Jerusalem Artichoke Mitochondria Research Articles

Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K (m) and V (max) values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min(-1) mg(-1) protein, respectively. The enzyme's pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.

Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that: (1) phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles. (2) Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation. (3) The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

Jerusalem artichoke mitochondria can export reducing equivalents in the form of malate as a result of d-lactate uptake and metabolism

We found that as a result of d-lactate uptake and metabolism by Jerusalem artichoke mitochondria, reducing equivalents were exported from the mitochondrial matrix to the exterior in the form of malate. The rate of malate efflux, as measured photometrically using NADP + and malic enzyme, depended on the rate of transport across the mitochondrial membrane. It showed saturation characteristics ( K m = 5 mM; V max = 9 nmol/min mg of mitochondrial protein) and was inhibited by non-penetrant compounds. We conclude that reducing equivalent export from mitochondria is due to the occurrence of a putative d-lactate/malate antiporter which differs from other mitochondrial carriers, as shown by the different inhibitor sensitivity.

Transport and metabolism of d-lactate in Jerusalem artichoke mitochondria

We report here initial studies on d-lactate metabolism in Jerusalem artichoke. It was found that: 1) d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S- d-lactoyl-glutathione. 2) Externally added d-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) d-lactate was metabolized inside mitochondria by a flavoprotein, a putative d-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added d-lactate by means of a d-lactate/H + symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial d-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.

Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus)

The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c(1)-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCl fractionation procedure, contained one Cyt of c type (formally c(1) with Em(7.0) of +240 millivolts), two b type Cyt with Em(7.0) values of +100 and -25 millivolts, ferredoxin-like centers presumably linked to succinic- and NADH-dehydrogenases, and a Rieske-type iron sulfur center (g(y) = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-1 showed sensitivity to antimycin A, myxothiazol, and n-2-hepthyl-1-hydroxyquinoline N-oxide with I(50) of 12 nanomolar, 30 nanomolar, and 0.1 micromolar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined.

Properties of the oxidation of exogenous NADH and NADPH by plant mitochondria. Evidence against a phosphatase or a nicotinamide nucleotide transhydrogenase being responsible for NADPH oxidation

(1) The optimum pH for the oxidation of exogenous NADH by mitochondria from both Jerusalem artichoke ( Helianthus tuberosus) tubers and Arum maculatum spadices was 7.0–7.1. NADPH oxidation had a lower optimum pH of 6.6 in Arum and 6.0 in Jerusalem artichoke mitochondria. In both types of mitochondria the rates of NADH and NADPH oxidation were identical below pH 6.0–5.5. (2) It is shown conclusively that neither a phosphatase converting NADPH to NADH nor a nicotinamide nucleotide transhydrogenase was involved in the oxidation of NADPH by these mitochondria. (3) Palmitoyl-CoA, an inhibitor of transhydrogenase activity in mammalian mitochondria, inhibits both NADH and NADPH oxidation by plant mitochondria with a K i of about 10 μM. (4) It is concluded that the known properties of NAD(P)H oxidation are best explained by assuming the presence of a second dehydrogenase specific for NADPH. At low pH, electron flow from the two dehydrogenases to oxygen shares a common rate-limiting step.

The stimulation of exogenous NADH oxidation in Jerusalem artichoke mitochondria by screening of charges on the membranes

The ability of plant mitochondria to oxidize exogenous NADH is of great significance in the energy economy of the cell and it follows that the means of regulating such an oxidation must attract interest. The stimulation of exogenous NADH oxidation by various inorganic monovalent and divalent salts has been reported extensively [ 11, where divalent cations were noted to be effective at much smaller concentrations than monovalent cations, whereas the nature of the anion appeared to be unimportant. More recently, evidence presented in [2,3] was interpreted to indicate that the stimulation of exogenous NADH oxidation may be specifically dependent on Ca2+ (with Mn2+ and Sr2+ being much less effective). In the present study, using a range of salts which include inorganic and organic cations, the oxidation of exogenous NADH has been re-examined in conjunction with an investigation into the behaviour of cations in the diffuse layer associated with the mitochondrial membrane using the fluorescent monovalent cation 9-aminoacridine (9-AA). The results presented here are consistent with the view that electrical charges at the membrane surface influence the ease with which the substrate NADH may approach the membranebound dehydrogenase.

The increase of hydroxyproline-containing proteins in Jerusalem artichoke mitochondria during the development of cyanide-insensitive respiration

Mitochondria purified from freshly cut and from aged slices of Jerusalem artichoke tubers contain hydroxyproline proteins. These proteins are present in much higher amounts in mitochondria with KCN-insensitive respiration, i.e. in those from aged slices. It is suggested that the dependence of KCN-insensitive respiration on Vit. C is due to the fact that the development of this alternate oxidase requires the ascorbic acid-dependent synthesis of a hydroxyproline-containing mitochondrial protein.

The Influence of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Malate by Jerusalem Artichoke Mitochondria

Mitochondria isolated from Jerusalem artichoke tubers oxidized endogenous NADH by both a piericidin A-sensitive and -resistant dehydrogenase if the level of oxaloacetate was kept low. In washed mitochondria the addition of NAD+ stimulated respiration in the presence of a variety of NAD+-linked substrates. In mitochondria purified through a sucrose density gradient exogenous caused a substantial stimulation of respiration only in the presence of malate. The apparent Km for malate was 20 ml in the absence of NAD4 and 2 mM in the presence of exogenous NAD4. The products of malate oxidation were altered by the addition of exogenous NAD4. Oxaloacetate and pyruvate were produced in equal amounts in the absence of added NAD4, but in the presence of exogenous NAD4 more pyruvate was formed. The rapid oxidation of malate in the absence of added required phosphate while the NAD4-stimulated component was not affected by the absence of phosphate. Phenylsuccinate inhibited the reduction of exogenous NAD+ by malate ; this compound was found to inhibit isolated malate dehydrogenase and malic enzyme. Several lines of evidence suggest that electron flux through one of the NADH dehydrogenase systems may directly affect the flow through the other dehydrogenases.

Stimulation of electron transport and activation of reduced nicotinamide--adenine dinucleotide dehydrogenase in Jerusalem-artichoke mitochondria.

Stimulation of electron transport and activation of reduced nicotinamide--adenine dinucleotide dehydrogenase in Jerusalem-artichoke mitochondria.

Purification and properties of succinyl-CoA synthetase from Jerusalem artichoke mitochondria.

Summary A direct spectrophotometric method for measurement of the activity of succinyl-CoA synthetase (succinate:CoA ligase (ADP), EC 6.2.1.5) having wider applications than those previously used has been developed. Succinyl-CoA synthetase was isolated from mitochondria and the resulting enzyme preparation had a specific activity more than 15-fold greater than that previously reported with the enzyme isolated from plant tissue. The apparent Km values for the substrates necessary for both the forward and reverse reactions were determined. The enzyme was shown to be highly specific for succinic acid and adenine nucleotide phosphates; in the reverse direction malonic acid was shown to act as a competitive inhibitor with succinic acid. The enzyme was less specific in its requirement for the metal-ion cofactor. Mn2+ and Co2+ could replace Mg2+, Zn2+ and Ca2+ could not.

Detection of phosphohistidine in nucleoside diphosphokinase isolated from Jerusalem artichoke mitochondria

Detection of phosphohistidine in nucleoside diphosphokinase isolated from Jerusalem artichoke mitochondria