The spectrophotometric phenazine methosulfate assay of succinate dehydrogenase was adapted to use with cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria with suitable modifications to overcome the permeability barrier to the dye. Procedures in the literature for the isolation and sonic disruption of mitochondria from these sources were modified to assure maximal yield and stability of the enzyme. In tightly coupled mung bean mitochondria, as isolated, about half of the succinate dehydrogenase is in the deactivated state, and the enzyme is further extensively deactivated on sonication or freeze-thawing. In cauliflower mitochondria most of the enzyme is in the deactivated form, and little or no further deactivation occurs on sonication or freeze-thawing. Incubation of mitochondria from either source with succinate leads to full activation of the enzyme. The energy of activation for the conversion of the deactivated to the activated form in membranal preparations under the influence of substrate is about 30,000 cal/mole, essentially the same value as in animal tissues. Activation of the enzyme also occurs under the influence of a variety of other agents, among which the action of anions as activators is documented in the present paper. Activation is accompanied by the release of very tightly bound oxaloacetate. As in animal tissues, the enzyme appears to contain covalently bound flavin (histidyl 8a-FAD), and the turnover number is 19,400 moles of succinate oxidized/ mole of histidyl flavin at pH 7.5, 38 C.While the known flavoproteins of the mammalian respiratory chain were isolated and characterized many years ago, characterization of the flavoproteins of mitochondria of higher plants has only recently begun (28). To the authors' knowledge only one attempt to purify a respiratory chain-linked flavoprotein (succinate dehydrogenase) from higher plants has been reported (6).Because (20), it was decided to explore to nature of this enzyme in representative higher plants, particularly in regard to its regulatory properties. The present paper is concerned with the assay of the membrane-bound enzyme in plant material, the demonstration that it contains covalently bound flavin as in animal tissues (8) and yeast (26), and its activation by substrates. The following paper (16) represents a detailed study of the activation of the plant enzyme by a variety of agents. Future reports will be concerned with the isolation and further characterization of succinate and NADH dehydrogenases from higher plant mitochondria.Since a large body of literature exists on the fine regulation of mammalian succinate dehydrogenase, it may be useful to summarize the salient facts, in order to permit comparison with the findings reported for the dehydrogenase in cauliflower and mung bean mitochondria.The properties of succinate dehydrogenase vary with the physiological role of the enzyme (20). In all obligate aerobes, where the enzyme is part of the terminal respiratory apparatus, the kinetic properties of the ...

The effect of various agents on the activation of succinate dehydrogenase in cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria and in sonicated particles has been investigated. Reduced coenzyme Qio, inosine diphosphate, inosine triphosphate, acid pH, and anions activate the enzyme in mitochondria from higher plants in the same manner as in mammalian preparations. Significant differences have been detected in the behavior of plant and animal prepa.rations in the effects of ATP, ADP, NADH, NAD-linked substrates, and of 2, 4-dinitrophenol on the state of activation of the dehydrogenase. In mammalian mitochondria ATP activates, whereas ADP does not, and the ATP effect is shown only in intact mitochondria. In mung bean and cauliflower mitochondria, both ATP and ADP activate and the effect is also shown in sonicated and frozen-thawed preparations. In sonicated mung bean mitochondria NADH causes complete activation, as in mammalian submitochondrial particles, but in sonicated cauliflower mitochondria activation by NADH is incomplete, as is also true of intact, anaerobic cauliflower mitochondria. Moreover, neither NAD-linked substrates nor a combination of these with NADH can fully activate the enzyme in cauliflower mitochondria. In contrast to mammalian mitochondria, succinate dehydrogenase is not deactivated in cauliflower or mung beam mitochondria under the oxidized conditions brought about by uncoupling of oxidative phosphorylation by 2, 4-dinitrophenol.It was shown in the previous paper (9) that succinate dehydrogenase in cauliflower and mung bean mitochondria is activated by substrates with the same kinetic characteristics and same high activation energy as in animal tissues. Activation by succinate appears to be accompanied by the release of tightly bound oxaloacetate. After full activation, membrane-bound succinate dehydrogenase in higher plants has the same turnover number as in heart mitochondria.The present paper extends these observations on the fine regulation of the enzyme from higher plants to a study of the characteristics of activation by CoQ,J-L2, adenine and inosine 'This work was supported in part by Grant GB-36570X from the National Science Foundation. Activation of succinate dehydrogenase in sonicated mung bean mitochondria by CoQ,0H2 is shown in Figure 1. The experiment was performed by incubating the particles at 29 C with chemically reduced CoQ,o under anaerobic conditions and in the presence of antimycin A to minimize oxidation of the reduced quinone. Aliquots were removed at varying intervals, chilled to 15 C and immediately assayed at that temperature for succinate dehydrogenase activity by the PMS-DCIP assay (9). At concentrations of the order of 150 to 250 jM CoQ,0H, the same activation was reached as with succinate as activator (Fig. 1A). Figure 1B shows the kinetics of activation by CoQ,oH2 at 29 C.These results on the activation of the enzyme in sonicated mung bean mitochondria by added CoQ,0H2 parallel the behavior of the mammalian enzyme (5). Similar...

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Reports in the literature on the number of isoenzymes of lipoyl dehydrogenase in mammalian heart mitochondria vary from 2 to 13 and one report claims that no isoenzymes occur in vivo. The present paper provides evidence that 6 main isoenzymes and 2 minor ones of lipoyl dehydrogenase occur in beef and pig heart, 3 of the main components being associated with 2‐oxoglutarate dehydrogenase, 3 with pyruvate dehydrogenase. Failure to detect these in some previous studies has been explained in terms of the experimental conditions used. Heart and liver mitochondria from several mammalian species present the same isoenzyme pattern. Evidence is presented that “free” (uncomplexed) lipoyl dehydrogenase detected in extracts of mitochondria by several investigators originates largely from the pyruvate dehydrogenase complex.