Phosphorylative fumarate reduction in Vibrio succinogenes: Stoichiometry of ATP synthesis

1. Cells of Vibrio succinogenes, treated with EDTA at pH 8, catalyze the phosphorylation of their endogenous ADP and AMP as a function of the electron transport from formate to fumarate. The P/fumarate ratio obtained from the initial velocity of the phosphorylation on initiation of the electron transport and from the activity of fumarate reduction in the steady state was 0.90. The phosphorylation was prevented by 10μmol/g protein carbonylcyanide-3-chlorophenylhydrazone.
2. The esterification of external phosphate in the presence of ADP, hexokinase and glucose is catalysed by a membrane preparation of V. succinogenes in the steady state of fumarate reduction by H₂. The phosphorylation was fully abolished by either 5μmol/g protein carbonylcyanide-4-trifluoromethoxyphenylhydrazone or 30μmol/g protein carbonylcyanide-3-chlorphenylhydrazone. Phosphorylation was blocked also by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent membrane bound ATP synthase, and by low concentrations of the inhibitors of electron transport 2-(n-nonyl)-4-hydroxyquinoline-N-oxide or 4-chloromercuriphenylsulfonate.
3. The P/fumarate ratios, measured with the membrane preparation, were found to increase with progressive inhibition of the electron transport from hydrogen to fumarate by means of 4-chloromercuriphenylsulfonate. The extrapolated ratio at vanishing electron transport activity was 0.47.
4. About 50% of the membrane preparation was found to consist of inverted vesicles with the hydrogenase and formate dehydrogenase oriented to the inside. The residual part is considered as being incapable of performing energy transduction. The extrapolated P/fumarate ratio valid for the inverted vesicles was 0.94.

     

Membrane-bound phosphate as driving force for ATP synthesis in chromatophores ofRhodospirillum rubrum

Chromatophores ofRhodospirillum rubrum preilluminated in the presence of unlabelled phosphate form labelled ATP in the dark after being separated from the preincubation mixture by gel-filtration and incubated with ADP and³²P_i. The driving force for the synthesis of this labelled ATP was previously shown to be ATP firmly bound to the membrane. The amount of labelled ATP produced is determined by measuring the incorporated³²P_i and extrapolation of the values towards zero time incubation and is shown to correlate with the phosphorylation activity of the chromatophores used. 2.5 nmoles ATP correspond to a phosphorylation activity of 100 moles ATP/mg Bchl. h. The results were compared with those from assays in which chromatophores were preincubated with pyrophosphate in the dark. The results strongly support the hypothesis that the XP formed in the light does not transfer its P to ADP in solution and therefore give further evidence to a double sited coupling factor as proposed previously.     

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.     

A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.     

Properties of CAR-kinase: The enzyme that phosphorylates the cAMP chemotactic receptor ofD. discoideum

The cell surface cAMP chemotactic receptor ofD. discoideum can be phosphorylated in partially purified plasma membrane preparations in a ligand-dependent manner. CAR-kinase, the enzyme responsible for receptor phosphorylation, was shown to be an integral membrane protein. It could utilize either ATP or GTP to phosphorylate the receptor, although ATP was much more efficient. The apparent affinity constant for ATP was approximately 20–25 µM. Maximum CAR-kinase activity was observed betweenpH 6.5 andpH 7, and required the presence of Mg²⁺. Neither Mn²⁺ nor Ca²⁺ could substitute for that divalent cation. The enzyme was found to be sensitive to the ionic strength and temperature of the incubation reaction. Dephosphorylation of the receptor was not observed in the membrane preparations, indicating that the enhanced level of receptor phosphorylation that occurred upon ligand binding was not an indirect reflection of receptor dephosphorylation and subsequent incorporation of radiolabeled phosphate.     

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP,K+, Na+, and strophanthidin in vitro

The effect of lipid peroxidation on the affinity of specific active sites of Na⁺, K⁺-ATPase for ATP (substrate), K⁺ and Na⁺ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl₂ at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na⁺, K⁺-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K⁺ and Na⁺ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na⁺, K⁺-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K⁺ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na⁺, K⁺-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.     

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.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes k ₁ for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients k ₁ probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/L) and DMSO (2250 mOsm/L) the filtration coefficient L _p in the presence of a penetrating cryoprotectant (L _pDMSO = (4.45 ± 0.04) · 10^?14 m³/Ns) is 3 orders lower compared to the case with electrolyte (L _pNaCl = (2.25 ± 0.25) · 10^?11 m³/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.     

Mechanism of proanthocyanidins-induced alcoholic fermentation enhancement in Saccharomyces cerevisiae

Our previous work revealed proanthocyanidins (PAs) could pose significant enhancement on the activity of H⁺-ATPase and fermentation efficiency after a transient initial inhibition (Li et al in Am J Enol Vitic 62(4):512–518, 2011). The aim of the present work was to understand the possible mechanism for this regulation. At Day 0.5 the gene expression level of PMA1 in AWRI R₂ strain supplemented with 1.0 mg/mL PAs was decreased by around 54 % with a 50 % and a 56.5 % increase in the concentration of intracellular ATP and NADH/NAD⁺ ratio, respectively, compared to that of control. After the transient adaptation, the gene expression levels of PMA1 and HXT7 in PAs-treated cells were enhanced significantly accompanied by the decrease of ATP contents and NADH/NAD⁺ ratio, which resulted in the high level of the activities of rate-limiting enzymes. PAs could pose significant effects on the fermentation via glucose transport, the energy and redox homeostasis as well as the activities of rate-limiting enzymes in glycolysis.     

Evidence for multiple phosphorylation sites in rat liver 3-hydroxy-3-methylglutaryl Coenzyme A reductase

Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase (EC 1.1.1.34) was inactivated by [γ-³²P]ATP in the presence of endogenous reductase kinases, solubilized, and purified 575-fold with retention of³²P to a state where phosphoreductase was the only³²P-labeled protein present.³²P comigrated with reductase activity under nondenaturing conditions (polyacrylamide gets, isoelectric focusing gels) and with reductase monomer under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gels). Polyfunctional antibody to homogeneous reductase precipitated all of the³²P present. The phosphate-reductase bond was acid-stable and base-labile. Following acid hydrolysis and high-voltage electrophoresis,³²P label migrated solely with phosphoserine and inorganic orthophosphate. Exhaustive (>100 h) tryptic digestion of phosphoreductase denatured in 2 M urea yielded two major phosphorylated components as judged by high-performance liquid chromatography or Sephadex G-25 chromatography. 3-Hydroxy-3-methylglutaryl Coenzyme A reductase inactivated in the microsomal state by [γ-³²P]ATP is thus phosphorylated exclusively at seryl residues and contains two structurally distinct phosphorylation sites.     

Studies on the regulatory complex of rabbit skeletal muscle: Contributions of troponin subunits and tropomyosin in the presence and absence of Mg2+ to the acto-S1 ATPase activity

The regulatory roles of the components of the troponin-tropomyosin complex in the presence and absence of Mg²⁺ on the acto-S1 ATPase have been examined. The effect of free Mg²⁺ on the inhibition of the acto-S1 ATPase by rabbit skeletal troponin (Tn) was studied at S1 to actin ratios ranging from 0.17:1 to 2.5:1. These studies were performed using two Mg²⁺ concentrations: 2.5 mM Mg²⁺-2.5 mM ATP, conditions considered to have low free Mg²⁺; and 5.0 mM Mg²⁺-2.5 mM ATP, conditions providing a high free Mg²⁺ concentration of ～2.5 mM. In the presence of high free Mg²⁺ (2.5 mM ATP-5.0 mM MgCl₂) the Tn inhibition of acto-S1-TM ATPase increased by approximately 40–50% over a range of S1 to actin ratios of 0.17:1 to 2.5:1. The effect of free Mg²⁺ on increasing quantities of Tn in the absence or presence of tropomyosin was studied independently at two S1 to actin ratios (1:1 and 2:1). In the absence of TM, at 5 mM Mg²⁺ there is an additional 38% (1:1 S1 to actin) or 37% (2:1) decrease in the ATPase activity by Tn compared to 2.5 mM Mg²⁺. Similarly, in the presence of TM and Tn, Mg²⁺ exerts its effect at both S1 to actin ratios. Significantly, the inhibition by the IT complex in the presence of TM is unaffected by free Mg²⁺. Furthermore, ultracentrifugation binding studies using¹⁴C-iodoacetamide-labeled Tn and TM established that the Tn-TM regulatory complex was firmly bound to F-actin at both Mg²⁺ concentrations, indicating that faciliation of binding to F-actin by Mg²⁺ is not responsible for the increased inhibition. Hence, it is concluded from the data that Mg²⁺ binding and by analogy Ca²⁺ binding to the Ca²⁺-Mg²⁺ sites of TnC promotes muscle relaxation by inducing inhibition of the actomyosin ATPase, whereas Ca²⁺ binding to the Ca²⁺-specific sites promotes contraction by potentiating the ATPase. The inhibition of the acto-S1-TM ATPase by TnT has also been further examined. The data indicate that TnT exerts the same level of inhibition upon the ATPase as TnI or Tn. The inhibitory activity requires TM, and occurs to the same extent under conditions where TM alone would have either a potentiating (2:1 S1 to actin) or an inhibitory (1:1 S1 to actin) effect upon the ATPase. In the presence of TM the IT complex is a more effective inhibitor than either TnI, TnT, or Tn. The inhibitory activity of the IT complex is partially released by TnC in the absence of Ca²⁺. These observations, in conjunction with those by Chong, Asselbergs, and Hodges, which showed that the inhibition by TnT is partially released by TnC plus Ca²⁺, indicate that the role of TnT involves more than anchoring Tn to the thin filament.     

Energy conservation in Bacillus megaterium

1. The respiratory chain energy conservation systems of Bacillus megaterium strains D440 and M have been investigated following growth in batch and continuous culture. Respiratory membranes from these strains contained cytochromes b, aa ₃ , o and b, c, a, o, respectively; both readily oxidised NADH but neither showed any pyridine nucleotide transhydrogenase activity.
2. Whole cells of both strains exhibited endogenous →H^-/O ratios of approximately 4; when loaded with specific substrates the resultant →H⁺/O ratios indicated that proton translocating loops 1 and 2 were present in strain D440 and that loops 2 and 3 were present in strain M.
3. In situ respiratory activities were measured as a function of dilution rate during growth in continuous culture. True molar growth yields with respect to oxygen (Y _{O 2}) of approximately 50 g cells·mole oxygen^-1 were obtained for most of the nutrient limitations employed. Average values for Y _ATP of 12.7 and 10.8 g cells·mole ATP equivalents^-1 were subsequently calculated for strains D440 and M respectively.
4. Energy requirements for maintenance purposes were low in energy-limited cultures but were substantially increased when growth was limited by nitrogen source (NH ₄ ⁺ ). Under the latter conditions there is probably a partial uncoupling of energy-conserving and energy-utilising processes leading to energy wastage.

     

The crucial elements of the ‘last step’ of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings

Key message

Kinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process.

Abstract

The last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4′,6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A₃ (CMA₃)-stained chromocenters, (3) increase in fluorescence intensity of CMA₃-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA₃-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn²⁺ and Ca²⁺/Mg²⁺-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification “in Byczkowska et al. (Protoplasma 250:121–128, 2013)”], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.     

ATP significantly increases P2Y2-dependent RANTES secretion and overexpression in human airway epithelial cells

Uncontrolled inflammation is frequently observed in human respiratory diseases. Extracellular ATP can induce a number of physiological phenomena via binding to purinergic receptors. In spite of the fact that ATP has long been known as a proinflammatory mediator in the airway, the signaling pathway mechanism is still unclear. Here we show that ATP increases RANTES secretion and overexpression in a time-dependent manner and siRNA-P2Y₂ significantly decreases RANTES secretion and overexpression. These results suggest that ATP can induce secretion and overexpression of the RANTES chemokine via a P2Y₂ Gαq coupled receptor-dependent manner. In addition, pharmacological inhibition of ERK1/2 MAPK by U0126 suppressed ATP/P2Y₂-induced RANTES overexpression in the human airway epithelium. These results show that RANTES secretion and overexpression are regulated by a P2Y₂ receptor and the ATP/P2Y₂ signaling complex may be critical for airway inflammation in respiratory diseases. Taken together, our investigation provides novel insight into the physiological functions of the P2Y₂ receptor and enhances our understanding of the inflamed microenvironment in the airway.     

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.

     

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.