Adenylate Cyclase in Skeletal Muscle

Abstract

Abstract Adenylate cyclase was studied in plasma membranes prepared from rabbit skeletal muscle. Such preparations represented an increase in specific activity of 10- to 20-fold over the whole homogenate with a yield of activity of approximately 30%. Various parameters, such as phase contrast microscopy, marker enzyme activities, and chemical composition, suggested that the preparation constituted sarcolemma in a high degree of purity. The Km for substrate (MgATP) was 0.3 to 0.5 mm. The Ka for Mg2+ was 3 to 5 mm. Binding of Mg2+ to a second site resulted in increased reactivity of the catalytic site for substrate. Stimulation by fluoride resulted from an increase in maximal velocity; the Ka for Mg2+ and the Km for substrate were not appreciably altered. The effect of F- was markedly temperature-sensitive and partially irreversible. Fluoride-stimulated activity was particularly sensitive to inhibition by pyrophosphate and this inhibition was competitive with respect to ATP (Ki, 0.45 mm). Catecholamines stimulated the enzyme in a typical β adrenergic fashion. The prominent kinetic effect of epinephrine was (like F-) to increase reaction velocity without affecting affinity for Mg2+ or ATP. The regulation of adenylate cyclase in skeletal muscle may be classified as a V allosteric system since metal ions, F-, and epinephrine all result in increased maximal velocity of the enzyme reaction.