JBC, Vol. 254, Issue 2, 255-258, Jan, 1979
Solubilization and reconstitution of dopamine-sensitive adenylate cyclase from bovine caudate nucleus
F. M. Hoffmann
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
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.
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