PREPARATION AND PROPERTIES OF A CELL‐FREE, HORMONALLY RESPONSIVE ADENYLATE CYCLASE FROM GUINEA PIG BRAIN¹

Abstract

Abstract —The accumulation of cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) was studied in cell‐free homogenates of guinea pig brain. Homogenates, prepared in Krebs‐Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [ 3 H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [ 32 P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg 2+ and Ca 2+ , resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH) 2 ‐ZnSO 4 mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell‐free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.