Reconstitution of Na+/H+-Antiporter of Bovine Renal Brush-Border Membrane into Proteoliposomes and Detection of a 110kDa Protein Cross-Reactive with Antibodies against a Human Na+/H+-Antiporter Partial Peptide in Antiport-Active Fractions after Partial Fractionation.

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  • Yamashita Tetsuro
    Zoological Institute, Faculty of Sciences, The University of Tokyo
  • Kawakita Masao
    Department of Pure and Applied Sciences, College of Arts and Sciences, The University of Tokyo

説明

Bovine renal brush-border membranes were solubilized by 1.6% sodium cholate. Na+/H+-antiporter was recovered in the supernatant after centrifugation at 160, 000×g for 1h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H+-antiporter showed a pH-gradient dependent and amiloridesensitive 22Na+ uptake very similar to that of brush-border membrane vesicles. Factors affecting the efficiency of reconstitution as well as the stability of the solubilized antiporter at various temperatures were studied. Sodium cholate-solubilized brush-border membrane proteins were fractionated by Sephacryl S-400 and DEAE-Toyopearl chromatography, and fractions containing reconstitutively active Na+/H+-antiporter were identified. A 110 kDa peptide cross-reactive with a polyclonal antibody against a C-terminal peptide (22-amino acid residues) of human Na+/H+-antiporter was consistently found on the immunoblot of the active fractions. A closely similar peptide was also detected in human placental membranes by this antibody. These results strongly suggest that the 110 kDa protein is responsible for Na+/H+-antiporter activity.

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