Solubilization and reconstitution of dopamine-sensitive adenylate cyclase from bovine caudate nucleus
Journal of Biological Chemistry
Dopamine-sensitive adenylate cyclase was reconstituted from the cholate-soluble components of caudate nucleus homogenate. Biological function was restored by precipitating the components from cholate and phospholipid with ammonium sulfate, dialyzing the resuspended precipitate, and activating the particulate complex with phospholipid in the assay. The reconstituted adenylate cyclase was stimulated 3- to 4-fold by dopamine and 8- to 12-fold by guanyl-5'-yl imidodiphosphate. The catecholamine
... e catecholamine stimulation was specific for dopamine and required the addition of GTP. The cholate-soluble component(s) of the basal adenylate cyclase were separated from the component(s) that conferred dopamine sensitivity by gel filtration chromatography. Dopamine-sensitive adenylate cyclase was also reconstituted from digitonin-soluble components. These were resolved into two fractions by DEAE-cellulose chromatography: one fraction contained adenylate cyclase, but both fractions were required for reconstitution of dopamine-sensitive adenylate cyclase.