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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
1
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pubmed:dateCreated |
1988-5-6
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pubmed:abstractText |
We have used a radiolabelled, benzophenone analog of bumetanide, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na + K + Cl) cotransport, and [3H]BSTBA binds to intact duck red cells with a high affinity similar to that of [3H]bumetanide (K 1/2 congruent to 0.1 microM). We incubated duck red cells with [3H]BSTBA, then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0 degree C to prevent dissociation of the [3H]BSTBA. The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H2O2-digested gel slices revealed [3H]BSTBA to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by [3H]BSTBA in a manner highly consistent with labelling of the (Na + K + Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na + K + Cl)-cotransport system in these cells: (1) Photolabelling of this protein by [3H]BSTBA is blocked when 10 microM unlabelled bumetanide is included in the initial incubation medium with [3H]BSTBA; (2) Photoincorporation of [3H]BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na + K + Cl) cotransport and saturable [3H]bumetanide binding in duck red cells; (3) The photolabelling of this protein shows a saturable dependence on [3H]BSTBA concentration, with a K1/2 (0.06 microM) similar to that for the reversible, saturable binding of [3H]BSTBA and [3H]bumetanide to duck red cells; and (4) [3H]BSTBA photoincorporation into the 150 kDa protein, like saturable [3H]bumetanide binding to intact cells, requires the simultaneous presence of Na+, K+, and Cl- in the medium containing the radiolabelled diuretic.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/4-benzoyl-5-sulfamoyl-3-(3-thenyloxy...,
http://linkedlifedata.com/resource/pubmed/chemical/Affinity Labels,
http://linkedlifedata.com/resource/pubmed/chemical/Benzophenones,
http://linkedlifedata.com/resource/pubmed/chemical/Bumetanide,
http://linkedlifedata.com/resource/pubmed/chemical/Carrier Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Chlorides,
http://linkedlifedata.com/resource/pubmed/chemical/Potassium,
http://linkedlifedata.com/resource/pubmed/chemical/Sodium,
http://linkedlifedata.com/resource/pubmed/chemical/Sodium-Potassium-Chloride Symporters,
http://linkedlifedata.com/resource/pubmed/chemical/Sulfanilamides
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0006-3002
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
22
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pubmed:volume |
939
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
131-44
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:3349075-Affinity Labels,
pubmed-meshheading:3349075-Animals,
pubmed-meshheading:3349075-Benzophenones,
pubmed-meshheading:3349075-Bumetanide,
pubmed-meshheading:3349075-Carrier Proteins,
pubmed-meshheading:3349075-Chlorides,
pubmed-meshheading:3349075-Dogs,
pubmed-meshheading:3349075-Ducks,
pubmed-meshheading:3349075-Erythrocyte Membrane,
pubmed-meshheading:3349075-Kidney,
pubmed-meshheading:3349075-Potassium,
pubmed-meshheading:3349075-Sodium,
pubmed-meshheading:3349075-Sodium-Potassium-Chloride Symporters,
pubmed-meshheading:3349075-Sulfanilamides
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pubmed:year |
1988
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pubmed:articleTitle |
Photoaffinity labelling of a 150 kDa (Na + K + Cl)-cotransport protein from duck red cells with an analog of bumetanide.
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pubmed:affiliation |
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
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pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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