Conversion of fructose to glucose in the rabbit small intestine. A reappraisal of the direct pathway.

Gopher et al [Gopher, A., Vaisman, N., Mandel, H. & Lapidot, A. (1990) Proc. Natl Acad. Sci. USA 87, 5449-5453] recently reported that about 50% of the glucose formed from [U-13C]fructose infused nasogastrically in children contained 13C3 adjacent to 13C4. Assuming a high isotopic dilution of the triosephosphate pool, the authors concluded that about 50% of the fructose converted to glucose in liver and intestine bypassed the classical aldolase pathway, utilizing a hypothetical direct pathway that would involve the phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate. The present work was undertaken in order to establish to what extent the conversion of fructose to glucose in the intestine could account for this unexpected isotopic distribution. The technique of everted sleeves was used to define the rate of conversion of [U-14C]glucose and [U-14C]fructose in the small intestine of 24-h-fasted rabbits. It appeared that, at the low concentration of fructose used by Gopher et al., almost as much fructose was converted to glucose as remained unmodified in the tissue. Fructose uptake was not inhibited by glucose, and the presence of all the necessary enzymes in the tissue indicated that the fructose to glucose conversion occurred by the aldolase pathway. Remarkably, this conversion operated with an isotopic dilution not exceeding 25%, due to the low rate of glucose metabolism and the near absence of gluconeogenesis from lactate. It can, therefore, be postulated that, in the presence of pure [U13C]fructose, the triosephosphate pool is highly enriched in 13C with little dilution by 12C, essentially giving rise to [U-13C]glucose, as reported by Gopher et al. There is, therefore, no need to postulate the participation of a direct pathway.

The control of trehalose biosynthesis in Saccharomyces cerevisiae: evidence for a catabolite inactivation and repression of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.

During diauxic growth of yeast in glucose-rich medium, the accumulation of trehalose started well after complete exhaustion of glucose from the medium. The accumulation of the disaccharide was concomitant with a resumption of cell growth on the ethanol accumulated in the medium, but not with a degradation of glycogen which occurred as soon as glucose had been consumed. In contrast, in a mutant deficient in phosphoenolpyruvate carboxykinase, the synthesis of trehalose coincided exactly with the degradation of glycogen. Upon inoculation of stationary phase wild-type cells into a glucose medium, the activities of trehalose-6-phosphate (Tre6P) synthase and Tre6P phosphatase dropped in parallel to reach only 15% of their initial values after 3 h, and only recovered their original values as cells re-entered stationary phase. In the presence of cycloheximide, the decrease in Tre6P synthase and Tre6P phosphatase activities was restricted to 50-60%, the remaining decrease being inhibited by the drug. Furthermore, the reappearance of the enzyme activities following transfer of cells to an acetate medium was blocked by cycloheximide. It was also shown that loss of activity of these two enzymes required a combination of metabolizable sugars together with a nitrogen source. Low activities of Tre6P synthase and Tre6P phosphatase were measured in mutants with increased adenylate cyclase activity (RAS2ala18val19 mutants). Moreover, derepression of these enzymes at the approach of stationary phase was prevented in a pde2 mutant when it was cultivated in the presence of exogenous cyclic nucleotide. The mechanism of this effect is not clear, but may involve a transcriptional regulation by cAMP of the genes encoding these proteins.

Induction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase by anoxia in rice seedlings.

Rice (Oryza sativa) seeds were imbibed for 3 days and the seedlings were further incubated for 8 days in the presence of either air or nitrogen. In aerobiosis, the specific activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase and that of the ATP-dependent phosphofructokinase increased about fourfold. In anaerobiosis, the specific activity of ATP-dependent phosphofructokinase remained stable, whereas that of pyrophosphate:fructose 6-phosphate 1-phosphotransferase increased as much as in the presence of oxygen and there was also a fourfold increase in the concentration of fructose 2,6-bisphosphate, a potent stimulator of that enzyme. These data suggest a preferential involvement of pyrophosphate:fructose 6-phosphate 1-phosphotransferase rather than of ATP-dependent phosphofructokinase in glycolysis during anaerobiosis.

Mechanisms of blood glucose homeostasis.

The mechanisms by which glycogen metabolism, glycolysis and gluconeogenesis are controlled in the liver both by hormones and by the concentration of glucose are reviewed. The control of glycogen metabolism occurs by phosphorylation and dephosphorylation of both glycogen phosphorylase and glycogen synthase catalysed by various protein kinases and protein phosphatases. The hormonal effect is to stimulate glycogenolysis by the intermediary of cyclic AMP, which activates directly or indirectly the protein kinases. The glucose effect is to activate the protein phosphatase system; this occurs by the direct binding of glucose to glycogen phosphorylase which is then a better substrate for phosphorylase phosphatase and is inactivated. Since phosphorylase a is a strong inhibitor of synthase phosphatase, its disappearance allows the activation of glycogen synthase and the initiation of glycogen synthesis. When glycogen synthesis is intense, the concentrations of UDPG and of glucose 6-phosphate in the liver decrease, allowing a net glucose uptake by the liver. Glucose uptake is indeed the difference between the activities of glucokinase and glucose 6-phosphatase. Since the Km of the latter enzyme is far above the physiological concentration of its substrate, the decrease in glucose 6-phosphate concentration proportionally reduces its activity. The control of glycolysis and of gluconeogenesis occurs mostly at the level of the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate under the action of phosphofructokinase 1 and fructose 1,6-bisphosphatase. Fructose 2,6-bisphosphate is a potent stimulator of the first of these two enzymes and an inhibitor of the second. It is formed from fructose 6-phosphate and ATP by phosphofructokinase 2 and hydrolysed by a fructose 2,6-bisphosphatase. These two enzymes are part of a single bifunctional protein which is a substrate for cyclic AMP-dependent protein kinase. Its phosphorylation causes the inactivation of phosphofructokinase 2 and the activation of fructose 2,6-bisphosphatase, resulting in the disappearance of fructose 2,6-bisphosphate. The other major effector of these two enzymes is fructose 6-phosphate, which is the substrate of phosphofructokinase 2 and a potent inhibitor of fructose 2,6-bisphosphatase; these properties allow the formation of fructose 2,6-bisphosphate when the level of glycaemia and secondarily that of fructose 6-phosphate is high.

Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii.

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.

Fructose 2,6-bisphosphate hydrolyzing enzymes in higher plants.

The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases I, II, and III, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a K(m) value of 30 micromolar, to a mixture of fructose 2-phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the beta-anomeric form. Its apparent K(m) for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described by F. D. Macdonald, Q. Chou, and B. B. Buchanan ([1987] Plant Physiol 85: 13-16). Phosphatase III copurified with phosphofructokinase 2 and corresponded to the specific, low-K(m) (24 nanomolar) fructose-2,6-bisphosphatase purified and characterized by Y. Larondelle, E. Mertens, E. Van Schaftingen, and H. G. Hers ([1986] Eur J Biochem 161: 351-357). Three similar types of phosphatases were present in a crude extract of Jerusalem artichoke (Helianthus tuberosus) tuber. The concentration of fructose 2,6-bisphosphate decreased at a maximal rate of 30 picomoles per minute and per gram of fresh tissue in slices of Jerusalem artichoke tuber, upon incubation in 50 millimolar mannose. This rate could be accounted for by the maximal extractable activity of the low-K(m) fructose-2,6-bisphosphatase. A new enzymic method for the synthesis of beta-glucose 1,6-bisphosphate from beta-glucose 1-phosphate and ATP is described.

Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae.

The properties of yeast trehalose-6-phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C., de Araujo, P. S., Moura-Neto, V. and Panek, A. D. (1987) Curr. Genet. II, 459-465] that the enzyme would be stimulated by ATP and partially inactivated by cAMP-dependent protein kinase. Trehalose-6-phosphate synthase activity was measured by the sum of [14C]trehalose 6-phosphate and [14C]trehalose formed from UDP-[14C]glucose and glucose 6-phosphate. The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP-Mg, cAMP and bovine heart cAMP-dependent protein kinase. The activity was also not significantly different in several mutants affected in the cAMP system. The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki = 2 mM). The activity of trehalose-6-phosphate phosphatase was measured by the amount of [14C]trehalose formed from [14C]trehalose 6-phosphate. The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6-phosphate. It was Mg dependent and its kinetics for trehalose 6-phosphate was hyperbolic. Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP-dependent change in its activity. Remarkably, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein. A 50-fold purification of the two enzymes could be achieved with a yield of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.

D-xylulose-induced depletion of ATP and Pi in isolated rat hepatocytes.

Xylitol is known to cause hepatic ATP catabolism by inducing the trapping of Pi in the form of glycerol 3-P as a consequence of an increase in the NADH:NAD+ ratio, resulting from the oxidation of xylitol to D-xylulose. The question was therefore raised whether D-xylulose also depletes hepatic ATP. In isolated rat hepatocytes, 5 mM D-xylulose decreased ATP by 80% within 5 min compared to 40% with 5 mM xylitol. Intracellular Pi decreased by 70% within the same time interval with both compounds, but was restored three-fold faster with D-xylulose. The rate of utilization of D-xylulose reached 5 mumol.min-1.g-1 of cells, as compared with 1.5 for xylitol, indicating that reduction of xylitol into D-xylulose is a rate-limiting step in the metabolism of the polyol. D-Xylulose barely modified the concentration of glycerol 3-P but increased xylulose 5-P from 0.02 to 0.5 mumol/g within 5 min. The main cause of the ATP- and Pi-depleting effects of D-xylulose was found to be an accumulation of sedoheptulose 7-P from a basal value of 0.1 to 5 mumol/g of cells after 10 min. Ribose 5-P increased from 0.03 to 0.5 mumol/g at 5 min. Ribose 1-P also accumulated, albeit outside of the cells. This extracellular accumulation can be explained by the release of intracellular purine nucleoside phosphorylase from damaged hepatocytes acting on inosine that had diffused out of the cells. Smaller increases in the concentrations of sedoheptulose 7-P and pentose phosphates were recorded after incubations of the cells with xylitol.

Increase in phosphoribosyl pyrophosphate induced by ATP and Pi depletion in hepatocytes.

A series of compounds that induce depletion of ATP and Pi when added to isolated rat hepatocytes were found to cause a remarkable, although transient, elevation in the concentration of phosphoribosyl pyrophosphate (PRPP) in these cells. After the addition of 5 mM fructose, xylitol, tagatose, or D-xylulose, PRPP increased from a basal value of 6 +/- 1 nmol/g of cells to, respectively, 68 +/- 11, 42 +/- 11, 67 +/- 22, and 530 +/- 50 nmol/g of cells (means +/- SEM of 3-9 experiments). In each case, the increase in PRPP was preceded by a latency period of 5-10 min. PRPP reached maximal levels 15 min after the addition of fructose and 30 min after that of xylitol and D-xylulose, but continued to increase for as long as 60 min after the addition of tagatose. Most striking was that the increase in PRPP closely paralleled the restoration of intracellular Pi. Ribose 5-P increased about two- to fivefold after the addition of fructose, xylitol, and tagatose, and approximately 12-fold after D-xylulose. The mechanism by which ATP- and Pi-depleting compounds stimulate the activity of PRPP synthetase in isolated rat hepatocytes is discussed.

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers.

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.

The control of glycogen metabolism in yeast. 2. A kinetic study of the two forms of glycogen synthase and of glycogen phosphorylase and an investigation of their interconversion in a cell-free extract.

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate-cyclase-deficient mutant that had been preincubated at the restrictive temperature of 35 degrees C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20-fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6-phosphate, the two forms of enzyme had a similar affinity for UDP-Glc but differed essentially by their Vmax. Glucose 6-phosphate had no effect on synthase a, but increased both Vmax and Km of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20-fold more active than synthase b. When an extract of cells that had been preincubated in the absence of glucose was gel-filtered and then incubated at 30 degrees C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6-phosphate. When a gel-filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30 degrees C in the presence of ATP-Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6-phosphate and only slightly and irregularly stimulated by cyclic AMP.

5'-Nucleotidase activities in human erythrocytes. Identification of a purine 5'-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate.

A purine 5'-nucleotidase has been separated by DEAE-Trisacryl chromatography from other 5'-nucleotidase activities present in human haemolysates and purified approx. 30,000-fold by subsequent chromatography on Blue Sepharose. The enzyme has an Mr of around 250,000, displays hyperbolic substrate-saturation kinetics and hydrolyses preferentially IMP, GMP and their deoxy counterparts. It is much less active with AMP and dAMP. The purine 5'-nucleotidase is inhibited by Pi, and is strongly stimulated by ATP, dATP and GTP, and by glycerate 2,3-bisphosphate. Stimulators decrease Km and increase Vmax. Glycerate 2,3-bisphosphate is the most potent stimulator of the enzyme and, under physiological conditions, over-rides the influence of the other effectors. Glycerate 2,3-bisphosphate also influences the binding of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood. It is concluded that the glycerate 2,3-bisphosphate-stimulated purine 5'-nucleotidase is responsible for the dephosphorylation of IMP and GMP, but not of AMP, in human erythrocytes.

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast.

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.

Extracellular metabolites in suspensions of isolated hepatocytes.

The activity of lactate dehydrogenase and the concentration of several metabolites were measured in a suspension of isolated hepatocytes and in the extracellular medium, obtained after elimination of the cells by centrifugation for 15 s. The initial proportions of ATP, fructose 2,6-bisphosphate and glycogen present in the medium were similar to that of lactate dehydrogenase, and were therefore explained by unavoidable cell breakage occurring during resuspension of the hepatocytes. ATP disappeared from the medium in less than 10 min, being presumably destroyed by membrane nucleotidases. By contrast, the proportions of hexose 6-phosphates and of glycerol 3-phosphate in the medium were several-fold in excess over that of lactate dehydrogenase; under certain conditions, the extracellular value accounted for 80-90% of the metabolite present in the total suspension, and there was no relationship between the extra- and intracellular concentrations of these metabolites. A potential source of external glycerol 3-phosphate was the hydrolysis of glycerophosphocholine by membranous enzymes. The main conclusion of this work is that the measurement, in isolated hepatocytes, of hexose 6-phosphates, glycerol 3-phosphate and possibly other metabolites that were not investigated, requires the previous separation of the cells from the incubation medium. This conclusion may apply to other cellular suspensions.

Effect of ethylene treatment on the concentration of fructose-2,6-bisphosphate and on the activity of phosphofructokinase 2/fructose-2,6-bisphosphatase in banana.

Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phosphofructokinase 2/fructose-2,6-bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate-dependent and of the ATP-dependent 6-phosphofructo-1-kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6-bisphosphate and of fructose 1,6-bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100-fold greater than the first. The change in fructose 2,6-bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6-phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3-phosphate, a potent inhibitor of phosphofructokinase 2. The burst in fructose 2,6-bisphosphate and the activity of the pyrophosphate-dependent phosphofructokinase, which is in banana the only enzyme known to be sensitive to fructose 2,6-bisphosphate, can explain the well-known increase in fructose 1,6-bisphosphate which occurs during ripening.

Fructose 2,6-bisphosphate in germinating oat seeds. A biochemical study of seed dormancy.

When dormant oat seeds were imbibed at the non-permissive temperature of 30 degrees C, the concentration of phosphoenolpyruvate and of glycerate 3-phosphate, which are two inhibitors of phosphofructokinase 2, increased almost linearly during 30 h. By contrast, these metabolites increased only after a lag period of about 10 h in non-dormant seeds imbibed at the same temperature. As a consequence of this, the concentration of the C3 derivatives remained always remarkably lower in non-dormant than in dormant seeds. Accordingly, the concentration of fructose 2,6-bisphosphate, which increased similarly in the two types of seeds during the first 8 h after the start of inhibition, then reached a plateau in dormant seeds but continued to increase for another 8 h in non-dormant seeds, reaching a maximal value a few hours before the beginning of radicle protrusion. When the dormant seeds were imbibed at the permissive temperature of 10 degrees C, the evolution of all metabolites was slowed down but behaved like that of non-dormant seeds imbibed at 30 degrees C. Experiments in which the dormant seeds were submitted to a jump from 10 degrees C to 30 degrees C and vice versa, always provoked reverse changes in the concentration of the C3 derivatives and of fructose 2,6-bisphosphate, the latter being increased in all conditions that allowed germination. Dormant seeds were also allowed to germinate at 30 degrees C by imbibition during 24 h in the presence of 3% ethanol. Again, this permissive treatment caused an arrest in the accumulation of C3 derivatives and an increase in fructose 2,6-bisphosphate. Another, apparently unrelated, biochemical difference between dormant and non-dormant oat seeds was their inorganic pyrophosphate content, which was approximately five-fold higher in non-dormant than in dormant seeds. This difference was observed before and persisted during imbibition as long as measurement could be made and was not affected by the temperature jumps or by ethanol. In contrast to the phosphoric esters under investigation, pyrophosphate was not preferentially located in the embryo.