Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity.
N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with [3H]MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.
Wu
J S
JS
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Lever
J E
JE
D000345:Affinity Labels / Q000378:metabolism*; D000584:Amiloride / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D011485:Protein Binding; D017923:Sodium-Hydrogen Exchangers; D013552:Swine; D014316:Tritium
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D017923:Sodium-Hydrogen Exchangers; D014316:Tritium; D000584:Amiloride; C056369:5-(N-methyl-N-isobutyl)amiloride