Stimulation of the Alternative Pathway by Succinate and Malate

Stimulation of the cyanide-resistant oxidation of exogenous NADH in potato (Solanum tuberosum L. cv Bintje) tuber callus mitochondria was obtained with succinate, malate, and pyruvate. Half-maximal stimulation was observed at a succinate or malate concentration of 3 to 4 mM, which is considerably higher than that found for pyruvate (0.128 mM). No effect of succinate or malate addition was found when duroquinone was the electron acceptor. Duroquinol oxidation via the alternative pathway was poor and not stimulated by organic acids. Under stimulating conditions, no swelling or contraction of the mitochondria could be observed. Conversely, variation of the osmolarity did not affect the extent of stimulation. However, the assay temperature had a significant effect: no stimulation occurred at temperatures below 16 to 20deg]C. Membrane fluidity measurements showed a phase transition at about 17deg]C. Ubiquinone reduction levels were not significantly higher in the presence of succinate and malate, but the kinetics of the alternative oxidase were changed in a way comparable to that found for stimulation by pyruvate. At low temperatures the alternative oxidase displayed "activated" kinetics, and a role for membrane fluidity in the stimulation of the alternative pathway by carboxylic acids is suggested.