The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states

It has been shown that K_M values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The K_i values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 μM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adenienucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Study of the regulation of oxidation and CO2 assimilation in intact Nitrobacter winogradskyi cells

1. Changes of the adenine nucleotides in resting and growing Nitrobacter winogradskyi cells were measured in connection with regulating processes during nitrite oxidation and endogenous respiration.
2. After the addition of nitrite to endogenously respiring cells the ATP pool increased strongly during the first 60 sec at the expense of the ADP pool. At this point the energy charge was approx. 0.55. After the first 90 sec the ATP pool dropped, oscillating, to a lower level. The CO₂ assimilation began at this point.
3. Under a nitrogen atmosphere the AMP pool increased and the ATP pool decreased. With a value of approx. 0.17 the energy charge was extremely low. When oxygen was added the Nitrobacter cells began to oxidize stored NADH. The ATP pool increased in a few seconds whereas the AMP pool decreased. The P/O ratio of endogenously respiring cells equaled 0.6 under these conditions.
4. During the changeover from anaerobic to aerobic conditions and in the presence of nitrite the nitrite oxidation and CO₂ assimilation, opposed to aerobic conditions, were inhibited at first after the nitrite addition. The changeover of the respiratory chain enzymes from a reduced to an oxidized charge and the ATP increase were delayed in comparison with experiments without nitrite. According to these findings the endogenous respiration must be almost nil while nitrite oxidizing cells are growing.

     

Altered mechano-chemical environment in hip articular cartilage: effect of obesity

The production of extracellular matrix (ECM) components of articular cartilage is regulated, among other factors, by an intercellular signaling mechanism mediated by the interaction of cell surface receptors (CSR) with insulin-like growth factor-1 (IGF-1). In ECM, the presence of binding proteins (IGFBP) hinders IGF-1 delivery to CSR. It has been reported that levels of IGF-1 and IGFBP in obese population are, respectively, lower and higher than those found in normal population. In this study, an experimental–numerical approach was adopted to quantify the effect of this metabolic alteration found in obese population on the homeostasis of femoral hip cartilage. A new computational model, based on the mechano-electrochemical mixture theory, was developed to describe competitive binding kinetics of IGF-1 with IGFBP and CSR, and associated glycosaminoglycan (GAG) biosynthesis. Moreover, a gait analysis was carried out on obese and normal subjects to experimentally characterize mechanical loads on hip cartilage during walking. This information was deployed into the model to account for effects of physiologically relevant tissue deformation on GAG production in ECM. Numerical simulations were performed to compare GAG biosynthesis in femoral hip cartilage of normal and obese subjects. Results indicated that the lower ratio of IGF-1 to IGFBP found in obese population reduces cartilage GAG concentration up to 18 % when compared to normal population. Moreover, moderate physical activity, such as walking, has a modest beneficial effect on GAG production. The findings of this study suggest that IGF-1/IGFBP metabolic unbalance should be accounted for when considering the association of obesity with hip osteoarthritis.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O₂ uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O₂ uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (K_m 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (K_m 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.     

Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively.
2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10^-4 M and 3.39×10^-4 M, respectively. Variation of MgCl₂ at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl₂ was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg⁺⁺ was higher than 0.5, suggesting that ATP: 2Mg⁺⁺ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K⁺ or NH ₊ ⁴ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, P_i, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate.
3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, P_i, F-1-P, pyruvate, or citrate.

     

Temperature acclimation in brook trout muscle: Adenine nucleotide concentrations, phosphorylation state and adenylate energy charge

Summary Cold acclimation in fish is associated with an elevation in metabolic rate. The present study investigates the role of adenine nucleotides and related compounds in metabolic regulation following temperature acclimation. Brook trout (Salvelinus fontinalis) were acclimated for 10 weeks to either +4°C or +24°C. Both groups of fish were exercised at 2.5 body lengths s^–1 for 2 weeks prior to sacrifice in order to control for differences in spontaneous activity.Concentrations of ATP, ADP, AMP, P _i and PC were approximately 2-fold higher in white than red muscles. Temperature acclimation had little effect on total adenine nucleotide concentration in either muscle type. In white fibres acclimation to 4°C results in a 39% increase in [ADP] and [AMP], a 35% decrease in [PC] (phosphorylcreatine), and no significant change in [P _i ]. In contrast temperature has little effect on concentrations of these compounds in red muscle.Parameters of metabolic control — adenylate energy charge ([ATP]+0.5 [ADP]/[ATP]+[ADP]+[AMP]), phosphorylation state ([ATP]/[ADP]·[P _i ]), and the ratios [ATP][ADP] and [ATP][AMP] — were significantly lower in cold- than warm-acclimated white muscle. The observed changes in phosphorylation state and [ATP][AMP] are consistent with an increase in mitochondrial respiration and glycolysis, respectively.In conclusion, changes in metabolites may be an important factor in producing an enhanced metabolic rate in cold-acclimated fish.     

Calcium-independent uncoupling activity of palmitic acid in liver mitochondria is regulated by the ion fluxes causing the interconversion of Δψ and ΔpH across the inner membrane

The effect of ion fluxes across the inner membrane on calcium-independent uncoupling activity of palmitic acid was investigated in experiments on rat liver mitochondria energized by the oxidation of succinate. The following compounds were used as the inductors of ion fluxes: the K⁺/H⁺ antiporter nigericin causing transformation of ΔpH into electrical potential difference (Δψ) across the inner membrane; tetraphenylphosphonium (TPP⁺) that freely crosses phospholipid membranes; protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) that induces a flow of H⁺ from the intermembrane space into the matrix and reduces Δψ and ΔpH. It was found that nigericin at a concentration of 20 nM, which causes an increase in maximal Δψ, partially inhibits the ability of palmitic acid to reduce Δψ and stimulates mitochondrial respiration. A specific inhibitor of the ATP/ADP antiporter (carboxyatractylate) and a substrate of the aspartate/glutamate antiporter (glutamate) increase Δψ and partially inhibit mitochondrial respiration in the presence of palmitic acid. Under these conditions, 10 μM cyclosporin A also inhibits respiration but has no effect on Δψ. The specific uncoupling activity of palmitic acid (V _U) and its specific components that characterize participation of the ATP/ADP antiporter (V _Catr), aspartate/glutamate antiporter (V _Glu), and cyclosporin-A-sensitive system (V _CsA) in the palmitic acid-induced uncoupling were estimated. It was shown that nigericin substantially reduces V _U, V _Catr and V _Glu but increases V _CsA. TPP⁺ at a concentration of 20 μM increases V _U and V _Glu, does not affect V _Catr and reduces V _CsA. FCCP at concentrations of 20 and 40 nM reduces Δψ by not more than 17% but does not affect V _U, V _Catr, V _Glu and V _CsA. The results suggest that the calcium-independent uncoupling effect of palmitic acid in liver mitochondria is caused by the return of protons to the matrix with participation of ADP/ATP and aspartate/glutamate antiporters and owing to activation of cyclosporin A-sensitive electron transport along the respiratory chain without affecting Δψ. The induced ion fluxes across the inner mitochondrial membrane can be considered as a factor of the calcium-independent regulation of uncoupling activity of palmitic acid in liver mitochondria with participation of the ADP/ATP and aspartate/glutamate antiporters and of the cyclosporin A-sensitive electron transport system.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

ATP and ADP hydrolysis in cell membranes from rat myometrium

Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K _m values were 506.4?±?62.1 and 638.8?±?31.3?μM, with a calculated V _max (app) of 3,973.0?±?279.5 and 2,853.9?±?79.8?nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5?mM). According to similar apparent K_m values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology.     

Short term cadmium intoxication of the shrimp Palaemon serratus: Effect on adenylate metabolism

• 1.1. Shrimps were exposed for 96 hr to various concentrations of cadmium under laboratory conditions. The LC₅₀ was around 4 ppm Cd in water, which corresponded to 0.180 μg/g wet weight of cadmium in tail muscles.
• 2.2. The effect of various concentrations of cadmium on adenylates was analyzed in tail muscles: At subletal cadmium concentrations, no variation of ATP, ADP and of the adenylate sum occurred, while the AMP concentration began to decrease from 0.06 ppm.

At the LC₅₀, the ATP, ADP and AMP concentrations dropped acutely, the ATP/ADP ratio increased acutely.The apparent equilibrium constant of the adenylate kinase reaction increased significantly from 2 ppm Cd, indicating an impairment in energetic metabolism.Cadmium intoxication did not influence the value of the adenylate energy charge.     

Regulation of biosynthesis of secondary metabolites

Partially purified citrate synthase (EC 4.1.3.7) was isolated from submerged mycelium ofStreptomyces aureofaciens RIA 57, a strain producing chlortetracycline. Enzymatic activity was determined spectrophotometrically by means of 5,5′-dithio-bis-(2-nitrobenzoic acid). Citrate synthase was inhibited by ATP, inhibition being competitive with respect to acetyl coenzyme A. In 5mm concentration, ATP caused 55% inhibition of the enzyme. ADP, in the same concentration, caused 30% inhibition, while AMP caused none. Other natural nucleoside di- and triphosphates, in 1–5mm concentration, did not significantly affect citrate synthase inStreptomyces aureofaciens. Mg²⁺ inhibited the activity of the enzyme, but also reduced the negative effect of ATP. The role of ATP in regulation of the metabolic paths of acetyl CoA is discussed.     

Nucleotide effects on glucan-synthesis activities of particulate enzymes from Saprolegnia

Membrane-bound β-1-3- and β-1-4-glucan synthetases of Saprolegnia are affected in vitro by the presence of nucleotides. Both enzymatic activities are inhibited by uridine nucleotides. Guanosine 5′-triphosphate and ATP reduce β-1-3-glucan synthesis but stimulate β-1-4-glucan production; they also increase V _max without effect on the K _m for uridine 5′-diphosphate glucose. The stimulation by ATP could be the result of an activation or stabilization of the enzymes and might have implications for cell-wall construction during hyphal growth.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

Regulation of electron transport in isolated chloroplasts by sequential binding of adenine nucleotides to the coupling factor protein

Changes in the ferricyanide-reducing activity in isolated spinach chloroplasts by adenine nucleotides were measured in the presence or absence of phosphate, arsenate or pyrophosphate at 15°C. The activity changes were analyzed and ascribed to the interaction between the nucleotides and the chloroplast coupling factor (CF₁).ADP and ATP (but not AMP) partially inhibited ferricyanide reduction through a 11 binding to the inhibition site on CF₁.When the ferricyanide reduction was coupled to either phosphorylation or arsenylation, the inhibition by ADP was released through a 11 binding of the second ADP molecule to the coupling site on the CF₁ with which the first ADP had been associated.The association constants of ADP for the inhibition and the coupling site were found to be approximately 5×10⁵ and 6×10⁴ M^–1, respectively. TheKm value of ADP for arsenylation (pH 8.3) was around 17 M.The ADP-regulated electron transport was defined based on these results. The ADP-regulated ferricyanide reduction, when coupled with phosphorylation, revealed a stoichiometry of P/e=1 between the amounts of esterified phosphate and reduced ferricyanide.Presented to the annual meeting of the Japanese Society of Plant Physiologists, Sendai, April 1974.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity

The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H₂O₂ accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H₂O₂ accumulation in AMF-inoculated roots was decreased by 42.44 % compared with the control under LT. H₂O₂ predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H₂O₂ production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H⁺-ATPase, P-Ca²⁺-ATPase, V-type H⁺-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H₂O₂ accumulation in the roots of cucumber.     

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 µM) and one with lower affinity (K_d = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 µM). The binding of [³⁵S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 µM) and six at the liver enzyme (K_d = 12 µM). The Paracoccus enzyme had only one binding site (K_d = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)