ISOLATION AND IMMUNOLOCALIZATION OF A RAT RENAL CORTICAL MEMBRANE URATE TRANSPORTER

Two modalities of urate transport have been reported in rat kidney, a urate/anion exchanger and a potential sensitive, uricase-like uniporte r. As an initial attempt to isolate and characterize the responsible t ransport protein(s), rat renal cortical membranes were harvested, solu bilized, and subjected to affinity chromatography with urate or xanthi ne as the affinity ligand. Pig liver peroxisomal uricase was purified with the same system, and the enzymatically active protein was used to generate polyclonal antibodies in rabbit, Silver stain of SDS-polyacr ylamide gel electrophoresis gels of the eluted fraction containing the affinity-purified renal membrane protein(s) demonstrated bands at 25, 32, 36, and 41 kDa. On Western blot, two of these bands (32 and 36 kD a) were immunoreactive to the polyclonal antibody to pig liver uricase . In 6 of 10 studies, the affinity-purified renal membrane protein(s) also oxidized urate. Anti-pig liver uricase produced a selective and d ose-dependent inhibition of the uricase like urate uniporter in renal membrane vesicles, but did not affect the urate/anion exchanger or the sodium-dependent glucose transporter. Immunocytochemical studies of r at renal cortex with the same antibody indicated that the immunoreacti vity was localized to proximal tubules. These studies demonstrate that the renal cortical plasma membranes contain urate-binding proteins, w hich have some func tional and immunological homology to the hepatic p er oxisomal core protein, uricase. Within the renal cortex, these prot eins are localized to proximal tubules, the site of urate transport. S ince the antibody that reacts with the affinity-purified urate-binding proteins on Western blot selectively inhibits urate transport in inta ct membrane vesicles, it is concluded that at least one of the affinit y-purified urate-binding proteins is a uricase like urate transporter.