Reconstitution of Na+/H+-Antiporter of Bovine Renal Brush-Border Membrane into Proteoliposomes and Detection of a 110 kDa Protein Cross-Reactive with Antibodies against a Human Na+/H+-Antiporter Partial Peptide in Antiport-Active Fractions after Partial Fractionation1

摘要

Bovine renal brush-border membranes were solubilized by 1.6sodium cholate. Na+/H+-antiporter was recovered in the supernatant after centrifugation at 160,000×gfor 1 h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H+-antiporter showed a pH-gradient dependent and amiloridesensitive22Na+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 chromatog-raphy, 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