Abstract
From the particulate reduced nicotinamide adenine dinucleotide (DPNH) oxidase of beef heart, Mackler (3) has obtained a soluble DPNH dehydrogenase which was estimated to be approximately 90 to 95% homogenous when examined electrophoretically.l Similarly, when the enzyme was chromatographed on diethylaminoethyl cellulose with phosphate buffer, pH 7.5, in a gradient elution method (0.001 M to 0.01 M), only a single, symmetrical peak with respect to protein and flavin was observed in the elution profile (4). The enzyme contains bound flavin and nonheme iron in the ratio of 1: 2. The minimal molecular weight, calculated from the flavin content, is 100,000 which is in general agreement with a value of 80,000 to 90,000 obtained by sedimentation in the ultracentrifuge.’ The activity of the enzyme with 2,6-dichlorophenolindophenol as an electron acceptor is 40 pmoles of DPNH oxidized per minute per mg of protein. With ferricyanide and cytochrome c as acceptors, the DPNH oxidizing activities are 40 and 3, respectively, in the same units. Although this enzyme is similar to other DPNH dehydrogenases described previously (5-ll), it is difficult to compare the properties (molecular weight, pH optimum, turnover number, specificity for electron acceptors, etc.) of these various enzymes since, even when prepared from the same tissue, different methods of solubilization have been used. One of the principal discrepancies between the various DPNH dehydrogenases is the nature of the flavin component. It has been reported that only FAD is found in a soluble DPNH dehydrogenase obtained by treatment of beef heart electron transport particle with Naja nuja venom (7, 9), and FAD has also been identified in a lipoflavoprotein DPNH dehydrogenase from beef heart (12). Earlier, Mahler et al. (5) had reported that the prosthetic group of the soluble DPNH-cytochrome c reductase from pig heart is a flavin dinucleotide not identical with FAD. On the other hand, we have reported in preliminary communications (1, 2) that FMN2 is the flavin component of the soluble DPNH dehydrogenase prepared from DPNH osidase, and King3 has reached
Highlights
The present paper extends our observations in support of the contention that flavin mononucleotide (FMN) is the prosthetic group of the beef heart DPNH dehydrogenase
10 mpmoles of flavin per mg of protein were found in the DPNH-cytochrome c reductase from pig heart [16], whereas when the DPNH dehydrogenase is obtained by treatment of beef heart electron transport particle with snake venom, only 1 mpmole of flavin per mg of protein is present [22]
The activity of the enzyme with dichlorophenolindophenol as acceptor drops from 40 to 3 Fmoles of DPNH oxidized per minute per mg of protein upon removal of the flavin, and in contrast to the results with cytochrome c, this activity is not restored upon the addition of FMN or flavin adenine dinucleotide (FAD)
Summary
Mate&&-Chemicals and enzymes were obtained from the following commercial sources: DPN, TPN, DPNH, TPNH, and nucleotide pyrophosphatase (Aspergillusoryzae) from California. For the assay of FAD by the n-amino acid oxidase, our previously described procedure [14] was adapted for spectrophotometry by coupling the reaction product, pyruvate, with excess lactic dehydrogenase according to the method of DeLuca, Weber, and Kaplan [15]. The nonenzymatic reduction of ferricyanide was observed by the change in absorbancy at 410 rnp for 1 minute after the addition of DPNH, whereupon the enzyme was added and the reaction rate followed at 15-second intervals. Release of Enzyme-bound Flavin-The flavin of the DPNH dehydrogenase was completely released by heating the enzyme, approximately 6 mg per ml, in a boiling water bath for 6 minutes, followed by cooling to 5” and acidification with perchloric acid to a final concentration of 10%. The latter method involves application of the flavins to the circumference of a small circle with a radius of approximately 2 cm, the center of which coincides with the center of a circular filter paper 24 cm in diameter
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