Studies on choline dehydrogenase. I. Extraction in soluble form, assay, and some properties of the enzyme.

The majority of the dehydrogenases of mammalian mitochondria are linked to the respiratory chain (cytochrome system) by way of pyridine nucleotides. Although the intramitochondrial pyridine nucleotide appears to be tightly bound in intact mitochondrial preparations (1) and thus does not function as a mobile coenzyme, the requirement of these dehydrogenases for added diphosphopyridine nucleotide or triphosphopyridine nucleotide is readily demonstrable after their removal from the mitochondrial environment. In contrast, the linkage of certain mitochondrial dehydrogenases to the respiratory chain does not appear to involve dissociable coenzymes even after extraction from the mitochondria and they were classified as ‘Lcytochrome-reducing” dehydrogenases in the older literature (2). In mammalian cells three dehydrogenases in this category have been recognized for a long time: succinic, a-glycerophosphate, and choline dehydrogenases. Besides the criteria mentioned they exhibit two further properties: lack of autooxidizability and resistance to extraction in soluble form. Since the identification of their prosthetic groups and the determination of their mechanism of action requires highly purified preparations free from other components of the respiratory chain, in 1954 our laboratory undertook the solubilization and isolation of these enzymes. After the isolation of succinic (3) and a-glycerophosphoric (4) dehydrogenases, the preparation of soluble choline dehydrogenase was undertaken as a first step in the isolation and characterization of the enzyme. The extraction of choline dehydrogenasel in soluble form from acetone powders of rat liver mitochondria by means of choleate was claimed by Williams and Sreenivasan in 1953 (5). However, Ebisuzaki and Williams (6) later recognized that such preparations were dispersions of insoluble particles and that upon removal of the choleate the enzyme reverted to the insoluble form. They also surveyed a large variety of alternative methods for solubilization and found that of these only a combination of ultra-