UBC Theses and Dissertations
UBC Theses and Dissertations
Studies on adenyl cyclase in heart and skeletal muscle Severson, David Lester
The kinetic properties of adenyl cyclase in heart and skeletal muscle were examined to determine the mechanisms by which hormones and other regulatory agents stimulate the formation of adenosine 3',5’-cyclic monophosphate (cyclic AMP). Experiments were performed with washed particles and lithium bromide-extracted particulate enzyme preparations from guinea pig or rabbit heart. Myocardial adenyl cyclase was stimulated by concentrations of metal ions in excess of the ATP concentration. Metal ions apparently bind to some second site on the enzyme in addition to the catalytic site and as a consequence increase reaction velocity. Manganese was more effective than magnesium in activating the cardiac enzyme; cobalt was less effective. The K[sub a] for manganese was 0.7 mM in comparison to 3 mM for magnesium. The K[sub m] for ATP was 0.1 mM; metal ions increased maximal velocity without altering the substrate affinity of the enzyme. Concentrations of ATP in excess of the metal ion concentration resulted in inhibition of myocardial adenyl cyclase due to competition with the enzyme for metal ions. Adenyl cyclase was also studied in rabbit skeletal muscle plasma membrane preparations. Isolation of plasma membranes resulted in a ten to twenty-fold increase in specific enzyme activity with a yield of approximately 30 per cent of the adenyl cyclase activity of the whole homogenate. Phase contrast microscopy as well as the analysis of the chemical composition and various enzyme activities suggested that the membrane preparations consisted of highly purified plasma membranes. The K[sub a] for magnesium was 3 to 5 mM and the K[sub m] for ATP was approximately 0.3 mM. Magnesium increased the reaction velocity at all concentrations of substrate, presumably by binding to some second site. The stimulation of skeletal muscle adenyl cyclase by fluoride was markedly temperature sensitive and partially irreversible. Fluoride-stimulated enzyme activity was particularly sensitive to inhibition by pyrophosphate; pyrophosphate inhibition was competitive with respect to ATP (K[sub i], 0.45 mM). The relative potencies of catecholamines in stimulating enzyme activity and the inhibition of the epinephrine stimulation by propranolol indicated some relationship between adenyl cyclase and the beta-adrenergic receptor. The stimulation by epinephrine was enhanced by the addition of low concentrations of GTP or GDP; guanyl nucleotides had little or no effect on basal or fluoride-stimulated adenyl cyclase activity. The kinetic nature of the stimulation by fluoride and epinephrine was identical. Both epinephrine and fluoride increased maximal velocity without affecting the affinity of the enzyme for magnesium or ATP. Thus, the regulation of adenyl cyclase in heart and skeletal muscle can be described as a "V" allosteric system. The properties of adenyl cyclase are discussed in relation to the involvement of the enzyme in a hormone informational transfer unit.
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