Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger.
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.
Huot
S J
SJ
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Cassel
D
D
Igarashi
P
P
Cragoe
E J
EJ
Slayman
C W
CW
Aronson
P S
PS
D000584:Amiloride / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D007672:Kidney Cortex / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride