Abstract
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.
Highlights
Modulator is the low molecular weight heat-stable the activated receptor to specific regions onthe chromatin (1, inhibitorofglucocorticoid-receptorcomplex activa- 2)
Modulator preparationwas judged to be homogeneous The observation that partially purified glucocorticoidby analytical silica high-performance liquid chroma- receptor complexes activate more efficiently first suggested to tography, two-dimensional silica thin-layer chroma- Milgrom et al [8]that endogenous inhibitors of Activation tographya, ndprotonnuclearmagneticresonance may be present in cytosol
Chemical analysis of the purified had both of the activities attributable to exogenous sodium modulatorfollowing two-dimensional silica thin-layer molybdate (Na,MoO,) in this system [19,20,21].The chemical chromatography indicates that modulator is an ami- nature of the low molecular weight activation inhibitor is nophospholipid
Summary
We have purified modulator to apparent homo- formation of the receptor-nucleus complex [4], andit appears geneity from heated ralitver cytosol This was accom- to occur in two discrete events [5]. Bio-Gel P-4 columns coid-receptor complex andstabilizes the steroid bind- stimulated activation Wecalled this low molecularweight ing ability of the unoccupied rat liver glucocorticoid inhibitor “modulator” [11]. In addition to inhibiting activation, the partially pied receptor by 50%.modulator appears to be purified factor stabilized the steroid-binding ability of the endogenous factor that exogenous sodium molyb- the unoccupied glucocorticoid receptor.
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