Phosphorylation of formate dehydrogenase in potato tuber mitochondria

Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha-subunit of pyruvate dehydrogenase (PDH). Isoelectric focusing/SDS-PAGE two-dimensional gels separated FDH and PDH and resolved several different phosphorylated forms of FDH. By using combinations of matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry, several phosphorylation sites were identified for the first time in FDH and PDH. FDH was phosphorylated on Thr76 and Thr333, whereas PDH was phosphorylated on Ser294. Both Thr76 and Thr333 in FDH were accessible to protein kinases, as demonstrated by protein structure homology modeling. The extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants.     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Activation of F1 -ATPase isolated from potato tuber mitochondria

The ATP-hydrolyzing activity of F₁-ATPase purified from potato tubers mitochondria was stimulated 2- and 3.5-fold by anions, chloride and bicarbonate, respectively, and 5.5- and 6.5-fold by detergents, octyl glucoside and lauryl dimethylamine oxide (LDAO), respectively. The maximal specific activity of the activated F₁, 129 μmol/min per mg protein is the highest activity of plant mitochondrial F₁ hitherto reported and exceeds several-fold values reported earlier. In the absence of activators F₁-catalyzed ATP hydrolysis exhibits non-linear double-reciprocal plots of [ATP]⁻¹ vs v⁻¹ indicative of negative cooperativity, while in the presence of activators, linear plots are observed. It is suggested that the activators reduce the cooperativity originating from the interaction between different subunits of the enzyme.     

l-Tryptophan metabolism in wound-activated and Agrobacterium tumefaciens-transformed potato tuber cells

The in vivo metabolism of L-tryptophan in wound-activated and Agrobacterium tumefaciens , strain C 58, transformed tissues of white potato tubers ( Solanum tuberosum L. cv. Saskia) was investigated. The following metabolites of L-tryptophan were identified in both tissues by co-chromatography with authentic standards in several thinlayer chromotography (TLC) and high pressure liquid chromatographic (HPLC) systems: indole-3-acetic acid (IAA), indole-3-acetaldehyde, indole-3-ethanol, indole-3-acetamide and tryptamine. Labelled indole-3-acetaldoxime was only found in transformed tissue. Crown gall tissue generally incorporated [¹⁴C]-L-tryptophan into precursors of IAA at a distinctly higher rate than did wound tissue. Tryptamine and indole-3-ethanol accumulated about ten-fold more label in crown gall cells than in cells from wounded tissue. The incorporation of radioactivity into indole-3-acetamide as determined by 2 consecutive TLC systems followed by HPLC analysis was rather low, though consistently observed in both tissues. An indole-3-acetamide hydrolyzing enzyme, the putative product of gene 2 on the T-DNA, could be extracted from the transformed tissue only. The indole-3-ethanol level was 4.3 nmol (g dry weight)⁻¹ and 41 nmol (g dry weight)⁻¹ for wounded tissue and primary crown gall tissue, respectively, as determined by HPLC with a [¹⁴C]-labelled internal standard. The experiments are critically discussed in relation to recent reports on a T-DNA encoded enzyme of IAA biosynthesis in crown gall tumors.     

Effect of ageing on sterol metabolism in potato tuber slices

When fresh potato tuber slices were incubated with [1-¹⁴C]-sodium acetate, cycloartenol was heavily labelled but no radioactivity was recovered in 24-methylene cycloartanol and free sterols. If potato slices were aged for 0–24 hr before feeding with radioactive acetate, a rapid increase of the label in the sterol precursors and the free sterols was observed. The free sterol content was 5 × higher after ageing for 24 hr. Isofucosterol synthesis was especially stimulated. The synthesis of sterols during the ageing process seems to be related to the appearance of a cycloartenol C24-methylase and may be linked to a biogenesis of membranes.Nomenclature: (1) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-cholest-24-en 3β-ol; (2) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-ergost-24(28)-en 3β-ol; (3) 4α,14α-dimethyl 9β,19β-cyclo 5α-ergost 24(28)-en 3β-ol; (4) 4α, 14α-dimethyl 5α-ergosta 8.24(28)-dien 3β-ol; (5) 4α-methyl 5α-ergosta 7,24(28)-dien 3β-ol; (6) ergosta 5,24(28)-dien 3β-ol; (7) stigmasta 5,Z-24(28)-dien 3β-ol; (8) (24R)-24 methyl cholest 5-en 3β-ol; (9) (24R)-24 ethyl cholest 5-en 3β-ol; (10) (24S)-24 ethyl cholesta 5,E-22(23)-dien 3β-ol; (11) cholest 5-en 3β-ol.     

Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

Dioxins invade the body mainly through the diet, and produce toxicity through the transformation of aryl hydrocarbon receptor (AhR). An inhibitor of the transformation should therefore protect against the toxicity and ideally be part of the diet. We examined flavonoids ubiquitously expressed in plant foods as one of the best candidates, and found that the subclasses flavones and flavonols suppressed antagonistically the transformation of AhR induced by 1 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin, without exhibiting agonistic effects that transform AhR. The antagonistic IC(50) values ranged from 0.14 to 10 microM, close to the physiological levels in human.     

Induction and activation of the alternative oxidase of potato tuber mitochondria

Potato tubers ( Solanum tubersum L. cv. Grata) were stored for atleast 1 week at room temperature and then incubated with an equal amount of apples ( Malus domestica L.) for 2 days. After this treatment, intact tuber mitochondria isolated by Percoll gradient centrifugation showed a high degree of induction of the alternative oxidase, measured as cyanide-resistant, salicylhydroxamic acid-sensitive respiration. With succinate as substrate an activity of more than 130 nmol O₂(mg protein) ¹ min ^t was obtained. An assay of the alternative oxidase using duroquinol as an electron donor was developed. To become reliable the assay required the presence of defatted bovine serum albumin (BSA) and catalase (EC 1. 11. 1. 6). Furthermore, a lowering of the assay temperature to 15°C improved the stability of the duroquinol-based activity. One remarkable finding was that with duroquinol (or external NADH) as substrate the alternative oxidase was synergistically activated by succinate (as well as by malate) even in the presence of the succinate dehydrogenase inhibitor malonate. Our interpretation is that succinate and malate (indirectly) activate the alternative oxidase and that this activation is part of a physiological mechanism for regulation of the alternative oxidase.     

Localization of L-lactate dehydrogenase in mitochondria

Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.     

On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber

Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD⁺ or 3-acetylpyridine-NAD⁺ by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute·milligram protein at pH 5 to 6. The K_m values for 3-acetylpyridine-NAD⁺ and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity.     

Influence of ethanol on alternative oxidase in mitochondria from callus-forming potato tuber discs

Ethanol, when added to the incubation medium of callus-forming potato tuber discs, inhibits callus growth and causes an increase of the mitochondrial antimycin-A resistant respiration, expressed as a percentage of state III-respiration. This increase in resistance to antimycin-A is the result of a poor development of the cytochrome pathway in tissue discs treated with ethanol. The development of the antimycin-A resistant alternative oxidase sensitive to chelator is about the same for treated and untreated discs. The respiratory control (RC) ratio of the mitochondrial respiration increases after addition of a chelator, which inhibits the alternative pathway. The RC ratio of the uninhibited mitochondrial respiration appears to be inversely related to the capacity of the alternative pathway, when mitochondrial preparations with different capacities to transfer electrons via the alternative path are compared. From the experimentally observed relation between RC-ratio and alternative oxidase capacity, it was concluded that at least half of the capacity of the alternative path is used in uninhibited state IV respiration.     

Solanidine metabolism in potato tuber tissue slices and cell suspension cultures

Two metabolites have been isolated from potato tuber tissue slices or cell suspension cultures that have been incubated with labeled solanidine. Initially glucosyl solanidine is formed which subsequently is glycosylated to a diglucosyl solanidine.     

Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c₁ and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c₁a and a₃ were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a₃ were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.     

Biochemical studies on mitochondria formed during aging of sliced potato tuber tissue

Respiratory activities in mitochondria of potato tuber tissueincreased about 3-fold after aging sliced tissue for 1 day.Increased activity was insensitive to cyanide. The number ofmitochondrial particles also increased during aging. It seemslikely that newly formed mitochondria are heavier than pre-existingones and that respiration in the former is insensitive to cyanide. (Received January 26, 1970; )     

Action of the herbicides neburon and siduron on membrane permeabilities in potato tuber mitochondria

The herbicides neburon and siduron are uncouplers of oxidative phosphorylation in potato tuber (Solanum tuberosum L. cv. Bintje) mitochondria. Their effect on the ion permeabilities of the mitochondrial membrane was investigated using the acid-base pulse technique, swelling experiments and integrity tests. Both herbicides permeabilize the membrane to H⁺ ions. They have no action on the permeabilities of K⁺ and Fe(CN)^3?₆. The swelling observed with Ca²⁺ was better interpreted as an effect on membrane structure than as a true swelling. Diuron, a parent compound that does not uncouple oxidative phosphorylation, does not act on Ca²⁺-induced apparent swelling.     

L-lactate oxidation by skeletal muscle mitochondria

1. Mitochondria isolated from rat skeletal muscle possess lactate dehydrogenase which is involved in direct oxidation of L-lactate in the presence of external NAD. 2. L-lactate oxidation can be stimulated in a reversible manner by ADP. 3. Mitochondrial lactate oxidation is sensitive to oxamate-inhibitor of LDH, alpha-cyano-3-hydroxy-cinnamate-pyruvate translocase inhibitor and respiratory chain inhibitors (rotenone, antimycin A, KCN). 4. In the same conditions the mitochondria did not oxidize pyruvate in the absence of malate, whereas, oxidize pyruvate plus external NADH in an uncoupling manner.     

Mitochondria and L-lactate metabolism

Although mitochondria have been the object of intensive study over many decades, some aspects of their metabolism remain to be fully elucidated, including the L-lactate metabolism. We review here the novel insights arisen from investigations on L-lactate metabolism in mammalian, plant and yeast mitochondria. The presence of L-lactate dehydrogenases inside mitochondria, where L-lactate enters in a carrier-mediated fashion, suggests that mitochondria play an important role in L-lactate metabolism. Functional studies have demonstrated the occurrence of several L-lactate carriers. Moreover, immunological investigations have proven the existence of monocarboxylate translocator isoforms in mitochondria.     

Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.