Analysis of the distribution of Na+/H+ exchanger isoforms among the plasma membrane subfractions of bovine kidney cortex: reevaluation of methods for fractionating the brush-border and the basolateral membranes.
Distribution of the NHE1 and the NHE3 isoforms of Na+/H+ exchanger in the plasma membranes of bovine kidney cortex was analyzed. Fractionation of the plasma membranes by centrifugation on a Percoll density gradient resulted in clear separation of the basolateral membranes (BLM) from the brush-border membranes (BBM), with Na+,K+-ATPase and aminopeptidase M as their respective marker enzymes. Under these conditions, a 110 kDa protein cross-reactive with an anti-NHE1 antibody was detected exclusively in the BLM fractions, while a 90 kDa protein cross-reactive with an anti-NHE3 antibody was detected in the BBM fractions. A conventional Mg2+-precipitation method for obtaining the BBM, which is adequate with rabbit kidney as a starting material, turned out to be inadequate with bovine kidney cortex, since a considerable amount of the 110-kDa NHE1 protein was detected in the bovine kidney BBM fraction prepared by this procedure, together with the 90-kDa NHE3 protein. Percoll density gradient centrifugation is thus strongly recommended for the fractionation of BBM and BLM of bovine kidney cortex. The bovine NHE1 isoform was shown to be unique in that it is far less sensitive to the inhibition by ethylisopropylamiloride than that of other species.
Yoshioka
S
S
Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science (Rinshoken).
Suzuki
T
T
Kawakita
M
M
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000917:Antibody Formation; D000918:Antibody Specificity; D001485:Basement Membrane / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D003429:Cross Reactions; D006801:Humans; D007536:Isomerism; D007672:Kidney Cortex / Q000378:metabolism* / Q000648:ultrastructure*; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000276:immunology / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D000906:Antibodies; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D000584:Amiloride; D000254:Sodium-Potassium-Exchanging ATPase; C039614:ethylisopropylamiloride