity chromatography. Purification will be assessed with gel filtration, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), gradient PAGE, and isoelectric focusing. When purified, polyclonal antibodies will be raised and the effect of the antibodies on urate transport in membrane vesicles will be examined. Finally, purified uricase will be reconstituted into artificial lipid membranes or biological membranes devoid of uricase (red blood cell ghosts). Urate transport and kinetic constants of transport and oxidation will then be examined in the reconstituted system. This collective data should provide a solid base for understanding the mechanism(s) of urate transport, and perhaps the transport of other endogenous and exogenous organic anions within the renal proximal tubule.
Showing the most recent 10 out of 11 publications
