Adenyl Cyclase in Cardiac Tissue

Abstract

Abstract Experiments were performed on adenyl cyclase in washed particles of guinea pig ventricle and in particulate preparations extracted with LiBr. The Km for ATP was 0.08 mm. Since one Mg++ binds with ATP at pH 7.5, it was concluded that the true substrate is a Mg-ATP complex. Concentrations of Mg++ as high as 10 mm were required to saturate the enzyme; the Ka for this cation was 2 to 3 mm. Mg++ saturation curves were sigmoidal. Mg++ did not affect the Km for ATP. It was concluded that Mg++ binds to a second and possibly allosteric site. The consequence of this binding is significantly increased reactivity of the catalytic site for substrate. Fluoride profoundly activated the enzyme even in the presence of saturating amounts of Mg++ and was without significant effect on the affinity of ATP or of Mg++ for their binding sites. It was concluded that F- binds to the enzyme possibly as a magnesium-fluoride complex, the consequence of which is greater reactivity of the catalytic site than that produced by Mg++ alone. This activating effect was specific for F-; a variety of anions and organic fluorine compounds were inactive. The enzyme was activated by catecholamines but to a much lower degree than by F-. Ca++ was inhibitory both in the presence and absence of F-; the inhibitory action of Ca++ was found to be competitive with respect to Mg++. A Ki of about 0.3 mm for Ca++ was obtained. The possibility is considered that Mg++, by binding to a site other than the catalytic site, might serve in physiological regulation of the enzyme and that Ca++ by binding at this site may also have a modulating action.