Highly activatable adenylate cyclase in [2-3H]adenine-prelabeled vesicles prepared from guinea pig cerebral cortex by a simplified procedure.

ABSTRACT

An improved procedure utilizing a simple fractionation technique is described for the preparation and use of [2-3H]adenine-prelabeled vesicles from guinea pig cerebral cortex containing highly responsive adenylate cyclase activity. Adenosine consistently increased activity 1500--2000%, contrasted with activations of 200--300% previously reported by other investigators. Adenosine at 5 microM was more active in our system than at 20 times this concentration in studies by other investigators, increasing activity by 580--840%. Experimental conditions were explored, and Ca2+ was found to be necessary during tissue homogenization, but not during subsequent vesicle incubation. However, neither the higher Ca2+ concentration used by us (2.5 mM) nor the method of tissue homogenization could adequately explain the high activity of our preparations. The size of the incubation vessel was critical for both low basal activity and high activity in the presence of adenosine. Our preparations were similar to others in that combinations of neurohormones, which included histamine and epinephrine, elicited higher activities than individual neurohormones. Inspection of our vesicle preparations by scanning electron microscopy also showed them to be compatible with previously described preparations.