Concentrations Of Hexose Phosphates Research Articles

Mitochondrial Oxidative Phosphorylation Is Regulated by Fructose 1,6-Bisphosphate: A POSSIBLE ROLE IN CRABTREE EFFECT INDUCTION?

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

Mechanism of Up-regulation of Starch Synthesis in Mature Leaves of Transgenic Apple Trees with Decreased Sorbitol Synthesis

The reaction catalyzed by ADP-glucose pyrophosphorylase (AGPase) to form ADP-glucose is a regulatory and rate-limiting step in starch synthesis in plants. In response to decreased sorbitol synthesis, starch synthesis was up-regulated in the transgenic apple plants. In this study, we examined both redox and metabolite regulation of AGPase to understand the mechanism responsible for the up-regulation of starch synthesis. No difference in the monomerization/dimerization of apple leaf AGPase small subunits was observed between the transgenic plants and the untransformed control. NADP-dependent malate dehydrogenase, indicative of chloroplastic redox status, did not show significant change in the transgenic plants either. Determination of key metabolites with nonaqueous fractionation indicated that concentrations of hexose phosphates (mainly glucose-6-phosphate and fructose-6-phosphate) were higher in both the cytosol and chloroplasts of the transgenic plants than in the control, whereas 3-phosphoglycerate (PGA) concentration in the chloroplast was not higher in the transgenic plants. We conclude that accumulation of hexose-phosphates results in a decrease in inorganic phosphate (Pi) concentration and an increase in PGA/Pi ratio in the chloroplast, leading to up-regulation of starch synthesis via activating AGPase.

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.

Reduced inositol content and altered morphology in transgenic potato plants inhibited for 1D‐myo‐inositol 3‐phosphate synthase

Summary Myo ‐inositol is a precursor of many plant metabolites, including polyols, cell wall components and phosphoinositides. The first committed step in the de novo myo ‐inositol synthetic pathway is catalysed by the enzyme 1D‐ myo ‐inositol 3‐phosphate synthase (MIPS; EC 5.5.1.4 ), which converts D‐glucose 6‐phosphate to 1D‐ myo ‐inositol 3‐phosphate. Suppression of MIPS activity by an antisense RNA approach in transgenic potato ( Solanum tuberosum L.) plants to below 20% of the wild‐type level in leaves resulted in strongly reduced levels of inositol, galactinol and raffinose (approximately 7%, 5% and 12%, respectively, of wild‐type values). In contrast, increases were observed for concentrations of hexose phosphates (up to 1.7‐fold), sucrose (twofold) and starch (two‐ to fourfold). Transgenic plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence and a decrease in overall tuber yield. These observations indicate a crucial role for myo ‐inositol in plant physiology and development.

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers.

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa 1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571-581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa 1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa 1 tubers. Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20-30% in ppa 1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa 1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to three-fold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30-100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa 1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider-significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed.

Organ Metabolism and Cryoprotectant Synthesis during Freezing in Spring Peepers Pseudacris crucifer

The effects of freezing on organ metabolism were monitored over a 36-h time period at -4 C in spring-collected specimens of a freeze-tolerant frog, Pseudacris crucifer. Within 2 min after nucleation, glycogenolysis in liver was activated as indicated by elevated levels of hexose phosphates; and levels of the cryoprotectant, glucose, rose quickly thereafter. Freezing stimulated a 3.3-fold increase in liver glycogen phosphorylase a activity, and a rapid phase of liver glucose production was maintained for at least the first hour of freezing (21 imol ? g wet mass-' h-1), followed by a steady rate of glucose increase of about 3 pmol ? gwm-1 h-1. Final liver glucose levels reached 141 pmol gwm-1 after 36 h. Changes in the concentrations of hexose phosphates and fructose-1,6-bisphosphate in liver indicated that glucose production was promoted by an inhibitory block on glycolysis at the phosphofructokinase reaction. Other organs accumulated the glucose exported by liver and cryoprotectant asccumulation by heart paralleled the rise in liver glucose levels. Liver energetics were not disrupted during early freezing but with prolonged freezing (4-36 h), ATP content fell, ADP and AMP increased, and energy charge, [ATP + ADP/2]/[ATP + ADP + AMP], was reduced from 0.90 in controls to 0.57 after 36 h freezing. Liver metabolism during freezing was supported by fermentative reactions with anaerobic glycolysis leading to lactate and alanine accllmnlation; concomitantly, levels of fermentable-free amino acids (aspartate and glutamate) decreased. Changes in free amino acid patterns in skeletal muscle and heart also indicated a reliance on amino acid fermentation during freezing, but additional increases in the levels of several other amino acids suggested that freezing might also elevate proteolysis. The results show a role for amino acids in anuran freezing survival and demonstrate that the biochemical mechanisms involved in con-

Effects of Exposure of Wheat Ears to High Temperature on Dry Matter Accumulation and Carbohydrate Metabolism in the Grain of Two Cultivars. II. Carry-Over Effects

Yield of wheat exposed in the field during the grain-filling stage to brief episodes of high (but sub- lethal) temperature is reduced as a consequence of lower single grain weight due mainly to diminished accumulation of starch. To simulate these effects, ears of wheat were enclosed in a transparent box midway through the grain filling stage and heated (35�C day/25�C night) above ambient conditions (21�C day/16�C night) for periods of up to 7 days. Afterwards the ears were returned to ambient conditions. Two cultivars known to differ in their tolerance of high temperature were compared. Final single grain dry weight of the more tolerant cultivar (Kalyansona) was not significantly affected by exposure to heat, but it was reduced in cv. Sun 9E by approximately 15.5% after 7 days of exposure. Rate of grain filling in the cool conditions following previous exposure to high temperature was also substantially slower in both cultivars than it was in ears that had not been heated. Following transfer from hot to cool conditions, the amounts of fructose and the concentration of UDP-glucose returned to control levels. On the other hand, quantities of sucrose in the grain of ears that had been heated rose higher than in ears that had been kept continuously in cooler conditions. Thus any effects of high temperature at the sucrose synthase step appeared to be readily reversible, and both cultivars responded similarly. Cultivar-dependent responses were observed however in effects on the concentrations of hexose phosphates and ADP-glucose. After only one hot day the concentration of G-6-P, F-6-P and G-1-P returned to control levels following transfer to the cooler environment. After 2 or more days the concentrations of these metabolites did not recover in cv. Sun 9E but recovery was observed in cv. Kalyansona after 2 or 4 days' exposure, but not after 7 hot days. Last, concentrations of ADP-glucose in the cultivar of which starch deposition was reduced most at high temperature (Sun 9E) rose more after transfer from high to low temperature than was the case in the more tolerant cultivar (Kalyansona), and ADP-glucose concentrations fell more in the latter than in the former cultivar as a consequence of 7 days at high temperature. It is suggested that more than one action of temperature could be involved in the cultivar-dependent responses to high temperature: effects on starch synthase activity as well as on the metabolism and possibly transport of hexose phosphates are possibilities in accordance with the information available.

Metabolite concentrations in Alcaligenes eutrophus H 16 and a mutant defective in poly-?-hydroxybutyrate synthesis

Intracellular concentrations of hexose phosphates, phosphoenolpyruvate, pyruvate, NAD(H) and NADP(H) as well as the protein and poly-β-hydroxybutyrate (PHB) content were measured in suspensions of autotrophically grown cells of Alcaligenes eutrophus H 16 and compared with those in a mutant unable to synthesize poly-β-hydroxybutyrate. The parent strain was subjected to successive changes in conditions, and new steady states were rapidly (≃20 min) attained. When the parent strain was provided with carbon and energy but no nitrogen source, it fixed CO2 and accumulated large amounts of PHB. When the mutant PHB-4 was exposed to identical conditions, no accumulation of PHB occurred, but pyruvate, malate and citrate were excreted, and a 6-fold accumulation of hexose monophosphate (over the levels in the parent) was observed: in contrast, cofactors in intermediates between fructose-1,6-phosphate and phosphoenolpyruvate reached steady state as in the parent strain. When ammonium ion was then supplied, growth started and the metabolite concentrations in the mutant returned to the levels observed in the parent strain.

The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart.

1. The work of the perfused rat heart was acutely increased by raising the aortic pressure in the Langendorff preparation from 50 to 120mmHg; within 1 min in perfusions with media containing glucose or glucose+acetate, rates of oxygen consumption and tricarboxylate-cycle turnover increased 2.5-fold, glycolysis rate doubled and oxidation of triglyceride fatty acid was strikingly enhanced. 2. Increased cardiac work had no significant effects on the heart concentrations of creatine phosphate, ATP, ADP or 5'-AMP. The only significant changes in tricarboxylate-cycle intermediates were a decrease in malate in perfusions with glucose and decreases in acetyl-CoA and citrate and an increase in aspartate in perfusions with glucose+acetate. 3. Measurements of intracellular concentrations of hexose phosphates, glucose and glycogen indicated that work accelerated glycolysis by activation of phosphofructokinase and subsequently hexokinase; the activation could not be accounted for by changes in the known effectors of phosphofructokinase. 4. Acetate at either perfusion pressure increased heart concentrations of acetyl-CoA, citrate, glutamate and malate and decreased that of aspartate; acetate increased tricarboxylate-cycle turnover by 50-60% and inhibited glycolysis and pyruvate oxidation. 5. In view of the markedly different effects of acetate and of cardiac work on the concentrations of cycle intermediates the changes that accompany acetate utilization may be specifically concerned with the regulatory functions of the cycle in control of glycolysis and pyruvate oxidation and not with the associated increase in cycle turnover. It is suggested that the concentrations of key metabolites controlling the rate of cycle turnover may fluctuate with each heart beat and that this may explain why no significant changes (for example, in adenine nucleotide concentrations) have been detected with increased work in the present study.

Regulation at the Phosphoenolpyruvate Branchpoint in Azotobacter vinelandii: Pyruvate Kinase

Pyruvate kinase (EC 2.7.1.40) from Azotobacter vinelandii responds sharply to the adenylate energy charge, with a decrease in activity at high values of charge, as expected for an enzyme of an adenosine triphosphate-regenerating sequence. Glycolytic intermediates, especially glucose 6-phosphate, fructose 6-phosphate, and fructose-1,6-diphosphate, strongly stimulate the reaction and overcome the inhibition caused by high values of energy charge. Thus, the properties of this enzyme depend on interaction between energy charge and the concentrations of hexose phosphates. The properties of pyruvate kinase, together with those of phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase, seem adapted to provide appropriate partitioning of phosphoenolpyruvate between competing pathways in response to metabolic need.

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions.

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.

Metabolic actions in intestinal smooth muscle associated with relaxation mediated by adrenergic alpha- and beta-receptors.

AbstractIn colonic muscle from the rabbit a relaxing effect was induced by stimulation both of adrenergic α‐receptors with phenylephrine and of β‐receptors with isoprenaline. The α‐receptor induced relaxation was not accompanied initially by any metabolic effects. After a latency period of 3 min did a decrease in the phosphorylase α activity, a decrease in the concentrations of hexose phosphates and lactate and an increase in the concentration of high‐energy phosphate compounds (ATP and CrP) become evident. There was a decrease of the cyclic AMP content. These effects were inhibited by dibenamine. The relaxation induced via adrenergic β‐receptors was preceded by an increase in the cyclic AMP content and the phosphorylase α activity, an increase in the concentration of hexose phosphates and lactate and an initial decrease of the ATP and CrP concentrations, which after 10 min was succeeded by an increase. The magnitudes of the relaxing and metabolic responses following stimulation of preceptors were correlated with regard to time and dose. These responses were blocked by an adrenergic β‐receptor blocking agent (sotalol) and were also eliminated in carbohydrate‐poor muscle. The relaxing and metabolic effects of isoprenaline could be reproduced completely by cyclic AMP, only incompletely by 5'AMP and not at all by other tested cyclic nucleotides.

Regulation of glycolysis and L-glycerol 3-phosphate concentration in rat epididymal adipose tissue in vitro. Role of phosphofructokinase.

1. Attempts were made to define the role of phosphofructokinase in glycolytic control and the factors regulating the concentration of l-glycerol 3-phosphate in rat epididymal fat pads incubated in vitro. 2. Glycolysis rates were altered by anoxia or by additions of insulin, adrenaline or both to the incubation medium, and the changes in rate were related to changes in the steady-state concentrations of hexose phosphates, adenine nucleotides, l-glycerol 3-phosphate and citrate in the whole tissue. Measurements were also made of the lactate/pyruvate concentration ratio in the medium after incubation. 3. The mass-action ratios of phosphofructokinase, calculated from the whole-tissue concentrations of products and substrates, were less than 0.1% of the value of the ratio at pH7.4 at equilibrium. 4. Only in the presence of adrenaline could the observed stimulation of glycolytic flux be related to a possible activation of phosphofructokinase since, in this situation, the concentration of one substrate, fructose 6-phosphate, was not altered and the concentration of the other, ATP, was decreased. Increased glycolytic flux in the presence of insulin may be explained by an observed increase in the concentration of the substrate, fructose 6-phosphate. Under anaerobic conditions, glycolytic flux was decreased but this did not appear to be the result of inhibition of phosphofructokinase, since the concentrations of both substrates, fructose 6-phosphate and ATP, were decreased. The changes in glycolytic flux with insulin and anoxia may be secondary to changes in the rate of glucose uptake. 5. Changes in l-glycerol 3-phosphate concentration appear to be related both to changes in the concentration of dihydroxyacetone phosphate and to changes in the NADH/NAD(+) concentration ratio in the cytoplasm. They do not seem to be related directly to alterations in glycolytic rate.

Regulation of glucose uptake by muscle. 7. Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes, starvation, hypophysectomy and adrenalectomy, on the concentrations of hexose phosphates, nucleotides and inorganic phosphate in perfused rat heart.

Regulation of glucose uptake by muscle. 7. Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes, starvation, hypophysectomy and adrenalectomy, on the concentrations of hexose phosphates, nucleotides and inorganic phosphate in perfused rat heart.

CITRATE AS AN INTERMEDIARY IN THE INHIBITION OF PHOSPHOFRUCTOKINASE IN RAT HEART MUSCLE BY FATTY ACIDS, KETONE BODIES, PYRUVATE, DIABETES, AND STARVATION.

IN perfused rat heart, glycolysis is accelerated by anoxia and uncouplers of respiratory chain phosphorylation and inhibited by the respiration of fatty acids, ketone bodies and pyruvate1–4. Measurements of the concentrations of hexose phosphates have led to the suggestion that these agents change the rate of glycolysis by altering the rate of the phosphofructokinase step1–3. The rates of glycolysis and of the phosphofructokinase reaction are also diminished in hearts from alloxan diabetic or starved rats in which the release of fatty acids from glycerides for oxidation is enhanced4,5. In extracts of guinea pig heart6 and of rabbit skeletal muscle7 the activity of phosphofructokinase is inhibited by increasing concentrations of ATP and stimulated by increasing concentrations of 5′ AMP, cyclic 3′,5′ AMP, ADP and inorganic phosphate. These observations suggest that anoxia and uncouplers of respiratory chain phosphorylation increase the rate of the phosphofructokinase reaction in muscle (and hence of glycolysis) by increasing the concentrations of 5′ AMP, ADP and inorganic phosphate and diminishing that of ATP.

Regulation of glucose uptake by muscle. 5. Effects of anoxia, insulin, adrenaline and prolonged starving on concentrations of hexose phosphates in isolated rat diaphragm and perfused isolated rat heart.

Membranetransport ofglucose andintracellular phosphorylation ofthesugar are bothaccelerated by anoxiain theperfused isolated ratheart (Morgan, Randle& Regen,1959). Inisolated rat diaphragm, membranetransport ofglucose isaccelerated byanoxia, buttheeffect ofanoxiaon glucose phosphorylation inthistissue isnotknown (Randle & Smith,1958a, b).Crane& Sols(1953) haveshownthatmusclehexokinase isinhibited by glucose 6-phosphate andthisobservation suggested thatanoxiamightaccelerate glucose phosphorylation inmusclebylowering theintracellular concentration ofglucose 6-phosphate andthereby increasing theactivity ofhexokinase. Theresults of experiments recorded hereappear toshowthat anoxia can lowertheintracellular concentration of glucose 6-phosphate inheartanddiaphragm, and further measurements havebeenmadeoftheconcentrations offructose 6-phosphate and fructose 1:6-diphosphate inan attempt todefine theenzymic steporsteps whichareaffected byanoxiaandwhich bringaboutthischangeintheconcentration of glucose 6-phosphate. The possibility thatmembrane transport of monosaccharides inmusclemightberegulated by theintracellular concentration ofglucose 6-phosphatehasalsobeeninvestigated bystudying the effects ofagents whichaltertheintracellular concentration ofglucose 6-phosphate, on themembrane transport ofD-xylose andD-3-0-methylglucose in

Glycolysis and respiration in Ehrlich ascites tumour cells. II. Effect of IAA and DNP.

Summary.1. When glycolysis in Ehrlich ascites tumour cells was limited by different concentrations of either IAA, phosphate or glucose, no direct correlation was revealed between the Crabtree effect and the glycolytic rate, or the concentrations of hexosephosphates and acid labile phosphate esters which accumulated during aerobic glycolysis.2. In DNP inhibited cells, incubated with 0—20 μmoles of inorganic phosphate per ml, a competition existed between glycolysis and respiration for the inorganic phosphate. In the presence of DNP, excess phosphate in the medium completely abolished the Crabtree effect.3. Evidence has been presented showing that the rate of glucose uptake in the ascites cells is determined by the inorganic phosphate in the medium taken up through the glyceraldehyde‐3‐phosphate dehydrogenase reaction. This is explained by assuming that glycolytically generated ATP determines the rate of the hexokinase reaction.