Exogenous NAD+ Effects on Plant Mitochondria

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Abstract
Addition of NAD+ to purified potato (Solanum tuberosum L.) mitochondria respiring .alpha.-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O2 uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD+ (NAP4-NAD+), an inhibitor of NAD+ uptake, but not by 1 mM EGTA, an inhibitor of external NADH oxidation. NAD+-stimulated malate-cytochrome c reductase activity and reduction of added NAD+ by intact mitochondria could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD+ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD+ by storing on ice for 72 h was completely dependent on added NAD+ and the effect of NAD+ on these mitochondria was prevented by incubating them with NAP4-NAD+. External NAD+ reduction by these mitochondria was not affected by NAP4-NAD+. All effects of exogenous NAD+ on plant mitochondrial respiration can be attributed to net uptake of the NAD+ into the matrix space.