Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

S. J. Huot, D. Cassel, P. Igarashi, E. J. Cragoe, C. W. Slayman, P. S. Aronson

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.

Original languageEnglish (US)
Pages (from-to)683-686
Number of pages4
JournalJournal of Biological Chemistry
Volume264
Issue number2
StatePublished - 1989

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Sodium-Hydrogen Antiporter
Amiloride
Purification
Carrier Proteins
Brushes
Microvilli
Kidney
Membranes
Buffers
Sodium Dodecyl Sulfate
Proteins
glycyl-glycyl-glycine
Affinity chromatography
Electrophoresis
Affinity Chromatography
Silver
Adsorption
Polyacrylamide Gel Electrophoresis
Gels
Rabbits

ASJC Scopus subject areas

  • Biochemistry

Cite this

Huot, S. J., Cassel, D., Igarashi, P., Cragoe, E. J., Slayman, C. W., & Aronson, P. S. (1989). Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger. Journal of Biological Chemistry, 264(2), 683-686.

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger. / Huot, S. J.; Cassel, D.; Igarashi, P.; Cragoe, E. J.; Slayman, C. W.; Aronson, P. S.

In: Journal of Biological Chemistry, Vol. 264, No. 2, 1989, p. 683-686.

Research output: Contribution to journalArticle

Huot, SJ, Cassel, D, Igarashi, P, Cragoe, EJ, Slayman, CW & Aronson, PS 1989, 'Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger', Journal of Biological Chemistry, vol. 264, no. 2, pp. 683-686.
Huot, S. J. ; Cassel, D. ; Igarashi, P. ; Cragoe, E. J. ; Slayman, C. W. ; Aronson, P. S. / Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 2. pp. 683-686.
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