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Renal cortical basolateral Na+/HCO3- cotransporter: I. Partial purification and reconstitution
A A Bernardo1, F T Kear, O S Ruiz
1Section of Nephrology, University of Illinois at Chicago.
The Journal of Membrane Biology
|May 1, 1994
Summary
Researchers purified the renal sodium-bicarbonate cotransporter from rabbit kidneys. This protein is crucial for bicarbonate transport, and a 56 kD protein may be the transporter itself.
Area of Science:
- Nephrology
- Molecular Biology
- Biochemistry
Background:
- The renal basolateral sodium-bicarbonate cotransporter (NBCn1) is essential for bicarbonate reabsorption in proximal tubules.
- Understanding its purification and reconstitution is key to studying renal acid-base balance.
Purpose of the Study:
- To purify and functionally reconstitute the rabbit renal Na+/HCO3- cotransporter.
- To identify the molecular entity responsible for renal bicarbonate transport.
Main Methods:
- Basolateral membrane vesicles from rabbit renal cortex were solubilized and reconstituted into proteoliposomes.
- Na+/HCO3- cotransporter activity was measured via 22Na uptake.
- Purification involved octylglucoside solubilization, hydroxylapatite chromatography, and glycerol gradient centrifugation.
- Protein identification utilized SDS-PAGE.
Main Results:
- An 18-fold enhancement in cotransporter activity was observed after initial reconstitution.
- Further purification yielded a 124- to 300-fold increase in activity.
- The purified transporter exhibited similar kinetics, SITS inhibition, and electrogenic properties to the original.
- SDS-PAGE revealed a prominent 56 kD protein doublet in highly purified fractions.
Conclusions:
- The study successfully partially purified and functionally reconstituted the renal Na+/HCO3- cotransporter.
- The 56 kD protein doublet is a strong candidate for the renal Na+/HCO3- cotransporter.