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PubMed2001

Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling.

Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (DeltaPsim) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (>/=30%, P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by >/=26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased DeltaPsim (JC-1: simulated ischemia 90+/-3%, adenosine 82+/-7%, diazoxide 87+/-4%, versus control 100%, P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21+/-0.03 nmol/L ATP/microg protein, simulated ischemia 0.12+/-0.02, adenosine 0.15+/-0.02, diazoxide 0.11+/-0.02, P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3+/-0.1 nmol/L oxygen/min/1x10(6) cells, simulated ischemia 3.1+/-0.1, adenosine 3.1+/-0.3, diazoxide 2.6+/-0.2, P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K(ATP)-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.

Adenosine↗
PubMed1983

Low calcium reperfusion of ischemic myocardium.

The mechanisms underlying protection of ischemic myocardium by reperfusion with solutions containing reduced concentrations of calcium (CA) were studied in isolated vascularly perfused rabbit interventricular septa at 37 degrees C. After 45 min of total ischemia adenosine triphosphate (ATP), and phosphocreatine (PC) contents fell to 6.8 +/- 0.82, and 12.4 +/- 2.0 micrometer/g dry tissue (dt) (+/- S.E.M.). After 5 min of reperfusion with 0.75 mM Ca these values had recovered significantly to 10.3 +/- 1.0, and 33.3 +/- 3.6, (micrometer/g dt), while reperfusion with 2.5 mM Ca produced 5.9 +/- 1.0 and 22.0 +/- 2.6 (micrometer/g dt). The significantly greater recovery of ATP and PC after 0.75 mM Ca reperfusion persisted after an additional 25 min of reperfusion with 2.5 mM Ca when compared to septa reperfused for the entire 30 min with 2.5 mM Ca. When mechanical work was reduced by cessation of stimulation during the first 5 min of reperfusion, ATP, PC and total creatine (TC) recovery were significantly improved in muscles exposed to 2.5 mM Ca but showed no additional improvement in muscles reperfused with 0.75 mM Ca. A further reduction to 100 micrometers Ca reperfusion in quiescent muscles did significantly improve ATP, PC and TC recovery. Quiescence achieved with 16.0 mM K impaired ATP, PC and TC recovery. This effect was reversed by 100 micrometers CA reperfusion. Measurements of mitochondrial oxygen consumption, respiratory control and ADP/O showed results parallel to the ATP, PC and TC determinations. Both mechanical work and CA itself influence mitochondrial respiration, ATP, PC and TC content after ischemia. As previously demonstrated for mechanical function, metabolic recovery can be improved by interventions limited to the first 5 min of reperfusion.

Adenosine Triphosphate↗
PubMed1998

Effect of calcium on the energy status of rat brain synaptosomes under acidosis.

Incubation of rat brain synaptosomes at pH 6.0 in Ca2+-containing medium is associated with a decrease in the ATP content and the rate of oxygen consumption. ATP/ADP ratio decreased from 6.6 +/- 0.24 at pH 7.4 to 3.2 +/- 0.17 at pH 6.0. The content of 86Rb+ and [3H]tetraphenylphosphonium measured at pH 7.4 did not change after preincubation at pH 6.0, indicating the absence of lesion of synaptosomal plasma membranes and intrasynaptosomal mitochondria. Incubation with 1 mM EGTA in Ca2+-free medium as well as addition of 1 mM ouabain or 10 microM ruthenium red prevents the effect of acidosis. Similar results were obtained when 5 mM pyruvate was used as a mitochondrial substrate instead of glucose. It is suggested that acidosis-induced decrease in the ATP level is associated with the increase in Ca2+ concentration in the cytoplasm and its transport into mitochondria. Ouabain reverses this process due to activation of Na+/Ca2+ exchange.

Acidosis↗
PubMed1995

Interference of the sulphonylurea antidiabeticum gliquidone with mitochondrial bioenergetics in the rat under in vitro conditions.

The hypoglycaemic sulphonylurea gliquidone and glibenclamide exerted a partial uncoupling effect on mitochondrial respiration of liver under in vitro conditions using various citrate cycle intermediates as substrates. Besides the uncoupling effect, gliquidone and glibenclamide caused a direct inhibition of ATP-as well as DNP-stimulated oxygen consumption. Both phenomena proved to be dose dependent. Respiratory control ratio decreased progressively with increasing concentrations of sulphonylureas mainly through the inhibition of ADP-stimulated respiration. Basal and DNP-stimulated ATP-ase activity of isolated mitochondria changed similarly to the respiratory parameters. Changes in membrane permeability of mitochondria and the inhibition of substrate uptake further support the assumption of structural and functional alteration of mitochondria by the hypoglycaemic compounds tested.

Animals↗
PubMed1980

Coupling of active ion transport and aerobic respiratory rate in isolated renal tubules.

We report the results of studies in which the cytoplasmic coupling between Na+,K+-ATPase activity (presumably a measure of active transport) and the mitochondrial respiratory rate was investigated in a tubule suspension from the rabbit kidney cortex. Simultaneous measurements of the redox state of mitochondrial nicotinamide adenine dinucleotide (NAD) (performed fluorometrically), the cellular ATP and ADP concentrations, and the oxygen consumption rate (QO2) were made under conditions known to alter the Na+,K+-ATPase turnover. Ouabain (25 microM) caused: (i) a 54% inhibition of QO2, (ii) a net reduction of NAD, and (iii) a 30% increase in the ATP/ADP ratio. The addition of K+ (5 ?M) to K+-depleted tubules caused: (i) an initial 127% stimulation of QO2 followed by a new steady-state QO2 50% above control, (ii) an initial large oxidation of NAD followed by a new steady state more oxidized than the control level, and (iii) a 47% decrease in the cellular ATP/ADP ratio. These data indicate that the cellular ATP and ADP concentrations or the ATP/ADP ratio may be part of the coupling mechanism linking Na+,K+-ATPase turnover and the aerobic metabolic rate in kidney.

Adenosine Diphosphate↗
PubMed1999

Muscle ATP synthesis and utilisation, balanced during flow-induced increase of respiration.

To investigate the control of cell energetic metabolism, creatine charge, ATP/ADP ratio and oxygen consumption (as indicators of an energetic status, the balance between ATP synthesis and degradation and the aerobic ATP turnover, respectively) were evaluated in the rat gracilis muscle, perfused-superfused in vitro. During the perfusion rate of 70 microl/min the ATP/ADP ratio, as well as the creatine charge are kept at the in vivo level. With the decrease of the rate toward 54 microl/min (of an abundant oxygen delivery), the values of both parameters are lower than levels in vivo. With the increase of the rate up to 100 microl/min, both parameters are kept at the in vivo level, when respiration increases by 125%. The data demonstrate the 'unmatched' control of ATP utilisation and synthesis steady rates during the low perfusion rate; during the increasing steady ATP turnover following the increased perfusion rate, the two fluxes are strikingly 'matched', i.e. precisely balanced.

Adenosine Triphosphate↗
PubMed1984

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies.

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.

Adenine Nucleotides↗
PubMed2003

Time course of the response of mitochondria from oxidative muscle during thermal acclimation of rainbow trout, Oncorhynchus mykiss.

The time course of changes in the properties of mitochondria from oxidative muscle of rainbow trout was examined during warm (15 degrees C) and cold (5 degrees C) acclimation. Mitochondrial oxidative capacities showed a biphasic response during thermal acclimation: at a given assay temperature, capacities first increased and then decreased during warm acclimation and showed the inverse pattern during cold acclimation. This was most apparent for maximal rates of state 3 oxygen consumption expressed per mg mitochondrial protein. Rates expressed per nmol ADP-ATP translocase (ANT) showed this pattern during cold acclimation. A biphasic pattern was also apparent for state 4 and oligomycin-inhibited (state 4(ol)) rates of oxygen uptake expressed per mg protein. Changes in states 4 and 4(ol) were smaller during cold than warm acclimation. Warm acclimation reduced the proportion of cytochrome c oxidase and citrate synthase needed during mitochondrial substrate oxidation. Phospholipid concentrations per mg mitochondrial protein changed little with thermal acclimation. Mitochondrial properties changed more quickly during warm than cold acclimation. While the biochemical modifications during thermal acclimation may eventually compensate for the thermal change, compensation did not occur at its onset. Rather, the initial changes of mitochondrial oxidative capacity in response to temperature change accentuated the functional impact of the thermal change, and prolonged exposure to the new temperature was required to attain a degree of thermal compensation.

Acclimatization↗
PubMed1985

Regulation of the maximum rate of renal ammoniagenesis in the acidotic dog.

Metabolism of glutamine results in the net production of ATP; however, cells cannot sustain an ATP production rate greater than their rate of ATP utilization. The purpose of these studies was to determine whether the rate of ATP turnover in the kidney could set an upper limit on renal glutamine metabolism and thereby renal ammoniagenesis. The acidotic dog kidneys extracted glutamine, lactate, citrate, and oxygen from the arterial blood and added ammonium and alanine to the venous blood. Renal glutamine metabolism was responsible for almost all the ammonium production. Renal ATP production was estimated from the rate of oxygen consumption and appeared to be derived roughly equally from the oxidation of glutamine and lactate. There was no apparent renal glucose production from ATP balance calculations and this impression was supported when the inhibitor of gluconeogenesis, 3-mercaptopicolinate, did not inhibit ammoniagenesis. Approximately 90% of the ATP synthesized was utilized to reabsorb sodium. When the amount of ATP utilized for sodium reabsorption in the proximal convoluted tubule (assumed to be 60% of filtered sodium) was compared with the amount of ATP produced from glutamine metabolism, the values were similar despite the fact that the glomerular filtration rate in individual dogs varied more than fourfold. When the quantity of ATP expended for sodium reabsorption was decreased by the infusion of ouabain or by the constriction of one renal artery without reducing glutamine delivery, the kidney lowered its rate of ammoniagenesis to a quantitatively predictable amount.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMedDate not supplied

Glycolytic ATP production estimated from 31P magnetic resonance spectroscopy measurements during ischemic exercise in vivo.

In an oxygen-depleted muscle, glycolytically produced ATP is inversely related to the ([ATP]+ creatine phosphate [PCr]) decrease because ATP, PCr, and glycolysis are virtually the only energy sources under these conditions. In particular, the onset of glycolysis or any appreciable increase in the rate of glycolytic ATP production will lead to a slower rate of ([ATP]+ [PCr]) breakdown at a given energy consumption. To quantify this relationship, endurance athletes performed isometric foot plantar flexion (20% of a test force [TF], n = 10; 50% TF, n = 5) during local arterial occlusion. Parameters of energy metabolism were measured with 31P magnetic resonance spectroscopy (31P-MRS). During exercise, [PCr] decreased to 80 +/- 10 (20% TF) and 11 +/- 4% (50% TF) of its resting concentration, and pH dropped from 7.04 +/- 0.01 to 6.98 +/- 0.10 (20% TF) and from 7.03 +/- 0.02 to 6.70 +/- 0.10 (50% TF). In both experiments, two phases of ([ATP]+ [PCr]) decrease were observed: an initial faster decrease was followed by a slower decline. The latter phase started at about the time when the pH began to drop. The difference between a line extrapolated from the slope of the initial phase and the measured ([ATP]+[PCr]) decrease was used as an estimate for glycolytically produced ATP. This estimate and pH were significantly correlated with r = -0.97 (20% TF) and r = -0.99 (50% TF). These results indicate that glycolytically produced ATP can be estimated from the ([ATP]+ [PCr]) decrease during exercise.

Adenosine Triphosphate↗
PubMed2005

DAF-2 pathway mutations and food restriction in aging Caenorhabditis elegans differentially affect metabolism.

In Caenorhabditis elegans, metabolism and life expectancy respond to environmental cues of food availability and temperature. Several genes act in a neuroendocrine, DAF-2, insulin/IGF-1 receptor-like pathway in which reduced signaling affects metabolism and increases longevity. Here we describe the effect of reduced DAF-2 signaling on several parameters of metabolism including rates of oxygen consumption and heat output, the calorimetric/respirometric ratio, ATP levels, XTT reduction capacity and accumulation of lipofuscin. We also asked whether the DAF-2 signaling pathway mediates the metabolic and longevity effects of axenic culture medium. We show that both interventions act either antagonistically or in concert, depending on the parameter examined and that axenic culture medium, unlike DAF-2 signaling, does not need DAF-16 for generating these effects. In addition, we provide evidence that DAF-2 signaling controls mitochondrial bioenergetics by adjusting the rate of ATP synthesis to the rate of ATP utilization and by regulating the heat-producing proton leak pathway.

Adenosine Triphosphate↗
PubMed2002

Thyroid status is a key regulator of both flux and efficiency of oxidative phosphorylation in rat hepatocytes.

Thyroid status is crucial in energy homeostasis, but despite extensive studies the actual mechanism by which it regulates mitochondrial respiration and ATP synthesis is still unclear. We studied oxidative phosphorylation in both intact liver cells and isolated mitochondria from in vivo models of severe not life threatening hyper- and hypothyroidism. Thyroid status correlated with cellular and mitochondrial oxygen consumption rates as well as with maximal mitochondrial ATP production. Addition of a protonophoric uncoupler, 2,4-dinitrophenol, to hepatocytes did not mimic the cellular energetic change linked to hyperthyroidism. Mitochondrial content of cytochrome oxidase, ATP synthase, phosphate and adenine nucleotide carriers were increased in hyperthyroidism and decreased in hypothyroidism as compared to controls. As a result of these complex changes, the maximal rate of ATP synthesis increased in hyperthyroidism despite a decrease in ATP/O ratio, while in hypothyroidism ATP/O ratio increased but did not compensate for the flux limitation of oxidative phosphorylation. We conclude that energy homeostasis depends on a compromise between rate and efficiency, which is mainly regulated by thyroid hormones.

Adenosine Triphosphate↗
PubMed1987

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells.

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.

Adenosine Diphosphate↗
PubMed1991

[Value of in vivo NMR spectroscopy in the study of cerebral metabolism under inhalation anesthesia].

NMR in vivo spectroscopy is one of the few methods available for non-invasive investigations of cerebral metabolism in animals and humans. 31P and 1H spectroscopy are particularly suitable for monitoring the cerebral energy metabolism by determining the cerebral levels of ATP, ADP, phosphocreatine (PCr), inorganic phosphate (Pi), lactate and intracellular pH (pHi). These techniques also seem to be suitable for studying the effects of anesthetics by directly comparing the anesthetized and unanesthetized states in the same subject. The effects of halothane and isoflurane on the changes elicited in the cerebral energy metabolism by experimental hypercapnia were investigated by in vivo NMR spectroscopy. Halothane was found to aggravate the decrease in PCr attributed to the shift in creatine kinase equilibrium induced by the cerebral acidosis associated to hypercapnia, while the level of cerebral ADP was decreased to a lesser extent than in unanesthetized animals. In contrast isoflurane did not modify the changes in cerebral energy metabolism elicited by hypercapnia except that the decrease in PCr was significantly slowed, suggesting a lower creatine kinase activity. These data indicate that isoflurane and halothane act by two different mechanisms to produce a decrease in oxygen consumption. Halothane could interfere with oxidative metabolism by disturbing ATP metabolism, while isoflurane could decrease oxygen consumption by a general sedative action, slowing both cerebral functional activity and cerebral energy homeostasis.

Anesthesia, Inhalation↗
PubMed1995

31P NMR kinetics study of cardiac metabolism under mild hypoxia.

The effects of mild hypoxia on the metabolic and mechanical functions of isovolumic perfused rat hearts have been studied. 31P NMR has been used to follow the metabolite concentrations as well as the intracellular pH. Additionally, the energy transfer through the creatine kinase reaction was estimated by the magnetization-transfer technique. The needs of myofibrillar energy and of mitochondrial ATP production have been assessed through mechanical activity and oxygen-consumption rate. It has been observed that mild hypoxia simultaneously impairs contractile and metabolic functions. The aerobic ATP production is maintained under these conditions while anaerobic energy metabolism seems accelerated. The accumulation of some metabolites (ADP and P(i)) and the decrease of creatine kinase forward flux (Vfor) tend, however, to prove that ATP availability for myofibrils is lowered. The large aerobic energy production observed must therefore be explained by an energy wastage in the mitochondria. In spite of normal ATP concentration, a contractile dysfunction is observed and can be explained by the P(i) accumulation, which is known to impair the use of the myofibrillar ATP. Another hypothesis supported by the magnetization-transfer experiments is the poor ATP availability resulting from the ATP wastage in the mitochondria and from the inefficient energy transport by the PCr-Cr shuttle.

Adenosine Triphosphate↗
PubMed1990

Effect of extracellular potassium on ouabain-sensitive consumption of high-energy phosphate by crayfish giant axons: a study of the energy requirement for transport in the steady state.

Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport "coupling ratio" and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady-state consumption of high-energy phosphate (approximately P) [ATP and phospho-L-arginine (Arg-P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total approximately P consumption (Q approximately P) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q approximately P averaged 52.8 +/- 4.7%/h (n = 44) of the initial [ATP] + [Arg-P]. Unlike total Q approximately P, the ouabain-sensitive portion of Q approximately P was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q approximately P to 8%/h, a result indicating that 85% of the total Q approximately P was ouabain sensitive. For 1.35 mM K+o, the ouabain-sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mM K+o, 41%. There was a small but significant increase in the ouabain-sensitive Q approximately P at 21.6 mM K+o, compared with Q approximately P at 5.4 mM K+o. The pattern of effect of [K+]o on Q approximately P was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/approximately P stoichiometry of 3, an actual ratio of JNa/approximately P stoichiometry of approximately 33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero-membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non-steady-state conditions.

Adenine Nucleotides↗
PubMed1984

Influence of fatty acids on energy metabolism. 1. Stimulation of oxygen consumption, ketogenesis and CO2 production following addition of octanoate and oleate in perfused rat liver.

Changes in metabolic rates (oxygen consumption, ketogenesis, 14CO2 production from labelled fatty acids, glycolysis) following the addition of octanoate or oleate were studied in isolated livers from fed and starved rats perfused with Krebs-Henseleit bicarbonate buffer in a non-recirculating system. The following results were obtained. The infusion of fatty acids caused a large increase in the rate of oxygen consumption. The effect was greater with octanoate than with oleate and was half-maximal with fatty acid concentrations (free plus albumin bound) around 0.1 mM. The effects of oleate were only partially suppressed when the perfusate contained albumin concentrations near the physiological range. When fatty acids were oxidized at high rates, the glycolytic rate was diminished by 50%. The increase in oxygen consumption could not be explained fully by the increased ATP demand for fatty acid metabolism or by a compensation for the diminished extramitochondrial ATP generation. In the presence of phenylalkyl oxirane carboxylic acid, an inhibitor of the transport of long-chain acyl-CoA derivates into the mitochondria, ketogenesis and 14CO2 production from labelled oleate were strongly inhibited, whereas the increase in oxygen consumption was only slightly affected. In the presence of antimycin A, the increase in oxygen consumption due to fatty acids was totally abolished. Following pretreatment of rats with ciprofibrate (induction of enzymes for peroxisomal beta-oxidation of long-chain fatty acids), ketogenesis (but not 14CO2 production) from oleate was enhanced threefold. The increase in oxygen consumption, however, was not affected. In conclusion, the increase in hepatic oxygen consumption due to addition of fatty acids reflects a mitochondrial process; it is, in part, independent of the ATP demand of the cell. An uncoupling-like effect of fatty acids on the respiratory chain and its possible physiological significance in ketogenesis are discussed.

Adenosine Triphosphate↗
PubMed1988

Kinetic studies on arachidonate 5-lipoxygenase from rat basophilic leukemia cells.

Arachidonate 5-lipoxygenase of a 10,000 x g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption measured is solely due to 5-lipoxygenation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10-20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 microM. Km(app) for arachidonic acid is 182 +/- 16 microM (mean +/- SD, n = 5) and Vmax(app) is 425 +/- 140 nmol O2/(min x mg protein) (mean +/- SD, n = 5).

Animals↗