Reoxidation Of NADH Research Articles

Production of aromatic α-hydroxyacids by epimastigotes ofTrypanosoma cruzi, and its possible role in NADH reoxidation

Epimastigotes of Trypanosoma cruzi in culture produce and excrete into the medium small amounts of phenyllactic acid and p-hydroxyphenyllactic acids, presumably arising from the catabolism of the aromatic amino acids phenylalanine and tyrosine, respectively. This production might constitute a minor pathway for the reoxidation of cytosolic NADH, through the concerted action of tyrosine aminotransferase and aromatic alpha-hydroxyacid dehydrogenase.

Intermediate metabolism in Trypanosoma cruzi.

Epimastigotes of Trypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of MH3, and glucose with production of reduced catabolites, chiefly succinate and L-alanine, even under aerobic conditions. This "aerobic fermentation of glucose" is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH, reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate or L-alanine, and L-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic alpha-hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH, NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8–9 U mg−1 protein (µmoles mg−1 min−1) for NADPH and 3–4 U mg−1 protein for NADH formation with reduced methylviologen (MV++) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg−1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.The Km-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg−1 protein of an NADP+-de...

Photosynthesis in Phosphoenolpyruvate Carboxykinase-Type C4 Plants: Mechanism and Regulation of C4 Acid Decarboxylation in Bundle Sheath Cells

The mechanism and regulation of C4 acid decarboxylation in phosphoenolpyruvate (PEP) carboxykinase-type C4 plants was examined in isolated bundle sheath cell strands. These cells decarboxylated added oxaloacetate to PEP at rates exceeding 2.5 μmol min−1 mg−1 chlorophyll when ATP was added. This requirement for ATP could be replaced by malate plus ADP; ander these conditions this cytosol-located decarboxylation of oxaloacetate via PEP carboxykinase was sustained by respiratory ATP. It was confirmed that respiratory ATP production was linked primarily to the oxidative decarboxylation of malate via NAD malic enzyme. This process, measured as pyruvate production, was highly dependent on Pi. Besides being required to generate ATP, Pi had a second role which was probably associated with the transport of malate into mitochondria. Maximum rates of malate decarboxylation via NAD malic enzyme substantially exceeded the minimum rates necessary for providing ATP for cytosolic oxaloacetate decarboxylation. When malate was added with oxaloacetate, ADP and Pi rates of malate decarboxylation of between 3 and 4 μmol min−1 mg−1 chlorophyll were recorded. About half of this activity was sustained by the reoxidation of NADH coupled to reduction of oxaloacetate via malate dehydrogenase. When malate was added without oxaloacetic acid, respiration by these bundle sheath cells was stoichiometrically linked with the oxidation of nialate to pyruvate. This malate-dependent respiration was stimulated by adding ADP or phosphorylation uncouplers; it was not significantly inhibited by including oxaloacetate. Possible mechanisms of regulation of the partitioning of C4 acid decarboxylation between PEP carboxykinase in the cytosol and mitochondrial NAD malic enzyme are discussed.

Pathway and regulation of erythritol formation in Leuconostoc oenos.

It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.

Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar diacetylactis

The production of aroma compounds (acetoin and diacetyl) in fresh unripened cheese by Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 was studied at 30 degrees C at different initial oxygen concentrations (0, 21, 50, and 100% of the medium saturation by oxygen). Regardless of the initial O(2) concentration, maximal production of these compounds was reached only after all the citrate was consumed. Diacetyl and acetoin production was 0.01 and 2.4 mM, respectively, at 0% oxygen. Maximum acetoin concentration reached 5.4 mM at 100% oxygen. Diacetyl production was increased by factors of 2, 6, and 18 at initial oxygen concentrations of 21, 50, and 100%, respectively. The diacetyl/acetoin concentration ratio increased linearly with initial oxygen concentration: it was eight times higher at 100% (3.3%) than at 0% oxygen (0.4%). The effect of oxygen on diacetyl and acetoin production was also shown with other lactococci. At 0% oxygen, specific activity of alpha-acetolactate synthetase (0.15 U/mg) and NADH oxidase (0.04 U/mg) was 3.6 and 5.4 times lower, respectively, than at 100% oxygen. The increasing alpha-acetolactate synthetase activity in the presence of oxygen would explain the higher production of diacetyl and acetoin. The NADH oxidase activity would replace the role of the lactate dehydrogenase, diacetyl reductase, and acetoin reductase in the reoxidation of NADH, allowing accumulation of these two aroma compounds.

Inhibition of the fermentation process in soil by acetylene

The effect of acetylene on the fermentation of added carbon in anaerobic soil was examined. With glucose as the C source and in the absence of C 2H 2, the fermentation products, acetate and butyrate, were produced in nearly equimolar quantities along with copious quantities of CO 2 and H 2. In the presence of 10 kPa of C 2H 2, there was significant inhibition of acetate, butyrate, CO 2 and H 2 production. The inhibitory effect was significantly greater on butyrate production than acetate production. In the absence of an inorganic N source, added C 2H 2 was reduced to C 2H 4, indicating the activity of the saccharolytic N 2-fixing clostridia. In the presence of NH + 4 (nitrogenase repressed), the inhibition of fermentation due to C 2H 2 appeared to be alleviated. The decrease in butyrate formation is suggested to result from reoxidation of NADH through the reduction of C 2H 2 to C 2H 4 by nitrogenase, rather than by acetyl CoA reduction to butyrate. The potential implications of the use of C 2H 2 in conjunction with N transformations by heterotrophic organisms, in light of these data, are discussed.

Oxidation of cytosolic NADH by the malate-aspartate shuttle in HOH13 human hepatoma cells

1. 1. The reoxidation of cytosolic NADH was studied in a line of human hepatoma cells (HuH13) whose mitochondria preferentially utilized glutamine for ATP formation. 2. 2. The tumor cells showed mitochondrial reoxidation of NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with l-lactate. The involvement of the respiratory chain was demonstrated by the addition of specific inhibitors. 3. 3. Glutamine oxidation proceeded in the tumor mitochondria exclusively via a pathway involving transamination. Malate stimulated aspartate production from glutamine. 4. 4. When the tumor cells were cultured in Eagle's medium with aminooxyacetate or in the absence of glutamine, a marked reduction in the cellular NAD/NADH ratio was observed. 5. 5. These results indicate that the malate-aspartate shuttle was functioning in the tumor cells.

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes ofTrypanosoma cruzi

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation. A and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of l-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biocbem. 192 (1990) 363–368).

Dynamic regulation of yeast glycolytic oscillations by mitochondrial functions

The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investigated in starved yeast cells that were metabolizing glucose semianaerobically. Glycolytic oscillations were triggered after a pulse of glucose by inhibition of mitochondrial respiration with KCN, myxothiazol and antimycin A or in mutants in the bc1 complex (ubiquinol:cytochrome c reductase) that were largely deficient in respiratory capacity. Inhibition of the adenine nucleotide translocator by preincubation with bongkrekic acid also triggered a train of damped sinusoidal oscillations after glucose addition. The oscillations consisted of cycles of reduction and oxidation of the intracellular pool of nicotinamide nucleotides with periods of 45 s to 1 min and amplitudes of 0.8 mM or lower. Preincubation with the uncoupler carbonyl cyamide p-(trifluoromethoxy)phenylhydrazone (FCCP) annihilated cyanide-induced oscillations of NAD(P)H. Evidence for de-energization of mitochondrial membranes in vivo was obtained by mitochondrial staining with dimethylaminostyryl-methyl-pyridiniumiodine (DASPMI) of starved cells. The low rates of NADH reoxidation shown by respiratory mutants and the FCCP-treated X2180 strain open up the possibility that mitochondrial dehydrogenases also control glycolytic oscillations. Low rates of cytosolic NADH reoxidation induced by pyrazole, an inhibitor of alcohol dehydrogenase, were also associated with the disappearance of glycolytic oscillations. From experimental evidence and model calculations we conclude that the modulation of the levels of cytosolic ATP by mitochondrial functions in turn modulates the approach of the dynamic behavior of glycolysis to an oscillatory domain. The mitochondrial NADH dehydrogenase and the glycolytic steps associated with NADH reoxidation downstream from pyruvate appear to provide another control level of glycolysis dynamics in vivo.

Properties and possible role of malate dehydrogenase in the foot muscle of the sea mollusc Concholepas concholepas (Gastropoda: Muricidae)

1. 1. Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase and significant activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were observed in the deeper foot muscle tissue of Concholepas concholepas. 2. 2. Malate dehydrogenase activity was resolved into two molecular forms, which differ in K m values for oxaloacetate and malate, and sensitivity to inhibition by α-ketoglutarate. 3. 3. In addition to reoxidation of NADH generated from glycolysis, the function of malate dehydrogenase is discussed in connection with octopine synthesis and oxidation in the foot muscle of C. concholepas. 4. 4. The results of initial rate and product inhibition studies of both forms of malate dehydrogenase are consistent with an Ordered Bi Bi kinetic mechanism, with NADH adding first and NAD + leaving last. Oxaloacetate substrate inhibition is suggested to result from formation of an enzyme-NAD +-oxaloacetate dead end complex.

Effect of epinephrine on ethanol metabolism by isolated rat hepatocytes

The effect of epinephrine on ethanol metabolism was determined in isolated rat hepatocytes. Epinephrine (10 μM) enhanced an initial rapid rate of ethanol elimination observed in the first 5 min. Thereafter, between 5 and 90 min, the rate of ethanol elimination was slower and not affected by epinephrine. Epinephrine resulted in higher acetaldehyde concentrations at 2 min, but not thereafter. Acetaldehyde production in the presence and absence of epinephrine was inhibited by 4-methylpyrazole, by a low free extracellular calcium concentration, and by the α 1-adrenergic blocker prazosin. Ethanol alone and epinephrine alone increased oxygen consumption, but the effects were not additive. The ethanol-induced decreases in the cytosolic NAD +/NADH and NADP +/NADPH ratios and in the mitochondrial NAD +/NADH ratio were delayed by the presence of epinephrine. An accelerated initial alcohol dehydrogenase activity sufficient to account for the rapid initial rate of ethanol elimination shown with epinephrine was demonstrated by coupling ethanol oxidation with lactaldehyde reduction, a system which increases the rate of dissociation of NADH from the enzyme and its oxidation back to NAD +. The findings in this study indicate that an increased reoxidation of NADH during ethanol oxidation by alcohol dehydrogenase is the basis for the rapid transient increase in ethanol elimination produced by epinephrine.

Intermediates of peroxisomal β-oxidation. A study of the fatty acyl-CoA esters which accumulate during peroxisomal β-oxidation of [U-14C]hexadecanoate

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation.

Evidence for Metabolic Domains within the Matrix Compartment of Pea Leaf Mitochondria

The simultaneous oxidation of malate and of glycine was investigated in pea (Pisum sativum) leaf mitochondria. Adding malate to state 4 glycine oxidation did not inhibit, and under some conditions stimulated, glycine oxidation. State 4 oxygen uptake with glycine is restricted because of the control exerted by the membrane potential but reoxidation of NADH by oxaloacetate reduction can still occur. Thus, malate addition stimulates glycine metabolism by producing oxaloacetate. The malate dehydrogenase (EC 1.1.1.37) enzyme fraction remote from glycine decarboxylase (EC 2.1.2.10) oxidizes malate whereas that closely associated with it produces malate, i.e. they function in opposite directions. It is shown that these opposing directions of malate dehydrogenase activity occur within the same mitochondrial matrix compartment and not in different mitochondrial populations. It is concluded that metabolic domains containing different complements of mitochondrial enzymes exist within the one mitochondrial matrix without physical barriers separating them. The differential spatial organization within the matrix may account for the previously reported limited access of some enzymes to the respiratory electron transport chain. The implications for leaf mitochondrial metabolism are discussed.

Oxidation of cytosolic NADH by the malate-aspartate shuttle in MC29 hepatoma cells

The malate-aspartate shuttle activity for the reoxidation of cytosolic NADH was studied in MC29 avian hepatoma cells whose mitochondria preferentially utilized glutamine and produced aspartate for ATP formation. The tumour cells showed reoxidation of NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with L-lactate. The involvement of the respiratory chain and transaminase in the process was demonstrated by the addition of specific inhibitors. When the tumour cells were cultured in Eagle's medium with aminooxyacetate or in the absence of glutamine, a marked reduction in the cellular NAD NADH ratio was observed. These results indicate that the malate-aspartate shuttle was actively functioning in the tumour cells and that this hepatoma may provide a suitable system for the investigation of the bioenergetics of malignant cells.

Decreased in vivo rate of ethanol metabolism in the suckLing rat.

Blood concentrations of ethanol and acetaldehyde were determined in suckling rats after a single oral ethanol gavage. These results were compared with the hepatic activities of alcohol and aldehyde dehydrogenase. After intragastric administration of 3 g/kg body weight of ethanol, ethanol concentrations were much higher in suckling rats than in adult animals, especially at 90, 120, and 180 min after its administration. In addition, acetaldehyde concentrations were undetectable in suckling rats as opposed to adult rats, in whom micromolar concentrations were detected. Thus, 5- to 30-day-old rats seem to have a limited capacity for in vivo ethanol metabolism. The analysis of hepatic alcohol dehydrogenase activity revealed that it was very low at birth and it increased progressively with time attaining adult levels after 20 days of life. The alcohol dehydrogenase activity present in the liver of suckling rats presented similar Km values and sensitivity to pyrazole as adult rat liver. Thus, the pattern of in vivo ethanol elimination during the suckling period is not explained by hepatic alcohol dehydrogenase activity. Whether that diminished ethanol metabolism is due to slower intestinal ethanol absorption, different ethanol distribution in the body, or diminished hepatic capacity for NADH reoxidation remains to be studied. At birth, hepatic aldehyde dehydrogenase activity was low and it increased reaching adult levels during the suckling period. Adult levels for the component of low Km were attained earlier than for the component of high Km. The low affinity hepatic aldehyde dehydrogenase component in the newborn was different from that in the adult as assessed by kinetic studies and by its sensitivity to disulfiram.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-Glycerophosphate shuttle in a clonal beta-cell line.

It has been proposed that the alpha-glycerophosphate (alpha-GOP) shuttle plays a crucial role in regulation of glycolysis in beta-cells by linking reoxidation of cytosolic NADH to formation of ATP in the electron transport chain (J. Biol. Chem. 265: 8287, 1981). Direct evidence for this suggestion is still lacking, however. In this work the operation of the alpha-GOP shuttle was investigated in the insulin-secreting cell line HIT-T15. The constituent enzymes of the pathway were found to be present in HIT cells. Flavin-linked alpha-GOP dehydrogenase was associated with the mitochondrial fraction, whereas NAD+-dependent alpha-GOP dehydrogenase was localized in the cytosol. In the presence of amobarbital (used to preserve the function of the alpha-GOP shuttle under conditions where oxidation of NADH by the respiratory chain was blocked), glucose increased insulin secretion, O2 consumption, and the cell [ATP]/[ADP] when compared with amobarbital alone. These results indicate that the alpha-GOP shuttle contributes to ATP generation in HIT cells and that its activation may be necessary for the initiation of insulin secretion by glucose.

Mechanism and regulation of ethanol elimination in humans: intermolecular hydrogen transfer and oxidoreduction in vivo.

Ethanol metabolism was studied in four healthy volunteers by intravenous infusion of a mixture of [1,1-2H2]ethanol (1.0 mmol/kg) and [2,2,2-2H3]ethanol (1.0 mmol/kg) followed by blood sampling at 10-min intervals. The concentrations of ethanols labeled with 1, 2, 3, and 4 deuterium atoms were determined by gas chromatography/mass spectrometry of the 3,5-dinitrobenzoates. During the first 30 min mono- and tetradeuteriated molecules appeared rapidly, which indicates that a fraction of the ethanol was formed from acetaldehyde by exchange. This fraction was calculated to be 38-58% and the hydrogen incorporated during the reduction was mainly (63-82%) derived from C-1 of ethanol, indicating slow exchange of enzyme-bound NADH. After 30 min the elimination followed first-order kinetics with t1/2 of 18-31 min and with a small primary isotope effect (1.05-1.11). This indicates almost complete removal of ethanol from blood passing through the liver when the concentration is low (below 1 mM). The results indicate that as long as hepatic blood flow is not limiting, the rate of alcohol dehydrogenase-catalyzed elimination of a small dose of ethanol in vivo is limited by the dissociation of NADH from the enzyme and by the rates of oxidation of acetaldehyde and reoxidation of NADH.

Lactate dehydrogenase M-subunit deficiency: a new type of hereditary exertional myopathy

Three families with a complete deficiency of the lactate dehydrogenase M subunit show exertional myoglobinuria. The response to ischemic forearm work is characteristic in these three families: an increase of venous lactate concentration after ischemic work was not observed and a marked increase of venous pyruvate was found. Glycolysis was markedly retarded in the patient's muscle in the glyceraldehyde 3-phosphate dehydrogenase (GA3PD) step. A significant increases in glyceraldehyde 3-phosphate, dihydroxyacetone phosphate and fructose 1, 6-diphosphate were observed. The glycolysis retardation may be attributed to the impaired reoxidation of NADH produced by GA3PD action. The cytosolic fraction of skeletal muscle is rich in alpha-glycerophosphate dehydrogenase. This enzyme reoxidizes the excess NADH and drains triose phosphates from the glycolytic pathway under anaerobic conditions. For this reason, ATP production was significantly impaired and muscle cells were damaged in these patients. Consequently, the cytosolic enzymes and proteins such as creatine kinase and myoglobin were released into the blood stream. Otherwise, patients with a lactate dehydrogenase M-subunit deficiency do not show muscle stiffness and myoglobinuria under ordinary circumstances. They complain of muscle rigidity and sudden myoglobinuria after strenous exercise under anaerobic conditions. Thus, the lactate dehydrogenase M-subunit deficiency does not show any symptoms under ordinary circumstances, but is a latent hereditary disorder, now recognized as a new type of hereditary exertional myoglobinuria.

Properties of mitochondria isolated from greening soybean and lupin tissues

In germinating soybean and lupin seedlings differing in their amount of lipid reserves, the activity of cotyledon mitochondria was found to be successively associated with the glyoxysomal and the peroxisomal systems. However, the level of glyoxysomal activity was lower in lipid poor lupin tissues. A peak in succinate oxidation by purified mitochondria was observed in the fully green cotyledons of the two species. A marked cyanide resistance developed in soybean cotyledon mitochondria during the greening of the tissue whereas lupin cotyledon mitochondria remained highly cyanides sensitive. This difference between the two species was also found in purified leaf mitochondria oxidizing succinate, malate or glycine. Moreover, the reoxidation of NADH provided during malate and glycine oxidation mainly used the rotenone and cyanide-resistant pathways in soybean mitochondria whereas the complex I and the cytochrome pathway were largely involved in lupin mitochondria.