title: Purification of the glucocorticoid receptor-mineralocorticoid receptor modulator-2 from rabbit liver.
abstract: Modulators-1 and -2 are endogenous low-mol-wt regulators of glucocorticoid and mineralocorticoid receptors and protein kinase C. Structural analysis of apparently purified modulators suggested that these molecules were novel ether aminophosphoglycerides. Subsequent X-ray crystallography and NMR spectroscopy indicated that the ultra-large scale modulator preparations were contaminated with glutamate and aspartate, although these amino acids lacked modulator activity. In this article, we describe the purification of modulator-2 from rabbit liver cytosol and the separation of this phosphoglyceride from these amino acids. This purification was similar to the ultra-large scale version (Bodine, P.V. and Litwack, G. [1990] J. Biol. Chem. 265, 9544-9554), but involved the chromatography of trypsinized rabbit liver cytosol on the 7-L bed volume Sephadex G-15 gel-filtration column. As before, two peaks of modulator activity (modulator-1 and -2), as well as a DNA-binding inhibitor (peak-3), eluted from the gel-filtration column. The resulting modulator-2 pool was incubated with glutamate decarboxylase and treated batch-wise with Dowex-50W cation-exchange resin and Chelex-100 resin. This enzyme/resin-treated modulator-2 preparation was then chromatographed on a Dowex-1 anion-exchange column. Finally, modulator-2 was purified by preparative silica TLC. This last purification step resulted in the separation of modulator-2 from glutamate, aspartate, and gamma-aminobutyrate. In summary, rabbit liver cytosol appears to be a reasonable source of modulator-2. In addition, treatment of the preparation with glutamate decarboxylase seems to facilitate the subsequent separation of modulator-2 from the contaminating amino acids.
authors.affiliation: Women's Health Research Institute, Wyeth-Ayerst Laboratories, Radnor, PA 19087, USA.
authors.forename: P V
authors.initials: PV
authors.lastname: Bodine
authors.forename: G
authors.initials: G
authors.lastname: Litwack
mesh_terms: D000315:Adrenalectomy; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014222:Triamcinolone Acetonide / Q000378:metabolism
chemical_list: C490662:Gmeb2 protein, mouse; C490661:Gmeb2 protein, rat; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D014157:Transcription Factors; D005968:Glutamate Decarboxylase; D014222:Triamcinolone Acetonide