Mass Action Ratio Research Articles

Regulation of glycolysis in the land snail Oreohelix during estivation and artificial hypercapnia

Respiration rates, hemolymph acid-base variables, and metabolite levels were measured in the land snail Oreohelix during a brief period of estivation (4 days) and during exposure of non-estivating snails to elevated levels of ambient CO2 (34 and 58 mmHg). Respiration rate dropped during entry into estivation reflecting decreased glycolytic flux. Analyses of metabolite levels in foot muscle and digestive gland tissues demonstrated that glycolytic control during early estivation was primarily vested in pyruvate kinase. Relative to control values, the mass action ratio (MAR) of this reaction decreased by a factor of 4 in the foot muscle and by a factor of 2.7 in the digestive gland, reflecting inhibition of this enzyme in both tissues. No other glycolytic reaction appeared to be inhibited. Exposure of non-estivating snails to artificial hypercapnia resulted in extracellular acid-base conditions similar to those seen during estivation and also promoted a reduction in respiration rate. However, the points of glycolytic control were different: artificial hypercapnia resulted in inhibition of phosphofructokinase in foot muscle and hexokinase in digestive gland. Furthermore, elevated ambient CO2 had no effect on the MAR of the pyruvate kinase reaction in non-estivating snails. These results suggest that the action of extracellular acid-base variables alone cannot fully explain the down-regulation of glycolysis which occurs during estivation in this land snail.

Lactate metabolism in inactive skeletal muscle during lactacidosis

Contributions of carbohydrate and fat metabolism to the removal of a lactate (Lac-) load were quantified in inactive soleus (SL), plantaris (PL), and white gastrocnemius (WG) rat hindlimb muscle. Male Sprague-Dawley rats were perfused for 60 min with normal perfusate (NP, n = 8) or a high-lactate perfusate (LP, n = 8), simulating ionic conditions found in arterial blood and plasma after intense exercise: Lac- = 11.0 mM, K+ = 7.88 mM, and pH = 7.15. Metabolite fluxes across the hindlimb were calculated from blood flow and arteriovenous differences. In NP, Lac- was continuously released (2.9 +/- 0.2 mumol.min-1 x 100 g-1). However, in LP, a rapid and significant uptake of Lac- increased muscle Lac- fivefold to 39.6 +/- 1.1, 33.1 +/- 2.2, and 28.8 +/- 1.7 mumol/g dry wt in SL, PL, and WG, respectively. Glucose and O2 uptakes were similar during LP and NP perfusion. Glycerol release increased eightfold to 3.3 +/- 0.7 mumol.min-1 x 100 g-1 in response to LP. Muscle ATP, creatine phosphate, glycogen, glycolytic intermediate, and triacylglycerol concentrations did not change. However, muscle lactate-to-pyruvate ratios were elevated in all muscles of the LP group postperfusion, indicating changes in the mass action ratio at the pyruvate dehydrogenase reaction. In LP, of 80 mumol of Lac- taken up, 11% was accounted for by increased muscle Lac-, 12-24% was oxidized, and 5% may have been involved in glycerol release. The remaining Lac- may have been involved in metabolic cycling along the glyconeogenic-glycolytic pathway and/or in triacylglycerol-free fatty acid substrate cycling.

Control of the Krebs Cycle in Arum Spadix

Summary Measurements of total tissue activity, and of the proportion of that activity confined to mitochondria, gave estimates of the maximum catalytic activities of citrate synthase, aconitase, isocitrate dehydrogenase (NAD + ), fumarase, and malate dehydrogenase (NAD + ) involved in the Krebs cycle in the developing spadices of Arum maculatum . Isocitrate dehydrogenase had the lowest activity and malate dehydrogenase the highest. The activity of each enzyme rose markedly during spadix development: comparison of these activities with flux in vivo showed that the cycle is subject to coarse control during development. Preparations of mitochondria from Arum spadix and from florets of cauliflower ( Brassica oleracea ) were used to determine the mass-action ratios of citrate synthase, aconitase, and fumarase during the oxidation of pyruvate in the presence of malate. Collectively, the measurements of enzymes and substrates suggest that in the mitochondria the reactions catalysed by citrate synthase and isocitrate dehydrogenase (NAD + ) are markedly displaced from equilibrium in vivo , whereas those catalysed by aconitase, fumarase and malate dehydrogenase (NAD + ) are close to equilibrium.

Correlations between cardiac protein synthesis rates, intracellular pH and the concentrations of creatine metabolites

We have examined in detail the correlations between protein synthesis rates, intracellular pH (pHi) and the concentrations of creatine metabolites in the rat heart perfused anterogradely in vitro. Using perfusion buffers ranging from pH 7.2 to 8.2 at 37 degrees C, we were able to manipulate pHi from between 7.24 to 7.66, i.e. from the slightly acidotic to the alkalinotic as compared with the physiological values of pHi (about pH 7.29). The dependence of pHi on extracellular pH (pHo) was linear, with the value of delta pHi/delta pHo being 0.4-0.5. Protein synthesis rates were significantly stimulated when pHi was increased above its physiological value, and they were strongly correlated with pHi. They were also strongly correlated with phosphocreatine concentrations (and with creatine concentrations and phosphocreatine/creatine concentration ratios). Adenine nucleotide (ATP, ADP and AMP) concentrations and the ATP/ADP concentration ratio were not systematically altered by manipulating pHi, and protein synthesis rates showed only a relatively weak dependence on these variables. Since creatine kinase catalyses a reaction that is close to equilibrium in the perfused heart, and since phosphorylation of creatine involves release of a proton, we argue that the changes in phosphocreatine and creatine concentrations are manifestations of alterations in pHi. In this regard, we show that [log[( phosphocreatine]/[creatine]) + log [( ADP]/[ATP])] [the value of which gives [pHi--log (mass action ratio)]] is positively correlated with pHi, although the slope of the line is 0.7, as opposed to the ideal value of unity. We discuss three hypotheses to account for our observations: (i) protein synthesis rates are influenced directly by pHi, (ii) pHi affects the concentrations of creatine metabolites, which in turn affect protein synthesis rates, and (iii) pHi affects the value of an unidentified co-variable, which in turn affects protein synthesis.

Effects of Exposure of Wheat Ears to High Temperature on Dry Matter Accumulation and Carbohydrate Metabolism in the Grain of Two Cultivars. I. Immediate Responses

Ears of wheat were exposed for up to 7 days during the grain-filling stage to high temperature (35�C day/25�C night) and metabolic responses in the grain were compared to those in ears maintained at lower temperatures (21�C day/16�C night). Two cultivars of wheat known to differ in their post-anthesis tolerance of high temperature were compared. Raising the temperature resulted in a small increase in the rate of dry matter accumulation: both cultivars responded similarly. Sucrose content of the endosperm was either not affected or increased by raising the temperature. Raising the temperature had differential effects on glucose and fructose content: fructose was substantially reduced while glucose was either unaffected or slightly increased. After raising the temperature the concentrations of all three hexose phosphates measured, glucose-6-phosphate (G-6-P), glucose-1-phosphate (G-1-P) and fructose-6-phosphate (F-6-P), were reduced similarly on a percentage basis and to about the same extent as fructose. The concentration of the sugar nucleotide (UDP-glucose) resulting from the breakdown of sucrose by sucrose synthase was also reduced at high temperature. Judging from calculated mass-action ratios, all three catalytic steps involved in the interconversion of the metabolites mentioned above were close to equilibrium, and only one mass action ratio (for sucrose synthase) was affected by heating: it was doubled. Although temperature clearly resulted in changes in the reaction catalysed by sucrose synthase, it was not clear how temperature had acted. Concentration of the precursor for starch synthesis (ADP-glucose) was slightly lower in both cultivars at the higher temperature. Taken together the responses could provide at least a partial explanation for the smallness of the increase in starch deposition with increase in temperature, but do not explain the different responses of these two cultivars to high temperature.

Identification of non-equilibrium glycolytic reactions in suspension-cultured plant cells

Levels of all enzymes and metabolites involved in glycolysis were determined in suspension-cultured Catharanthus roseus cells. From both the maximum catalytic activities of the enzymes and comparisons of mass-action ratios with the apparent equilibrium constants for the reactions, it is concluded that the reactions catalysed by hexokinase, fructokinase, phosphofructokinase and pyruvate kinase are far from equilbrium, whereas the other reactions including that catalysed by pyrophosphate-fructose-6-phosphate 1-phosphotransferase, are close to equilibrium in vivo.

Apparent equilibrium constant and mass-action ratio for sucrose-phosphate synthase in seeds of Pisum sativum

The aim of this work was to use preparations from germinating seeds of Pisum sativum to determine the apparent equilibrium constant of the reaction catalysed by sucrose-phosphate synthase (EC 2.4.1.14) and to compare this with the mass-action ratio of the reaction in the seeds. The apparent equilibrium constant ranged from 5.3 at 0.25 mM-MgCl2, pH 7.0, to 62 at 10 mM-MgCl2, pH 7.5. The sucrose phosphate content of the seeds, 23 nmol/g fresh wt., was determined by separating sucrose phosphate from sucrose by ion-exchange chromatography and then measuring the sucrose released by alkaline phosphatase. Comparison of equilibrium constants and mass-action ratios in the cotyledons of 38 h-germinated seeds showed that the reactions catalysed by glucose-6-phosphate isomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase are close to equilibrium, and those catalysed by sucrose-phosphate synthase and sucrose phosphatase are considerably displaced from equilibrium in vivo.

The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp,Crangon crangon

The mobilization of glycogen and phosphoarginine during work and their resynthesis during periods of recovery were investigated in abdominal muscles of the shrimpCrangon crangon. All parameters, metabolite levels as well as glycogen phosphorylase (EC 2.4.1.1) and synthase (EC 2.4.1.11) activities were determined in each individual shrimp investigated. At the onset of work both glycogen and phosphoarginine were degraded with the rate of phosphoarginine utilization being more than 80-fold faster than glycogen. After exhaustive work phosphoarginine stores were replenished within 30 min and seemed to exceed the resting level thereafter. In contrast, glycogen was not resynthesized immediately after work, but was further degraded during recovery leading to the accumulation of lactate. Only when the phosphagen level had reached the resting level did glycogenolysis shift to its resynthesis. The shift is characterized by: (1) a change in the mass action ratio of phosphoglucomutase from values below the equilibrium constant to values above the constant, (2) a dramatic decrease in the ratio fructose 1,6-bisphosphate/fructose 6-phosphate indicating phosphofructokinase inhibition, (3) an increase in the glucose concentration, and (4) an increase in the proportion of glycogen synthase I. The inactivation of glycogen phosphorylase by dephosphorylation during recovery was 2.4-fold. 36±8% (n=5) of total activity remained in the phosphorylated form. It is proposed that this part of the enzyme was inactivated by the drop in inorganic phosphate level due to the restoration of phosphoarginine.

Studies on the pigeon red blood cell metabolism

1. Pigeon erythrocyte was found to depend on the glycolytic and pentose phosphate pathway for most of its energy production in the form of adenosine triphosphate and reducing potential, since there was no detectable activity of any of the citric acid cycle (TCA) cycle enzymes measured. 2. The absence of detectable amounts of 2,3-diphosphoglyceric acid (2-3-DPG) indicated that there is no direct relationship between the active glycolytic system and the function of these cells. 3. A comparison of the mass action ratios with the equilibrium constants of the glycolytic reactions showed that hexokinase, phosphofructokinase and pyruvate kinase reactions are displaced from equilibrium, implying that these are the key regulatory enzymes of glycolysis in pigeon erythrocytes. 4. The changes in the concentrations of the glycolytic metabolites under hypoxic conditions that stimulate the flux through the glycolytic pathway were found to be consistent with the above hypothesis. 5. Flux measurements of the pentose phosphate pathway showed that it metabolizes only 3.4% of the total glucose consumed by the resting erythrocyte. 6. Hypoxic conditions resulted in a stimulation of the pentose phosphate pathway by as much as four-fold, whilst the glycolytic pathway was not stimulated by more than about twice.

Effect of acute administration of a large dose of ethanol on hepatic contents of adenine nucleotides and their degradation products in rats.

A single oral administration of ethanol (5g/kg) to fasted rats significantly increased the hepatic content of ATP and of total adenine nucleotides after a lag period of 1h. The ADP content gradually decreased, and the AMP content increased. There was no change in the energy charge. The phosphorylation state significantly increased at 3h and thereafter, and the adenylate kinase mass-action ratio was elevated during the intoxication. The IMP content significantly decreased at 3h and thereafter. A small but significant increase in the hypoxanthine content was noted only in the early phase during the intoxication. This early increase after the ethanol administration was not enhanced by the pretreatment with allopurinol, by which the hepatic activities of xanthine oxidase and xanthine dehydrogenase were inhibited by more than 90%. Xanthine was detected only in the liver extracts from rats pretreated with allopurinol, but there was no significant effect of the ethanol loading on the xanthine content.These results demonstrate that, during acute intoxication with a large dose of ethanol, the overall metabolism of adenine nucleotides shifts toward their accumulation rather than degradation, that the increase in hypoxanthine is rather small and transient, and that xanthine is produced from metabolites other than hypoxanthine.

Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.

1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.

The use of the creatine kinase reaction to determine free energy change of ATP hydrolysis in anoxic cardiomyocytes

In isolated cardiomyocytes from adult rat heart the free energy change of ATP hydrolysis (dG) was determined under conditions of substrate-free anoxia. Changes of free cytosolic ADP concentrations, needed for the calculation of dG, were determined by two indirect methods since a direct measurement is not feasible: (i) via the mass action ratio of the creatine kinase reaction (CK) assuming near equilibrium conditions, and (ii) via quantification of the net hydrolysis of ATP to ADP by a detailed balancing of possible contribution to Pi production. Both approaches gave virtually identical results, showing that in anoxia only 6% of the ATP hydrolysed are hydrolysed to ADP and 94% completely to adenosine and further degradation products. The convergence of both methods also indicates that in this model the CK reaction is indeed catalysed near its equilibrium. Therefore estimations of free ADP and dG using its mass action ratio are valid. In anoxic cardiomyocytes dG values fell from 57 kJ/mol in normoxia to 42 kJ/mol after 120 min anoxia, corresponding to a decrease of ATP contents from 24 to 4 nmol/mg protein.

Estimation of the mitochondrial redox state in human skeletal muscle during exercise

The mitochondrial redox (NAD+/NADH) state can be used as a reflection of oxygen availability within the mitochondrion. Previous studies using isolated muscle preparations suggest that active muscle is not hypoxic during lactate production, whereas experiments with humans come to the opposite conclusion. Six men exercised for 5 min at 75% maximal O2 consumption (VO2max) and then at 100% VO2max to exhaustion. Ammonia, oxoglutarate (alpha-ketoglutarate), and glutamate, as well as lactate, were measured in biopsies (vastus lateralis) taken at the end of each exercise. The three former metabolites were used to determine the mass action ratio of glutamate dehydrogenase and thus were used as an estimate of the mitochondrial redox state. Muscle lactate increased (P less than 0.05) to 14.5 and 24.5 mmol/kg wet wt after 75 and 100% VO2max, respectively. At both exercise intensities, muscle ammonia rose (P less than 0.05), glutamate fell (P less than 0.05) to only 30-35% of rest levels, and oxoglutarate declined (P less than 0.05). Despite the high levels of muscle lactate accumulation, the estimated mitochondrial redox rate rose 300% (P less than 0.05) in both exercise bouts. This response should increase the activity of key oxidative enzymes and promote increased VO2. Furthermore the data do not support the concept that muscle lactate is formed because of tissue hypoxia.

Pyruvate carboxylation in the rat heart. Role of biotin-dependent enzymes.

Pyruvate carboxylation in the isolated perfused rat heart was studied under steady-state conditions. A biotin deficiency resulting in a 90% decrease in myocardial pyruvate carboxylase left the pyruvate carboxylation rate unchanged. Pyruvate carboxylation in heart muscle must therefore take place by means of an enzyme which does not contain biotin. The kinetic properties and mass-action ratio of the NADP-linked malic enzyme in heart muscle can be taken as circumstantial evidence in favour of the role of malic enzyme in pyruvate carboxylation in myocardium.

Regulatory steps of glycolysis during environmental anoxia and muscular work in the cockle,Cardium tuberculatum: control of low and high glycolytic flux

Concentrations of glycolytic intermediates, end products of anaerobic metabolism and the adenylates have been determined in the foot muscle and in the whole soft body tissue of the cockle,Cardium tuberculatum, after anoxic incubation and after the performance of vigorous escape movements. Comparison of the mass action ratios (MAR) with the equilibrium constants (Keq) showed that the reactions catalyzed by glycogen phosphorylase, hexokinase, phosphofructokinase (PFK) and pyruvate kinase (PK) were displaced from equilibrium under all physiological situations investigated.

Control of glycogenosis in rainbow trout muscle during exercise

The purpose of this study was to measure and compare the in vivo levels of glycolytic pathway intermediates during high-intensity, progressive exercise in rainbow trout red and white muscle. The mass action ratios of those reactions catalyzed by the enzymes hexokinase, phosphofructokinase, and pyruvate kinase appear to be displaced far from thermodynamic equilibrium (greater than log 2) under all experimental conditions in both tissue types. Furthermore, the near-equilibrium glyceral-dehyde 3-phosphate dehydrogenase – phosphoglycerate kinase complex deviated from thermodynamic equilibrium during the exhaustive swim for white muscle and for all exercise intensities in red muscle. The mass action ratio of the combined glyceraldehyde 3-phosphate dehydrogenase – phosphoglycerate kinase/lactate dehydrogenase demonstrated displacements from thermodynamic equilibrium similar to those of the glyceraldehyde 3-phosphate dehydrogenase complex. Potential control sites were identified as phosphorylase for white muscle and hexokinase and glyceraldehyde 3-phosphate dehydrogenase – phosphoglycerate kinase for red muscle. No alterations in potential control sites were found in white muscle with changing flux rates (sustained and burst swims) provided sufficient glycogen was available as precursor. Phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase only demonstrated potential control in this tissue when glycogen content and flux rates were low (exhaustive swim). These changes coincided with substrate depletion to low levels and large declines in the ATP/ADPf ratio. Therefore, it would appear that glycogen content is the primary factor regulating glycolytic flux and control sites via alterations in adenylate levels.

Carbohydrate metabolism in Onchocerca gutturosa and Onchocerca lienalis (Nematoda:Filarioidea)

Dunn T. S., Raines P. S., Barrett J. and Butterworth P. E. 1988. Carbohydrate metabolism in Onchocerca gutturosa and Onchocerca lienalis (Nematoda:Filarioidea). International Journal for Parasitology 18: 21–26. Adult O. gutturosa and O. lienalis have a complete sequence of glycolytic and tricarboxylic acid cycle enzymes. The relative activities of the glycolytic enzymes are similar to those found in other glycolytic tissues. A comparison of the mass action ratios of the glycolytic enzymes with their apparent equilibrium constants indicates that phosphorylase, hexokinase, phosphofructokinase and pyruvate kinase are non-equilibrium regulatory enzymes. There is also some evidence that aldolase may be pseudo-regulatory under conditions of high flux. Both O. gutturosa and O. lienalis have measureable amounts of fumarate reductase activity as well as phosphoenolpyruvate carboxykinase and NADP-linked malic enzyme. The two filariids appear to be essentially homolactic fermentors, the only other end-product being traces of alanine and occasionally succinate. The rate of lactate production was unaffected by the presence or absence of oxygen or by the presence or absence of exogenous glucose. Similarly, oxygen uptake by the two worms was unaffected by the presence or absence of exogenous glucose.

Effect of acute ethanol on serine biosynthesis in liver

The effect of an acute intraperitoneal dose of ethanol (1 g/kg), glucose (7.2 g/kg), or the combination of the two on the metabolite pattern of the biosynthetic pathway of l-serine has been determined in rabbit liver in vivo as has the effect of 10 m m ethanol on the glucose-, fructose-, or pyruvate-stimulated accumulation of l-serine in rabbit hepatocytes in vitro. In vivo, the 50% increase in l-serine and 80% increase in l-phosphoserine content of liver following glucose injection was completely prevented by ethanol. In fact, the l-phosphoserine content fell to only 6% of the control value. In spite of these and other significant changes in the metabolite pattern of the pathway of l-serine biosynthesis ( d-3-phosphoglycerate dehydrogenase, l-phosphoserine aminotransferase (PSAT), and l-phosphoserine phosphatase), the mass action ratio of the combined reactions of the first two steps remained close to their equilibrium position. As a consequence it is estimated that the tissue content of phosphohydroxypyruvate fell to less than 2% of the control value, to approximately 0.3% of its K m for the PSAT reaction. The conclusion that acute ethanol blocks l-serine biosynthesis (presumably by redox effects) was supported by the prevention or inhibition of l-serine accumulation in hepatocytes metabolizing glucose, fructose, or pyruvate. Because l-serine is an important source of one-carbon fragments, the inhibition of its biosynthesis may be another mechanism by which ethanol interferes with folate and one-carbon metabolism.

The photosynthetic induction response in wheat leaves: net CO2 uptake, enzyme activation, and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO2 fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.

Role of NADP+ (corrected)-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart.

Cytosolic and mitochondrial concentrations of malate, 2-oxoglutarate, isocitrate and pyruvate in the isolated perfused rat heart were measured by non-aqueous tissue fractionation, taking the NADP-linked isocitrate dehydrogenase as indicator reactions for the free [NADPH]/[NADP+] ratios. The mass-action ratios of NADP-linked malic enzymes (EC 1.1.1.40) were found to be on the side of pyruvate carboxylation by more than one order of magnitude in both the cytosolic and the mitochondrial spaces in hearts perfused with glucose, whereas during propionate perfusion this ratio approached the equilibrium constant (Keq.) of malic enzyme. The results consequently indicate that the NADP-linked malic enzymes cannot be responsible for the feed-out (cataplerotic) reactions from the tricarboxylic acid cycle which occur during glucose perfusion. Only when other anaplerotic fluxes into the cycle are high, as during propionate oxidation, which results in accumulation of tricarboxylic acid-cycle intermediates, is a steady state reached which allows efflux via the malic enzyme.