Role of Specific Membrane Lipids in Modulating the Activity of Adenylate Cyclase

Abstract

Male Sprague-Dawley rats were decapitated and their brains removed; subsequent operations were performed at 0°C. The brain was homogenized in 8 vol (vol/wt) of 3 mM MgCl2, 3 mM DTT, 50 mM Tris-HCl pH 8.4 and centrifuged for 10 min at 40,000 g,,..,. This procedure was repeated once and the pellet was homogenized in 8 vol (vol/wt) of 1 mM MgCl2, I mM DTT, 10%o glycerol (vol/vol), 0.5% sodium deoxycholate (DOC), 50 mM Tris-HCI, pH 8.4 (Buffer A). The homogenate was centrifuged for 45 min at 300,000 g,..., and the supernatant, containing solubilized enzyme, retained. Enzyme in the supernatant was separated from solubilized lipid by chromatography on Ultrogel AcA34 equilibrated with buffer A. When required, phospholipid, dissolved in chloroform:methanol (2:1 vol/vol) and dried under a stream of N2, was dispersed in the solubilized enzyme preparation. Enzyme activity was measured at 300C (10).