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Abstract
The renal basolateral Na+/HCO3- cotransporter is the main system responsible for HCO3- transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na+/HCO3- cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in L-alpha-phosphatidylcholine (proteoliposomes). Na+/HCO3- cotransporter activity was assessed as the difference in 22Na uptake in the presence of HCO3- and gluconate. The activity of the Na+/HCO3- cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was electrogenic. The catalytic subunit of protein kinase A significantly inhibited Na+/HCO3- cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na+/HCO3- cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na+/HCO3- cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na+/HCO3- cotransporter and suggest that the 56 kD doublet protein may represent the Na+/HCO3- cotransporter.
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