Reconstitution of catecholamine-sensitive adenylate cyclase. Reconstitution of the uncoupled variant of the S49 lymphoma cell

The uncoupled (UNC) variant of the S49 lymphoma possesses receptors and other known regulatory components necessary for hormone-stimulated adenylate cyclase activity but fails to respond to β-adrenergic agonists or to prostaglandin E₁. A procedure is described for the reconstitution of responses to hormones in purified plasma membranes of these cells. This technique utilizes cholate extracts prepared from membranes of wild type S49 cells or from membranes of other cell lines that lack either β-adrenergic receptors or the catalytic subunit of adenylate cyclase. The procedure also restores adenylate cyclase activity to membranes of the adenylate cyclase-deficient (cyc^-) S49 cell variant. Hormone responses of reconstituted UNC and cyc^- membranes resemble those of wild type S49 cell membranes in their dependene on agonist concentration for both enzyme activation and binding to β-adrenergic receptors. Effects of guanine nucleotides on the binding of agonists, which are lost in UNC and cyc^- membranes, are also restored in the reconstituted preparations. The factors in the cholate extract that are required for reconstitution of the two variant membranes are similarly labile to heating at 37°C and to treatment with N-ethylmaleimide but are differentially sensitive to treatment with trypsin. A protein is thus required for the reconstitution of UNC membranes. This protein appears to be closely linked to and, at the present time, is not distinguishable from the protein necessary for the restoration of adenylate cyclase activity in cyc^-.