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PubMed1986

Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer.

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.

Adenosine Triphosphate↗
PubMed1992

Detection of the neor gene expression in animal cells after genetic transformation.

A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.

Animals↗
PubMed1987

ATPase activity and tension development in mechanically-skinned feline jaw muscle.

At a muscle length L0 (just taut), isometric tension and ATPase activity were measured at constant levels of various Ca2+ activations in muscle fibres (2-3 mm long and 34-66 microns in diameter) isolated from temporal, masseter and digastric (anterior belly) muscles. The isometric tension increased in a sigmoid fashion with increasing Ca2+ concentration from about pCa 6.60 to pCa 5.00 in the temporalis and from about pCa 6.37 to pCa 5.00 in both the masseter and digastric. The maximum isometric tension from four preparations of each muscle averaged 44.4 +/- 6.6 g/mm2 in the temporalis, 31.9 +/- 4.0 g/mm2 in the masseter, and 23.9 +/- 5.5 g/mm2 in the digastric. The ATPase activity also increased sigmoidally with increasing Ca2+ concentration from slightly below pCa 7.0 to pCa 5.0 in the temporalis, and from slightly above pCa 6.0 to pCa 5.0 in both the masseter and digastric. The maximum ATPase activity obtained from four preparations of each muscle averaged 0.35 +/- 0.06 mumol/mg protein per min in the temporalis, 0.29 +/- 0.06 mumol/mg protein per min in the masseter, and 0.18 +/- 0.04 mumol/mg protein per min in the digastric. The tension cost (ATPase activity/tension) estimated from these results was lower in the digastric than in the temporalis or masseter, indicating more economical ATP consumption in the digastric.

Adenosine Triphosphatases↗
PubMed1982

Compartmentation of citrate in relation to the regulation of glycolysis and the mitochondrial transmembrane proton electrochemical potential gradient in isolated perfused rat heart.

Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The delta pH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.

Animals↗
PubMed1982

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells.

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg2+, Mn2+, Co2+, Ni2+ and Fe2+. This activation is based on the formation of Me2+ -ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me2+ in this transport process. Higher Me2+ concentrations inhibit calcium transport with various efficiencies. Mn2+ directly competes with Ca2+ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [gamma-32P]ATP is induced by Ca2+ while rapid dephosphorylation requires the presence of Mg2+. At higher concentrations Mn2+ and Ni2+ inhibit predominantly the formation of EP, while Co2+ and Fe2+ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [gamma-32P]ATP is significantly stimulated by Mn2+ and Co2+, as compared to that produced by Mg2+, Fe2+ and Ni2+. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.

Biological Transport, Active↗
PubMed1996

The role of plasma membrane K+ and Ca2+ permeabilities for glucose induction of slow Ca2+ oscillations in pancreatic beta-cells.

In individual pancreatic beta-cells the rise of the cytoplasmic Ca2+ concentration ([Ca2+]i), induced by 11 mM glucose, is manifested either as oscillations (0.2-0.5 min-1) or as a sustained elevation. The significance of the plasma membrane permeability of Ca2+ and K- for the establishment of these slow oscillations was investigated by dual wavelength microfluorometric measurements of [Ca2+]i in individual ob/ob mouse beta-cells loaded with fura-2. Increasing the extracellular Ca2+ to 10 mM or the addition of Ca2+ channel agonist BAY K 8644 (1 microM) or K+ channel blocker tetraethylammonium+ (TEA: 10-20 mM) caused steeper rises and higher peaks of the glucose-induced oscillations. However, when extracellular Ca2+ was lowered to 0.5 mM the oscillations were transformed into a sustained suprabasal level. When the beta-cells exhibited glucose-stimulated sustained elevation of [Ca2+]i in the presence of a physiological Ca2+ concentration (1.3 mM), it was possible to induce slow oscillations by promoting the entry of Ca2+ either by raising the extracellular Ca2+ concentration to 10 mM or adding TEA or BAY K 8644. The results indicate that glucose-induced slow oscillations of [Ca2+]i depend on the closure of ATP-regulated K+ channels and require that the rate of Ca2+ influx exceeds a critical level. Apart from an inherent periodicity in ATP production it is proposed that Ca(2+)-induced ATP consumption in the submembrane space contributes to the cyclic changes of the membrane potential determining periodic entry of Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

ATP-producing and consuming processes of Ehrlich mouse ascites tumor cells in proliferating and resting phases.

The extents of ATP-yielding and consuming processes in Ehrlich mouse ascites tumor cells during the proliferating and resting growth phase were compared. In the resting phase the total ATP production was decreased by one-third. The ATP supply by oxidative phosphorylation was drastically reduced, whereas the rate of glycolysis stayed nearly constant. All ATP-consuming processes investigated, i.e., protein turnover, Na+/K(+)-ATPase, Ca2(+)-ATPase, and RNA synthesis, were decreased proportionally with the total ATP consumption.

Adenosine Triphosphate↗
PubMed1986

31P-NMR studies of respiratory regulation in the intact myocardium.

The mechanism by which mitochondrial respiration is coupled to ATP consumption in intact tissues is unclear. We determined the relationship between high-energy phosphate levels and oxygen consumption rate in rat hearts operating over a range of workloads and perfused with different substrates. With pyruvate +glucose perfusion, ADP levels were in general very low, and varied with MVO2 yielding an apparent Km of 25 +/- 5 microM, suggesting regulation of oxidative phosphorylation through availability of ADP. In contrast, with glucose perfusion in the presence or absence of insulin, ADP levels, ADP/ATP ratio or the phosphate potential were relatively constant over the workload range examined and generally not correlated with alterations in MVO2; it is suggested that under these conditions, carbon substrate delivery to the mitochondria may control mitochondrial respiration. The common feature of both of the suggested regulatory mechanisms is substrate limitation which, however, is exercised at different metabolic points depending on the carbon substrate available to the myocardium.

Adenosine Diphosphate↗
PubMed1988

Programmed cell death and adenine deoxynucleotide metabolism in human lymphocytes.

Agents that cause the accumulation of DNA strand breaks are directly cytotoxic to non-dividing normal human peripheral blood lymphocytes, and to chronic lymphocytic leukemia (CLL) cells. Activation of poly(ADP-ribose) polymerase (ADPRP), and the resultant consumption of NAD, play an essential role in mediating the toxicity of these agents. Human peripheral blood lymphocytes contain a substantial number of alkali-sensitive DNA sites, reflecting ongoing DNA strand breakage and repair. However, resting lymphocytes have a limited capacity to synthesize NAD. Pulse-chase experiments indicate that approximately 75% of their NAD turnover is due to ADPRP activity. Exposure of the cells in vitro to deoxyadenosine, or to 2-chlorodeoxyadenosine (CdA, an adenosine deaminase resistant deoxyadenosine congener), caused an increase in DNA strand breaks, rapid NAD consumption, ATP depletion and cell death. Supplementation of the medium with inhibitors of poly(ADP-ribose) polymerase blocks the fall in cellular NAD and ATP, and protects the lymphocytes from the toxicity of DNA damaging agents. Slowly dividing malignant lymphocytes from patients with CLL are also susceptible to lethal NAD depletion following DNA damage. 2-chlorodeoxyadenosine (CdA) induced massive DNA strand break formation in CLL cells in vitro and a fall in NAD and ATP pools. In an initial clinical trial, several CLL patients, and two patients with hairy cell leukemia, have responded to treatment with CdA, with minimal toxicity. Thus, the suicidal activation of ADPRP in response to DNA damage has been rationally exploited in the treatment of chronic lymphoid malignancies.

Cell Survival↗
PubMed1984

Platelet-activating factor phosphatidate, but not platelet-activating factor, is a powerful calcium ionophore in the human red cell.

Platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, PAF) is a potent inducer of shape-change, aggregation and secretion in platelets. PAF causes a rapid increase in intracellular calcium, but has no calcium gating effect in intact lipid bilayers. Human red cells (RBC) did not metabolize either PAF or PAF-phosphatidate (PAF-PA). While PAF (10 microM) was devoid of calcium ionophoretic activity, PAF-PA (1-5 microM) stimulated calcium influx into intact human RBC. In addition, PAF-PA (1-10 microM), but not PAF (10 microM), elicited a series of satellite effects related to the rise of intracellular calcium: 1) increased efflux of intracellular potassium (Gàrdos effect); 2) alkalinization of unbuffered RBC suspensions; 3) stimulation of ATP consumption and production, and enhancement of glycolytic flux with crossover at the glyceraldehyde 3-phosphate dehydrogenase step. These effects exactly duplicate those brought about by the calcium ionophore A23187. The ionophoretic potency of PAF-PA was about half that of A23187. Approximately the same concentrations of PAF-PA as those that stimulate calcium influx into RBC elicit full aggregatory response in human platelets. It is possible that transformation of PAF into PAF-PA by the combined action of phospholipase C and diacylglycerol kinase contributes to the increase of calcium influx in platelets.

Calcium↗
PubMed1994

Why does RecA protein hydrolyse ATP?

RecA is a DNA-dependent ATPase involved in DNA-strand repair. Most of the ATP hydrolysis that occurs in a RecA nucleoprotein filament is implicitly considered to be irrelevant in many current models for RecA-mediated DNA-strand exchange. However, preventing RecA from hydrolysing ATP alters its behavior, suggesting that ATP hydrolysis by RecA is more than incidental. This review explores recent results detailing the effects and rates of ATP hydrolysis by RecA, and models are proposed that permit us to account quantitatively for ATP consumption by this protein.

Adenosine Triphosphate↗
PubMed2005

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats.

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c1 and a + a3, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.

Adenylate Kinase↗
PubMed2006

Functioning of oxidative phosphorylation in liver mitochondria of high-fat diet fed rats.

We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24+/-6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N(epsilon)-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Deltapsi), ADP/O ratio, and the content of coenzyme Q(9), cytochromes b and a+a(3). Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=-0.56) ATP/ADP ratios, and Deltapsi (C=-0.11). We conclude that although HFD induces accumulation of LCAC and N(epsilon)-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.

Adenine Nucleotide Translocator 3↗
PubMed2005

Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium.

The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l(-1) of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l(-1)h(-1)) every 2 h to the production medium increased the production of methionine to 3.39 g l(-1). A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.

Adenosine Triphosphate↗
PubMed2004

In vivo approaches and rationale for quantifying kinetics and imaging brain lipid metabolic pathways.

Developing a kinetic strategy to examine rates of lipid metabolic pathways can help to elucidate the roles that lipids play in tissue function and structure in health and disease. This review summarizes such a strategy, and shows how it has been applied to quantify different kinetic aspects of brain lipid metabolism in animals and humans. Methods involve injecting intravenously a radioactive or heavy isotope labeled substrate that will be incorporated into a lipid metabolic pathway, and using chemical analytical and/or imaging procedures (e.g., quantitative autoradiography or positron emission tomography) to determine tracer distribution in brain regions and their lipid compartments as a function of time. From the measurements, fluxes, turnover rates, half-lives and ATP consumption rates can be calculated, and incorporation rates can be imaged. Experimental changes in these kinetic parameters can help to identify changes in the expression of regulatory enzymes, and thus aid in drug targeting. Cases that are discussed are arachidonic acid turnover and imaging of neuroreceptor-initiated phospholipase A2 activation, ether phospholipid biosynthesis, and kinetics of the phosphatidylinositol cycle.

Adenosine Triphosphate↗
PubMed2005

SAXS and the working protein.

In this issue of Structure, Davies et al., 2005, present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution.

Adenosine Triphosphatases↗
PubMed2000

Perturbed equilibria of myosin binding in airway smooth muscle: bond-length distributions, mechanics, and ATP metabolism.

We carried out a detailed mathematical analysis of the effects of length fluctuations on the dynamically evolving cross-bridge distributions, simulating those that occur in airway smooth muscle during breathing. We used the latch regulation scheme of Hai and Murphy (Am. J. Physiol. Cell Physiol. 255:C86-C94, 1988) integrated with Huxley's sliding filament theory of muscle contraction. This analysis showed that imposed length fluctuations decrease the mean number of attached bridges, depress muscle force and stiffness, and increase force-length hysteresis. At frequencies >0.1 Hz, the bond-length distribution of slowly cycling latch bridges changed little over the stretch cycle and contributed almost elastically to muscle force, but the rapidly cycling cross-bridge distribution changed substantially and dominated the hysteresis. By contrast, at frequencies <0.033 Hz this behavior was reversed: the rapid cycling cross-bridge distribution changed little, effectively functioning as a constant force generator, while the latch bridge bond distribution changed substantially and dominated the stiffness and hysteresis. The analysis showed the dissociation of force/length hysteresis and cross-bridge cycling rates when strain amplitude exceeds 3%; that is, there is only a weak coupling between net external mechanical work and the ATP consumption required for cycling cross-bridges during the oscillatory steady state. Although these results are specific to airway smooth muscle, the approach generalizes to other smooth muscles subjected to cyclic length fluctuations.

Adenosine Triphosphate↗
PubMed2001

Modulation of islet ATP content by inhibition or stimulation of the Na(+)/K(+) pump.

High (30 mM) K(+), known to cause beta-cell membrane depolarisation, significantly decreased the islet total ATP content, supporting the view that beta-cell membrane depolarisation can activate the ATP-consuming Na(+)/K(+) pump. Ouabain (1 mM) did not change the islet ATP content after 5-15 min of incubation in the absence or presence of 3 mM glucose but reduced it after 30 min, and in the presence of 20 mM glucose, the reduction by ouabain occurred already after 15 min. Incubation of islets with ouabain for 60 min decreased the islet ATP content in the presence of 3, 10 or 20 mM glucose or 30 mM K(+). Also, the islet glucose oxidation rate was decreased by ouabain. When K(+) deficiency was used to inhibit the Na(+)/K(+) pump, no change in ATP content was observed irrespective of glucose concentration, although K(+) deficiency caused a slight inhibition of the glucose oxidation rate. Diazoxide reduced the islet glucose oxidation rate and increased the islet ATP content in the presence of 20 mM glucose. There may exist a feedback mechanism decreasing the flow of glucose metabolism in response to reduced ATP consumption by the Na(+)/K(+) pump.

Adenosine Triphosphate↗