Photoaffinity labeling of the oxysterol receptor.
A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, can be labeled covalently by photoactivation of 7,7'-azo-[5,6-3H]cholestane-3 beta,25-diol. Other compounds tested for their potential as photoaffinity reagents were: 25-hydroxycholesta-4,6-dien-3-one, 3 beta,25-dihydroxycholest-5-en-7-one, and 3 beta-hydroxycholesta-8(14),9(11)-dien-15-one. These sterols did not bind to the receptor with adequate affinity, were not readily photolyzed, or did not react covalently with the receptor during photolysis. The successful photoaffinity label, 7,7'-azocholestane-3 beta,25-diol, binds to the receptor with high affinity (Kd = 9.1 nM). After activation of the partially purified oxysterol-receptor complex with UV light (greater than 300 nm), several covalently labeled proteins were found upon sodium dodecyl sulfate-gel electrophoresis. Labeling of one protein, Mr approximately 98,000, was much reduced when the binding reaction was carried out in the presence of an excess of unlabeled oxysterol. Under the reaction conditions investigated so far, approximately 1% of the specifically bound sterol was covalently linked after photolysis. These results are consistent with previous information suggesting that the Mr of the receptor subunit is approximately 97,000. The covalent labeling of the receptor reported herein should facilitate its further purification and characterization.
Taylor
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FR
Jackson Laboratory, Bar Harbor, Maine 04609.
Kandutsch
A A
AA
Anzalone
L
L
Phirwa
S
S
Spencer
T A
TA
D000345:Affinity Labels / Q000378:metabolism*; D001391:Azo Compounds / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002777:Cholestanols / Q000378:metabolism*; D002784:Cholesterol / Q000096:biosynthesis; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D010782:Photolysis; D011987:Receptors, Steroid / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D000345:Affinity Labels; D001391:Azo Compounds; D002777:Cholestanols; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; C054900:7,7'-azocholestane-25-diol; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases