A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein.
Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280). We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger.
Ross
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W
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Bertrand
W
W
Morrison
A
A
D000345:Affinity Labels; D000584:Amiloride / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents; D004285:Dogs; D007457:Iodine Radioisotopes; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D015394:Molecular Structure; D008970:Molecular Weight; D010777:Photochemistry; D017923:Sodium-Hydrogen Exchangers
D000345:Affinity Labels; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D017923:Sodium-Hydrogen Exchangers; C063513:N(5)-piperidine-amiloride; C063512:N(5)-piperazine-amiloride; C050819:5,6-dichloroamiloride; D000584:Amiloride