Photoaffinity labeling by ( sup 3 H)-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity

Journal Article · Tue Apr 04 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00433a036· OSTI ID:5405608

Wu, J S; Lever, J E ^[1]

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1. Univ. of Texas Medical School, Houston (USA)

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 ({sup 3}H)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 ({sup 3}H)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.

• OSTI ID: 5405608
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:7; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKALI METAL COMPOUNDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CELL CONSTITUENTS
CELL MEMBRANES
CHROMATOGRAPHY
DIURETICS
DOMESTIC ANIMALS
DRUGS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KIDNEYS
KINETICS
LABELLED COMPOUNDS
LABELLING
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MEMBRANES
MOLECULAR WEIGHT
ORGANIC COMPOUNDS
ORGANS
PROTEINS
REACTION KINETICS
SEPARATION PROCESSES
SODIUM COMPOUNDS
SWINE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES