Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride.
Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.
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Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Litwack
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D000818:Animals; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000032:analysis; D005938:Glucocorticoids / Q000378:metabolism; D006358:Hot Temperature; D008099:Liver / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D008982:Molybdenum / Q000494:pharmacology; D010742:Phospholipid Ethers / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D013055:Spectrophotometry, Infrared
D005938:Glucocorticoids; D010742:Phospholipid Ethers; D011965:Receptors, Glucocorticoid; C055044:glucocorticoid receptor complex modulator; D008982:Molybdenum; C024687:sodium molybdate(VI)