Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision

The dark respiration rate of discs from fully expanded tobacco leaves (Nicotiana tabacum) increased linearly with decreasing diameter, the relative increase being independent of leaf age. The wound respiration responsible for this situation reached a plateau within 15 minutes of excision. Metabolite analysis gave evidence for two independent effects, also unrelated to age. The first was a forward crossover between phosphoenolpyruvate and pyruvate which was found as early as 1 minute after excision and persisted for up to 40 minutes. It was attributed to activation of pyruvate kinase by a changed ionic balance resulting from membrane damage, was accompanied by a reverse crossover between triose phosphates and 3-phosphoglycerate, and was localized in the outer region of the discs. The second effect was a rapid rise in hexose monophosphate and ATP levels throughout the discs. After 1 to 10 minutes the ATP/ADP ratio rose strongly for at least 3 hours; after 20 to 40 minutes there was net synthesis of adenine nucleotide as ATP. These results indicate that extrapolation from leaf discs to intact leaves is highly inadvisable.     

Effect of experimental diabetes on glycolytic intermediates and regulation of phosphofructokinase in rat lens

The pattern of glycolytic intermediates in the lens of alloxan-diabetic rats was indicative of regulation at phosphofructokinase. The changes in metabolites influencing phosphofructokinase activity in the diabetic, relative to the normal, rat lens were: glucose 6-phosphate, 182%; fructose 6-phosphate, 107%; fructose diphosphate, 57%. There was also a marked decrease in phosphoenolpyruvate, pyruvate, lactate and ATP but no significant change in other triose phosphates or cyclic AMP. The resuts are considered in relation to the early changes in [Ca²⁺] known to occur in lens in diabetes and to the coordinating effect of fructose diphosphate on flux through the glycolytic route.     

Post-mortem glycolysis in ox skeletal muscle. Effect of pre-rigor freezing and thawing on the intermediary metabolism

1. Ox sternomandibularis muscle was ;slow-frozen' by placing it in air at -22 degrees or ;fast-frozen' by immersion in liquid air or acetone-solid carbon dioxide. In all cases muscles were frozen pre-rigor. Changes in length, pH and the concentrations of P(i), creatine phosphate, hexose monophosphate (glucose 1-phosphate+glucose 6-phosphate+fructose 6-phosphate), fructose diphosphate (fructose 1,6-diphosphate+(1/2) triose phosphate), lactate, ATP, ADP, AMP and NAD(+) during freezing and during subsequent thawing were determined. In addition some measurements were made of the changes in alpha-glycerophosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate concentrations during slow freezing. 2. Appreciable shortening and marked changes in chemical composition took place during slow freezing but not during fast freezing. 3. During slow freezing the hexose monophosphate concentration fell and fructose 1,6-diphosphate and triose phosphate increased substantially. Increases also took place in 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate, but not in pyruvate. 4. On thawing, most of the chemical changes were similar to those in unfrozen muscle post mortem, but took place much more rapidly; loss of NAD(+) was particularly rapid. Fast-frozen muscle metabolized at a faster rate on thawing than did slow-frozen muscle. 5. The overall changes in length during freezing and thawing were about the same in slow-frozen as in fast-frozen muscle.     

Reversal of glycolysis in yeast.

When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.     

Metabolic control of hepatic gluconeogenesis during exercise.

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.     

CONTROL OF AEROBIC GLYCOLYSIS IN GUINEA-PIG CEREBRAL CORTEX SLICES

—The effect of glutamate on aerobic glycolysis in guinea-pig cerebral cortex slices was analysed in comparison with that of high-potassium. In contrast to the increased glycolysis in 50 mm -potassium medium which was accompanied by increases of fructose diphosphate and triose phosphates in the slices, the addition of 5 mm -d -glutamate to the medium increased the rate of glycolysis without increasing these intermediates. When increasing the concentration of potassium in the medium up to 20 mm , the rate of aerobic glycolysis was not increased although fructose diphosphate and triose phosphates in the slices were increased. At this potassium concentration in the medium ATP in the slices was highest. At 30 mm -potassium the rate of glycolysis was increased significantly, but fructose diphosphate and triose phosphates were decreased. ATP was lower at 30 mm - than at 20 mm -potassium. By increasing potassium to 40 mm and above, the rate of glycolysis was further increased, and fructose diphosphate and triose phosphates were again increased. Between 5 and 20 mm -potassium in the medium the increasing effect of glutamate on glycolysis was very pronounced. d -Glutamate decreased the amounts of ATP, fructose diphosphate and triose phosphates at any concentration of potassium in the medium. When adding cyclic AMP and 5′AMP to the slices, fructose diphosphate and triose phosphates were increased, but the rate of glycolysis was not increased. On the basis of these observations mechanisms of the control over glycolysis in guinea-pig cerebral cortex slices are discussed. It is suggested that the glycolysis is controlled by the changes in ATP concentration through their action on the glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase system. The changed patterns of the glycolytic intermediate profile in the slices when adding ATP to the medium are consistent with this suggestion. The addition of l -phenylalanine to guinea-pig cerebral cortex slices did not inhibit the rate of glycolysis, although it inhibited the activity of pyruvate kinase.     

Enzymatic analysis of the pathways of glucose catabolism and gluconeogenesis in Pseudomonas citronellolis

Extracts of Pseudomonas citronellolis cells grown on glucose or gluconate possessed all the enzymes of the Entner-Doudoroff pathway. Gluconokinase and either or both 6-phosphogluconate dehydratase and KDPG aldolase were induced by growth on these substrates. Glucose and gluconate dehydrogenases and 6-phosphofructokinase were not detected. Thus catabolism of glucose proceeds via an inducible Entner-Doudoroff pathway. Metabolism of glyceraldehyde 3-phosphate apparently proceeded via glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. These same enzymes plus triose phosphate isomerase were present in lactate-grown cells indicating that synthesis of triose phosphates from gluconeogenic substrates also occurs via this pathway. Extracts of lactate grown-cells possessed fructose diphosphatase and phosphohexoisomerase but apparently lacked fructose diphosphate aldolase thus indicating either the presence of an aldolase with unusual properties or requirements or an alternative pathway for the conversion of triose phosphate to fructose disphosphate. Cells contained two species of glyceraldehyde 3-phosphate dehydrogenase, one an NAD-dependent enzyme which predominated when the organism was grown on glycolytic substrates and the other, an NADP-dependent enzyme which predominated when the organism was grown on gluconeogenic substrates.     

Glycolysis at the climacteric of bananas.

This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and PPi; and the maximum catalytic activities of pyrophosphate-fructose-6-phosphate 1-phosphotransferase, 6-phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12-day period that included the climacteric. Cytosolic fructose-1,6- bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6-phosphate and glucose 6-phosphate, and a rise in fructose 1,6-bisphosphate. No change in pyrophosphate-fructose-6-phosphate 1-phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6-phosphate into glycolysis at the climacteric.     

In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis.

Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates.Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

CONTROL OF AEROBIC GLYCOLYSIS AND PYRUVATE KINASE ACTIVITY IN CEREBRAL CORTEX SLICES

Abstract— —The concentrations of glycolytic intermediates were measured in aerobically incubated guinea pig cerebral cortex slices. Increasing the concentration of potassium in the medium increased fructose diphosphate ten-fold and triose phosphates three-fold. Omitting calcium increased all the glycolytic intermediates except pyruvate; triose phosphates were increased most. The changed patterns of the glycolytic intermediate profile in the slices are consistent with the previously proposed hypothesis that the phosphofructokinase is a main regulatory step in glycolysis. Glycolysis is also limited at the step of pyruvate kinase, which is inhibited in cerebral cortex slices. Calcium in the tissue and cellular organization of the slices were shown to be responsible for this inhibition. It was concluded that the effects of potassium and calcium on aerobic glycolysis in cerebral cortex slices are direct—on the pyruvate kinase—and also indirect. Calcium was shown to be inhibitive also to the activities of hexokinase, phosphoglucoisomerase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase and enolase of guinea pig cerebral tissue.     

The effects of adenine nucleotides on carbohydrate metabolism in pigeon-liver homogenates

1. The effects of added AMP on carbohydrate metabolism were investigated in pigeon-liver homogenates, which can degrade glucose and synthesize it from lactate. Suitable experimental conditions were established for studying such effects, including the addition of P(i) (20mm) to stabilize adenine nucleotides and supplementation with NAD(+) (0.5mm). 2. Lactate increased the rate of oxygen consumption and kept the concentration of ATP high and that of AMP relatively low. 3. Added AMP (1.25-5mm) raised the net rate of carbohydrate removal and inhibited the net formation of glucose from lactate, as well as the incorporation of lactate into glucose. These effects were accompanied by a fall in the concentrations of hexose 6-phosphates and a rise in those of fructose diphosphate and triose phosphates. When the activity of glyceraldehyde 3-phosphate dehydrogenase was limited experimentally by a low concentration of NAD(+) or when it was blocked by iodoacetate, the accumulations of fructose diphosphate and triose phosphates were large and accounted for most of the carbohydrate degraded in the presence of AMP. 4. AMP also inhibited the conversion of pyruvate into phosphoenolpyruvate. Data on the concentrations of pyruvate, phosphoenolpyruvate and intermediates of the tricarboxylic acid cycle, as well as on isotope distribution, suggest that the effect was due to inhibition of phosphoenolpyruvate carboxykinase. 5. The results indicate that in the homogenates phosphofructokinase and fructose diphosphatase, controlled in their activity by adenine nucleotides and other cell constituents, are enzymes which regulate the direction of carbohydrate metabolism (degradation or synthesis) in the liver. 6. It is suggested that active transport of adenine nucleotides, citrate, Mg(2+), Ca(2+), P(i) and other cell constituents may play a role in regulating the activity of enzymes which are affected by these substances. 7. A procedure is described for generating alkali in a closed manometer vessel, by mixing mercuric oxide and a solution of sodium iodide, for use in a method for measuring the oxygen consumption at physiological bicarbonate concentrations.     

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

Regulation of anaerobic glycolysis in Ehrlich ascites tumour cells.

1) In intact Ehrlich ascites tumour cells the anaerobic glycolytic flux rate and pattern of intermediates have been investigated at different pH values of the extracellular medium. 2) As predicted from the dependence of the lactic acid dehydrogenase equilibrium on pH a strong negative correlation between log ([lactate]/[pyruvate]) and pH has been found. 3) The steady state fluxes of glycolysis at pH 8.0 and 7.4 are rather equal, despite significant differences in the intracellular concentrations of glycolytic intermediates. At pH 8.0 the concentrations of ATP, glucose 6-phosphate, and fructose 6-phosphate are lower, and the concentrations of ADP, AMP, fructose 1,6-bisphosphate, triose phosphates, phosphoglycerates, and phosphoenolpyruvate are higher than at pH 7.4. 4) From the analysis of the pH dependent changes of metabolites it follows that different mechanisms are responsible for maintaining equal actual activities of hexokinase, phosphofructokinase and pyruvate kinase at pH 7.4 and 8.0. 5) From an application of the linear theory of enzymatic chains and a calculation of the control strength of the regulatory important enzymes results that hexokinase is evidently rate-limiting for glycolysis, and phosphofructokinase is also significantly influencing the glycolytic flux. Pyruvate kinase and glyceraldehyde phosphate dehydrogenase, on the other hand, do not significantly affect the rate of the overall glycolytic flux in ascites.     

Some relationships between contents of photosynthetic intermediates and the rate of photosynthetic carbon assimilation in leaves of Zea mays L.

The relationship between the gas-exchange characteristics of attached leaves of Zea mays L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of CO₂ and at different irradiances at a constant intercellular partial pressure of CO₂. (i) The behaviour of the pools of the C₄-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO₂. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3-phosphate exists under many different flux conditions, consistent with the notion that the pools of C₄ and C₃ cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO₂ and with irradiance are compared with results obtained in C₃ leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C₄ plants may differ from that in C₃ plants. (iv) The behaviour of the ribulose-1,5-bisphosphate pool with CO₂ and irradiance is contrasted with the behaviour of these pools measured in leaves of C₃ plants.Abbreviations P _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - PEP phosphoenolpyruvate - triose-P triose phosphates - PGA glycerate-3-phosphate     

Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados

A continuous exposure of intact avocados (Persea americana) to 400 μl/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3-phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase.     

The control properties of phosphofructokinase in relation to the respiratory climacteric in banana fruit

Glucose 6-phosphate, fructose 6-phosphate, fructose 1, 6-diphosphate, and triose phosphates, and the enzymes phosphofructokinase, aldolase, and glucose 6-phosphate dehydrogenase were extracted from banana fruit (Musa cavendishii, Lambert var. Valery) at the (a) preclimacteric, (b) climacteric rise, (c) climacteric peak, and (d) postclimacteric stages of ripening. The level of fructose 1, 6-diphosphate increased 20-fold whereas the concentration of other intermediates changed no more than 2.5-fold between stages a and c. For these same extracts, phosphofructokinase activity increased 2.5-fold whereas the activity of glucose 6-phosphate dehydrogenase and aldolase changed only fractionally. Substrate saturation studies (fructose 6-phosphate) of phosphofructokinase activity showed a decrease in the [S]_0.5 from 5.6 to 1.7 mM betwen stages a and c. The enzyme from both sources seems to be regulated by a negative cooperative effect with the control being more stringent in the enzyme from stage a. The difference in enzyme activity is consistent with the increase in respiratory activity between the two stages.     

Changes in the concentrations of some phosphorylated intermediates and stimulation of glycolysis in liver slices

1. The concentrations of some phosphorylated glycolytic intermediates and of NADH were measured in glycolysing rat liver slices. 2. In anaerobically incubated liver slices the concentration of hexose monophosphates decreases during the first 20min. of incubation, whereas the concentrations of fructose diphosphate and triose phosphates increase progressively. 3. In liver slices from fed rats, previously exposed to oxygen, the stimulated anaerobic glycolysis is accompanied by an increase in the concentration of hexose monophosphates; fructose diphosphate and triose phosphates maintain the concentrations reached at the end of the aerobic preincubation. 4. The same pattern in the concentration of glycolytic phosphorylated intermediates is seen under all conditions where aerobic preincubation brings about a stimulation of anaerobic glycolysis. A similar pattern is also found in liver slices from fed rats incubated anaerobically in the presence of fructose; these slices display a high glycolytic activity, which is not further affected by previous aerobic incubation. 5. The concentration of NADH decreases in liver slices during exposure to oxygen; during the subsequent anaerobic glycolysis the concentration increases but is always lower in preincubated than in non-preincubated liver slices. 6. The results of the present experiments suggest that the limiting step mainly affected by the preliminary exposure to oxygen might be at the level of the utilization of triose phosphates.     

The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.