The pyridine nucleotide metabolism of mitochondria incubated with and without added substrates and metabolic inhibitors

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Abstract
The effect of temperature, added substrate and metabolic inhibitors on the amounts of oxidized and reduced di and tri-phosphopyridine nucleotides was studied in rat-liver mitochondria suspended in a [image]saline[image] medium. The particles of the [image]fluffy layer[image] behaved essentially the same as the mitochondria. At 10[degree] and below the levels of reduced pyridine nucleotides increased slowly; reduced diphosphopyridine nucleotide (DPN) accumulated more rapidly than reduced triphosphopyridine nucleotide (TPN) during incubation with added substrate. Net oxidation (sometimes after an initial reduction) of coenzyme was detected only when the temperature was 15[degree] and above, the rate of disappearance of reduced TPN being greater than that of reduced DPN. In more concentrated (2-fold) suspensions of mitochondria the initial reduction was more pronounced. Without substrate, the QO2 of the preparations (measured after 15 min. at 25[degree]) was usually about 3 and the respiration rate declined during further incubation for 1 hr. Added [alpha]-oxoglutarate, citrate, pyruvate plus malate, pyruvate, [beta]-hydroxybutyrate and malate increased both the levels of reduced DPN and reduced TPN (maximum increase in ratio of reduced/oxidized coenzymes from 1.55 to 4.80, with added [alpha]-oxoglutarate; minimum increase from 1.55 to 1.80 with added malate) and the rates of oxygen uptake (maximum increase 8.5-fold, with [alpha]-oxoglutarate; minimum increase 3-fold, with malate), above the values found with freshly isolated mitochondria. All the substrates except citrate, pyruvate and malate reduced the nucleotides appreciably in less than 2 min. Succinate produced the highest amounts of reduced coenzyme observed in suspensions of mitochondria, altering the levels in less than 30 sec. The increase in the ratio of reduced/oxidized coenzyme was about 4-fold. If the reduced nucleotides of isolated mitochondria were partially oxidized by pre-incubation for about 30 min. at 25[degree] without added substrate, re-reduction of DPN (and, more slowly, TPN) could be demonstrated after the addition of pyruvate plus malate, [beta]-hydroxybutyrate and succinate. The reduction of mitochondrial pyridine nucleotides in the presence of pyruvate or without added substrate was less when dinitrophenol was added at concentrations of 10[mu][image] or greater. Dinitrophenol (0.1 m[image]) was less effective in blocking reduction if malate was added with the pyruvate. The reduction of coenzymes by malate and succinate was much less sensitive to dinitrophenol than that by pyruvate; relatively high ratios (2.5-18) of reduced to oxidized coenzyme could be obtained with concentrations of dinitrophenol of 10 [mu][image] and 0.1 m[image]. The maintenance of reduced coenzyme by endogenous'' substrate and pyruvate was enhanced for a short time by amytal, the effect usually being maximal after about 30 min. of incubation. Subsequently there was a rapid decline in the amount of reduced coenzyme. On the basis of the results obtained, it is proposed that the function of mitochondrial reduced TPN is that of a reserve of reducing power, which is supplemented when the oxidation of substrate exceeds the capacity of the electron-transport chain and which is utilized in the preservation of the level of reduced DPN when there is little oxidizable substrate. The significance of the data obtained with dinitrophenol for the formulation of the mechanism of the reduction of mitochondrial coenzymes by succinate and the evidence obtained for [image]com-partmentation[image] of these coenzymes is also discussed.