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 uniporter. As an initial attempt to isolate and characterize the responsible transport protein(s), rat renal cortical membranes were harvested, solubilized, and subjected to affinity chromatography with urate or xanthine 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-polyacrylamide 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 kDa) 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 dose-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 rat renal cortex with the same antibody indicated that the immunoreactivity was localized to proximal tubules. These studies demonstrate that the renal cortical plasma membranes contain urate-binding proteins, which have some functional and immunological homology to the hepatic peroxisomal core protein, uricase. Within the renal cortex, these proteins are localized to proximal tubules, the site of urate transport. Since the antibody that reacts with the affinity-purified urate-binding proteins on Western blot selectively inhibits urate transport in intact membrane vesicles, it is concluded that at least one of the affinity-purified urate-binding proteins is a uricase-like urate transporter.
Knorr
B A
BA
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Lipkowitz
M S
MS
Potter
B J
BJ
Masur
S K
SK
Abramson
R G
RG
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D007672:Kidney Cortex / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D013552:Swine; D014503:Urate Oxidase / Q000276:immunology / Q000302:isolation & purification; D014527:Uric Acid / Q000378:metabolism
D002352:Carrier Proteins; D007074:Immunoglobulin G; D014527:Uric Acid; D014503:Urate Oxidase