PURIFICATION OF THE GLUCOCORTICOID RECEPTOR-MINERALOCORTICOID RECEPTOR MODULATOR-2 FROM RABBIT LIVER

Modulators-1 and -2 are endogenous low-mel-wt regulators of glucocorti coid and mineralocorticoid receptors and protein kinase C. Structural analysis of apparently purified modulators suggested that these molecu les were novel ether aminophosphoglycerides. Subsequent X-ray crystall ography and NMR spectroscopy indicated that the ultra-large scale modu lator preparations were contaminated with glutamate and aspartate, alt hough these amino acids lacked modulator activity. In this article, we describe the purification of modulator-2 from rabbit liver cytosol an d 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 involve d the chromatography of trypsinized rabbit liver cytosol on the 7-L be d volume Sephadex G-15 gel-filtration column. As before, two peaks of modulator activity (modulator-1 and -2), as well as a DNA-binding inhi bitor (peak-3), eluted from the gel-filtration column. The resulting m odulator-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 chromatogr aphed on a Dowex-l anion-exchange column. Finally, modulator-2 was pur ified 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 reason able source of modulator-2. In addition, treatment of the preparation with glutamate decarboxylase seems to facilitate the subsequent separa tion of modulator-2 from the contaminating amino acids.