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 fractionation.
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 x g for 1 h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H(+)-antiporter showed a pH-gradient dependent and amiloride-sensitive 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.
Yamashita
T
T
Zoological Institute, Faculty of Sciences, University of Tokyo.
Kawakita
M
M
D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D005591:Chemical Fractionation; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D003429:Cross Reactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D008871:Microvilli / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017923:Sodium-Hydrogen Exchangers
D000906:Antibodies; D002352:Carrier Proteins; D002793:Cholic Acids; D008565:Membrane Proteins; D010455:Peptides; D017923:Sodium-Hydrogen Exchangers; D019826:Cholic Acid