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Studies on heart muscle lipases and studies on 3', 5'-cyclic nucleotide phosphodies-terase Yamamoto, Masanobu


PART I STUDIES ON HEART MUSCLE LIPASES The study of the role of lipids in supplying the energy requirements of the heart has attracted widespread attention, particularly within the past decade. It is now known that the heart, under normal conditions, oxidizes lipids as its main source of energy. Numerous investigators have studied the in vivo and in vitro uptake and utilization of exogenously supplied lipids in the form of triglycerides, free fatty acids and ketone bodies. However, very few have studied the utilization of endogenous lipids by the working heart. We have examined the relative importance of both endogenous glycogen and triglycerides for supplying the caloric needs of the isolated beating rat heart, and found that under the perfusion conditions used, endogenous glycogen appears to supply the initial source of energy. A lipase in rat cardiac tissue was also examined. The enzyme had a pH optimum near 6.8, and was strongly inhibited by 0.2 M NaF and by 2 x 10⁻⁴M diisopropylfluorophosphate. Most of the activity was found in the nuclear fraction of tissue homogenates. The enzyme hydrolyzed both monoolein and mono-stearin, and possessed much less activity against tripalmitin. The enzyme also rapidly hydrolyzed the monostearin component of Ediolʀ (a commercial coconut oil emulsion widely used in lipase studies), and the implications of these findings are discussed. It was concluded from these studies that a lipase other than lipoprotein lipase exists in rat myocardium. PART II STUDIES ON CYCLIC 3', 5'-NUCLEOTIDE PHOSPHODIESTERASE In recent years, the study of the role of cyclic 3', 5'-adenosine monophosphate (cyclic 3', 5'-AMP) in the regulation of several biological reactions and processes has received widespread attention. The presence of a physiological mechanism for terminating the action of cyclic 3', 5'-AMP in biological systems would therefore be expected. Indeed, an enzyme, cyclic 3', 5'-nucleotide phosphodiesterase has been shown to exist in most mammalian tissues which have been studied for its activity. The central nervous system, particularly the cerebral cortex, possesses a very high activity of this enzyme. In this study, cyclic 3', 5'-nucleotide phosphodiesterase was partially purified from rabbit brain and its properties were studied. The enzyme required Mg⁺⁺ions for activity and was inhibited by 2 x l0⁻⁴M theophylline. Cyclic 3', 5'-dAMP, cyclic 3', 5'-GMP and cyclic 3', 5'-dGMP were hydrolyzed by the brain diesterase at approximately one-half the rate at which cyclic 3', 5'-AMP was hydrolyzed. Little activity against cyclic 3', 5'-CMP, cyclic 3', 5'-dCMP and cyclic 3', 5'-TMP was detected, although cyclic 3', 5'-UMP was hydrolyzed at approximately 13% of the rate at which cyclic 3', 5'-AMP was hydrolyzed. The brain diesterase therefore possessed a high specificity for cyclic 3', 5'-nucleotides with purine bases. Optimum enzyme activity was observed near pH 7.0, and the activity was stimulated about 1.5-fold by 0.06 M imidazole. The Km value of the enzyme with cyclic 3', 5'-AMP as substrate was approximately 0.8 x 10⁻⁴M. The properties of the partially purified phosphodiesterase from brain were thus very similar to the diesterases which have been purified from beef and dog hearts. A study of the intracellular localization of the brain diesterase indicated that about 50% of the activity was located in the 105,000 x g supernate. The microsomal and mitochondrial fractions also contained considerable amounts of diesterase activity, but little activity was located in the nuclear fraction. A survey of cyclic 3', 5'-nucleotide phosphodiesterase activity in several available specimens of the plant kingdom indicated the absence of this enzyme activity in these organisms. However, appreciable levels of diesterase activity were detected in E. coli.

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