Characterization of the Na+/H(+)-antiporter of bovine renal cortex and its immunoaffinity purification in a reconstitutively active form.
In order to analyze the localization and pharmacological characteristics of the Na+/H(+)-antiporter of bovine kidney, renal cortex membranes were fractionated by 35-48% continuous sucrose density gradient centrifugation. Both the Na+/H(+)-antiporting activity and the 110-kDa peptide cross-reactive with a polyclonal antibody against a peptide representing the C-terminal 22 amino acid residues of human Na+/H(+)-antiporter (NHE1) were found in the same fractions as alkaline phosphatase, a marker enzyme of the brush-border membrane. Most of the Na+/H(+)-antiporter distributed among the fractions was found to be of the low amiloride-sensitive type. Brush-border membranes were solubilized with sodium cholate and the solubilized proteins were applied to an immunoaffinity matrix (Protein A Sepharose CL-4B coupled with the above antibody). The 110 kDa protein was adsorbed to the affinity matrix and then recovered in the fraction eluted from the column at pH 11.5. After reconstitution into proteoliposome, the fraction containing the 110 kDa protein was highly active in Na+/H(+)-antiport. A 125-fold increase in the specific activity of the Na+/H+ antiport reaction was attained with the present immunoaffinity purification procedure. This represents the highest degree of purification of the native Na+/H(+)-antiporter so far attained.
Yamashita
T
T
Zoological Institute, Faculty of Science, University of Tokyo.
Kawakita
M
M
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002096:C-Peptide / Q000276:immunology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D003429:Cross Reactions; D007425:Intracellular Membranes / Q000737:chemistry; D007672:Kidney Cortex / Q000737:chemistry*; D008861:Microsomes / Q000737:chemistry; D008871:Microvilli / Q000737:chemistry / Q000187:drug effects; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers
D002096:C-Peptide; D002352:Carrier Proteins; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers; C020485:proteoliposomes; D000584:Amiloride