Choline dehydrogenase: Assay, properties and inhibitors

Abstract

Although choline has proven useful in the treatment of neurological disorders, its duration of action is limited by its rapid rate of metabolism. In order to evaluate the role of choline dehydrogenase in regulation of choline metabolism, some of the properties of the enzyme were studied using a new radioisotopic assay that is more sensitive and specific than previous methods. Choline dehydrogenase activity was present predominantly in kidney and liver of all species examined. Rats, mice, toads, cats, dogs, monkeys and sheep had highest activities in liver, whereas humans, baboons, rabbits and guinea pigs had more enzyme in kidneys. Enzyme activity was found to increase with age, reaching a maximum concentration at 1 week in livers and 4 weeks in kidneys of mice. Choline dehydrogenase, partially purified from rat liver mitochondria, was optimally active at a pH between 7 and 9, at a temperature of 37°, and in the presence of phenazine methosulfate. The K m for choline was similar for both the soluble (5.7mM) and the particulate (5.3mM) enzyme but the V max was slightly higher for the soluble [77 nmoles/min · (mg protein) −1] as compared to the particulate [34 nmoles min · (mg protein) −1] preparation. When choline oxidation was studied in vivo, the concentration of free choline in liver (60 μM) and the rate of oxidation of [ 3H]-choline administered intravenously [0.29 nmoles/min · (mg protein) −1] were found to be markedly less than the respective K m and V max values determined in vitro. Thus, choline dehydrogenase may not be saturated with choline under physiologic conditions, which could explain the rapid rate of metabolism of exogenously administered choline. The product of the reaction, betaine aldehyde, was a potent competitive inhibitor of choline dehydrogenase.( K i = 0.6 mM), whereas other products of choline metabolism were weaker inhibitors ( K i values >10 mM). Dimethylaminoethanol (deanol; a cholinomimetic agent) and 2-amino-2-methylpropanol inhibited the enzyme in vitro with inhibitory constants of 1.8 and 1.4mM, respectively, and caused an increase in concentration of choline in kidney and liver when administered to mice, which suggests that choline dehydrogenase is an important enzyme in regulation of the concentration of free choline in tissues under physiologic conditions.