Adenyl Cyclase in Fat Cells

Abstract

Abstract Some of the characteristics of adenyl cyclase activity and its response to the stimulatory agents, adrenocorticotropin (ACTH) and fluoride ion, were investigated in rat fat cell ghosts. ACTH and fluoride ion stimulated the activity of the same enzyme. The membrane-bound enzyme was unstable at 0 and 37° and was inactivated by trypsin. Of several monovalent cations tested, lithium ion inhibited the basal, ACTH-, and fluoride-stimulated activities of the enzyme. Potassium ion, at 10 mm, specifically enhanced the response of the enzyme to ACTH; this effect of potassium was abolished by sodium ion (100 mm). The enzyme required a divalent ion for activity; among several divalent cations tested, Mn++, Mg++, and Co++ were effective. Mn++ (5 mm) stimulated basal and fluoride activities but not ACTH activity which showed a preference for Mg++. Ca++ inhibited adenyl cyclase activity; ACTH and fluoride ion reduced the concentration necessary to obtain 50% inhibition from 2.0 mm to 0.75 and 0.40 mm, respectively. The affinity of adenyl cyclase for Mg++ was increased in the presence of ACTH and fluoride ion, whereas the affinity for ATP was not changed. The kinetic characteristics of the basal, ACTH-, and fluoride-stimulated activities of the enzyme in the presence of Mg++ indicated that adenyl cyclase has two binding sites for Mg++, one of which is at the catalytic site and the other at some site that is altered by the actions of ACTH and fluoride ion and which enhances, allosterically, the reaction of the catalytic site with its substrate, MgATP. Although both ACTH and fluoride affected the same kinetic parameter (increased affinity for Mg++), the following differences were observed in their characteristics of activation: (a) Fluoride- and ACTH-stimulated activities were affected differently in the presence of potassium and manganous ions; (b) ACTH concentration curves were hyperbolae whereas fluoride concentration curves were sigmoids; (c) the concentration of ACTH giving half-maximal stimulation (apparent Km) was only slightly affected by temperature, whereas the apparent Km for fluoride activation was changed from 0.9 mm at 37° to 2.0 mm at 30°. It was concluded that ACTH and fluoride ion activate the same enzyme and alter the same kinetic parameter involved in activation, but do so by different means.