Identification of the human NHE-1 form of Na(+)-H+ exchanger in rabbit renal brush border membranes.
To study the relation between the human Na(+)-H+ exchanger (NHE-1) and the renal brush border membrane (BBM) Na(+)-H+ exchanger, polyclonal antibodies to synthetic peptides representing a putative external (Ab-E) and an internal cytosolic domain (Ab-I) of human NHE-1 were generated in rabbits. Western immunoblot analyses indicated that both antibodies recognized a 97-kD protein in rabbit renal BBM but not basolateral membranes (BLM). Octyl glucoside-extracted rabbit renal BBM proteins also contained the 97-kD polypeptide as did a fraction eluted from an anion-exchange column with 0.2 M NaCl (fraction A). A fraction eluting between 0.2 and 0.4 M NaCl (fraction B) did not contain this protein. Prior reconstitution studies have indicated that Na(+)-H+ exchange activity is higher significantly in fraction B than fraction A. Administration of NH4Cl for 3-7 d to rabbits, a stimulus known to increase renal BBM Na(+)-H+ exchange activity, did not result in a change in expression of the 97-kD protein in either renal BBM or BLM. The results indicate that affinity-purified polyclonal antibodies to two separate domains of the human Na(+)-H+ exchanger recognize a 97-kD protein in rabbit renal BBM but not BLM. The dissociation between recognition of the 97-kD protein using antibodies and the majority of functional Na(+)-H+ exchange activity after chromatographic fractionation of solubilized BBM proteins and in native BBM after administration of NH4Cl suggest that rabbit renal BBM contains more than one form of Na(+)-H+ exchanger.
Weinman
E J
EJ
Department of Medicine, University of California, Los Angeles 90024.
Steplock
D
D
Corry
D
D
Shenolikar
S
S
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D009928:Organ Specificity; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D000643:Ammonium Chloride