Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase. II. Isolation and molecular properties of the enzyme from beef heart.

Although, starting with Straub’s diaphorase (4), a number of enzymes capable of oxidizing reduced nicotinamide adenine dinucleotide (NADH) have been isolated from respiratory chain preparations during the past 25 years (5-9), it has not been possible to identify even tentatively any of these as a relatively unmodified, soluble form of the primary dehydrogenase of the mitochondrial NADH oxidase chain. The main reason for this uncertainty has been the fact that the isolation of this enzyme was attended by one major problem in addition to those usually encountered in investigations of respiratory chain-linked dehydrogenases in general (10) : the question of the identity of the enzyme. Mammalian heart and liver, the source materials most commonly used for the isolation of NADH-oxidizing enzymes from animal tissues, contain several flavoproteins for the oxidation of NADH. The characteristics of the respiratory chain-linked enzyme that serve to differentiate it from other NADH dehydrogenases in multienzyme preparations (mitochondrial localization; inhibition by Amytal, antimycin A, and other respiratory chain inhibitors; and generation of adenosine triphosphate during NADH oxidation) are not properties of the enzyme itself, but manifestations of its functional linkage to the electron transport system (10, II), and it might be expected that these features would be lost once the dehydrogenase is separated from the respiratory chain. The consequence of this fact and of the absence of a completely specific assay method for the respiratory chainlinked enzyme is that once an NADH dehydrogenase is removed from the mitochondrial environment, it is extremely difficult to pinpoint its identity. In order to circumvent this dilemma, it seemed logical to use, for the isolation of the enzyme, a specific starting material that was, insofar as possible, devoid of NADH-oxidizing enzymes other than the one of interest. Two very similar respiratory chain preparations described in the literature seemed to fulfill this requirement: the electron transport particle preparation of Crane, Glenn, and Green (12) and the NADH oxidase of Mackler