Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria.

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS.

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.The sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenyl sulfonic acid, and mersalyl inhibited the oxidation of exogenous NADPH by the mitochondria whereas NADH oxidation was unaffected at similar concentrations of inhibitor. The data suggest that exogenous NADPH is oxidized by potato mitochondria via a dehydrogenase primarily situated on the outer face of the inner mitochondrial membrane that is neither the dehydrogenase involved with endogenous NADH oxidation nor with exogenous NADH oxidation.

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria.

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.

Stoichiometry of carbon dioxide release and oxygen uptake during glycine oxidation in mitochondria isolated from spinach (Spinacia oleracea) leaves.

Mitochondria isolated from spinach (Spinacia oleracea) leaves oxidized glycine with a stoichiometry of CO2 evolution to O2 uptake of 2 : 1. In the absence of added substrate, the mitochondria exhibited an extremely low endogenous rate of O2 uptake.

Isolation and Oxidative Properties of Intact Mitochondria from the Leaves of Sedum praealtum: A Crassulacean Acid Metabolism Plant.

A procedure is described for preparing intact mitochondria from leaves of Sedum praealtum D.C., a plant showing Crassulacean acid metabolism. These mitochondria oxidized malate, pyruvate, alpha-ketoglutarate, succinate, NADH, NADPH, and isocitrate with good respiratory control and ADP/O ratios better than those observed in mitochondria from other photosynthetic tissues.Malate oxidation was very resistant to inhibition by rotenone. Glycine oxidation was very slow with poor respiratory control and was resistant to rotenone inhibition. Antimycin A completely inhibited the oxidation of both NADH and NADPH. The oxidation of isocitrate, malate, succinate, and alpha-ketoglutarate was partially inhibited by antimycin A and cyanide. Overall rates of substrate oxidation were slow on a protein basis, but purification of the mitochondrial preparations on a linear sucrose gradient removed a large amount of nonmitochondrial protein. The original mitochondrial preparations contained little glycolate oxidase activity, and most of this activity was removed by the sucrose gradient.

Effect of ethylene and carbon dioxide on potato metabolism: stimulation of tuber and mitochondrial respiration, and inducement of the alternative path.

The respiration of potato tubers (Solanum tuberosum var. Russet Burbank) which have been kept at room temperature for 10 days is stimulated upon subsequent treatment with C(2)H(4) (10 microliters per liter) and O(2). The respiratory rise reaches a peak in 24 to 30 hours and thereafter declines. Coincident with the rise in tuber respiration is an increase in the respiratory rates of fresh slices and isolated mitochondria. Slices and mitochondria from C(2)H(4)- and O(2)-treated tubers also display substantial resistance to CN, and the resistant respiration is inhibited by hydroxamates.The longer the tubers are stored after harvest, the less effective is C(2)H(4) in causing CN resistance in slices and mitochondria from treated tubers. Addition of 10% CO(2) to the C(2)H(4)-O(2) mixture, however, causes extensive CN resistance to develop, even in slices and mitochondria from old tubers. The results show that C(2)H(4), O(2), and CO(2) act synergistically to induce alternative path development in potatoes.