Coordinate control of sucrose formation in soybean leaves by sucrose-phosphate synthase and fructose-2,6-bisphosphate

Net photosynthesis (CER), assimilate-export rate, sucrose-phosphate-synthase (EC 2.4.1.14) activity, fructose-2,6-bisphosphate content, and 6-phosphofructo-2-kinase (EC 2.7.1.105) activity were monitored in leaves of soybean (Glycine max (L.) Merr.) plants during a 12:12 h day-night cycle, and in plants transferred, at regular intervals throughout the diurnal cycle, to an illuminated chamber for 3 h. In the control plants, assimilate-export rate decreased progressively during the day whereas in transferred plants, a strongly rhythmic fluctuation in both CER and export rate was observed over the 24-h test period. Two maxima during the 24-h period for both processes were observed: one when plants were transferred during the middle of the normal light period, and a second when plants were transferred during the middle of the normal dark period. Overall, the results indicated that export rate was correlated positively with photosynthetic rate and sucrose-phosphate-synthase activity, and correlated negatively with fructose-2,6-bisphosphate levels, and that coarse control and fine control of the sucrose-formation pathway are coordinated during the diurnal cycle. Diurnal changes in sucrose-phosphate-synthase activity were not associated with changes in regulatory properties (phosphate inhibition) or substrate affinities. The biochemical basis for the diurnal rhythm in sucrose-phosphate-synthase activity in the soybean leaf thus appears to involve changes in the amount of the enzyme or a post-translational modification that affects only the maximum velocity.Abbreviations FBPase fructose-1,6-bisphosphatase - SPS sucrose-phosphate synthase - F26BPase fructose-2,6-bisphosphatase - PGI glucose-6-phosphate isomerase - F6P fructose-6-phosphate - F26BP fructose-2,6-bisphosphate - G6P glucose-6-phosphate - CER net carbon exchange rate - Pi inorganic phosphate - DHAP dihydroxyacetone phosphate - PGA glycerate 3-phosphate - F6P,2-kinase 6-phosphofructo-2-kinase Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10503 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601     

Carbon-partitioning inArabidopsis is regulated by the fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase enzyme

To further elucidate the mechanisms underlying carbon-partitioning in plants, we established an experimental system by generating transgenicArabidopsis lines that overexpress both the fructose 6-phosphate, 2-kinase (F6P,2-K) and the fructose 2,6-bisphosphatase (F26BPase) domains. We also produced knockout transgenic plants for these domains via RNAi and T-DNA tagging. In F6P,2-K overexpressing transgenics, F6P,2-K activity increased slightly and Fru-2,6-P₂ levels were elevated by 80%, compared with the wild type (WT). F26BPase activity was similar between the WT and transgenic plants. However, when that domain was overexpressed, F26BPase activity was increased by 70% compared with the WT, whereas F6P,2-K activity was reduced to 85% of the WT level. In knockout and RNAi mutant lines that showed reduced F6P,2-K and F26BPase activities, levels of Fru-2,6-P₂ were only between 3 to 7% of those for the WT. In F6P,2-K overexpressing transgenic lines, the levels of starch, hexose, and triose phosphates slightly increased, while sucrose content was marginally reduced. In F26BPase overexpressing plants, however, the levels of soluble sugars and hexose phosphates were slightly increased, but starch and triose phosphate contents declined. Furthermore, compared with the WT, the levels of soluble sugars rose while starch and hexose phosphate quantities decreased in 2-kinase/fructose-2,6-bisphophatase knockout mutants. Therefore, our data reaffirms that Fru-2,6-P₂ contributes to the regulation of photosynthetic carbon-partitioning between starch and sucrose inArabidopsis leaves by limiting sucrose synthesis.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.     

Characterization of yeast fructose-2,6-bisphosphate 6-phosphatase

To obtain information on the biological significance of yeast fructose-2,6-bisphosphate 6-phosphatase, kinetic data of the purified enzyme [(1987) Eur. J. Biochem. 164, 27-30] have been measured. Maximal activity was found between pH 6 and 7, the apparent Michaelis constant with fructose 2,6-bisphosphate was 7.2 microM at pH 6.0 and 79 microM at pH 7.0. Concentrations required for 50% inhibition of the enzyme at pH 6.0 were 8 microM Fru2P, 45 microM G1c6P, 80 microM Fru6P and 200 microM inorganic phosphate. The known intracellular steady-state level of about 10 microM fructose 2,6-bisphosphate in the presence of glucose is likely to be the result of a balance between the rapid synthesis of fructose 2,6-bisphosphate catalyzed by 6-phosphofructose 2-kinase and a fructose 2,6-bisphosphate degrading activity. The biological function of fructose-2,6-bisphosphate 6-phosphatase with an apparent Michaelis constant between 7 and 79 microM fructose 2,6-bisphosphate at pH 6-7 is therefore suggested to participate in the maintenance of a steady-state level of fructose 2,6-bisphosphate in a concentration range that fits well with the Michaelis constant of the enzyme.     

Fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase during liver regeneration.

Glycogen and fructose 2,6-bisphosphate levels in rat liver decreased quickly after partial hepatectomy. After 7 days the glycogen level was normalized and fructose 2,6-bisphosphate concentration still remained low. The 'active' (non-phosphorylated) form of 6-phosphofructo-2-kinase varied in parallel with fructose 2,6-bisphosphate levels, whereas the 'total' activity of the enzyme decreased only after 24 h, similarly to glucokinase. The response of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from hepatectomized rats (96 h) to sn-glycerol 3-phosphate and to cyclic AMP-dependent protein kinase was different from that of the enzyme from control animals and similar to that of the foetal isoenzyme.     

Ethylene-induced increase in fructose-2,6-bisphosphate in plant storage tissues

Treatment of carrot roots with ethylene led to: (a) a doubling of the fructose-2,6-bisphosphate content; (b) a general increase in the concentration of glycolytic intermediates; and (c) an increase in the extractable activity of fructose-6-phosphate,2-kinase, the enzyme synthesizing fructose-2,6-bisphosphate from fructose-6-phosphate and adenosine triphosphate.     

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.     

Quasi-stationary concentrations of fructose-2,6-bisphosphate in the phosphofructokinase-2/fructose-2,6-bisphosphatase cycle

The cooperation of phosphofructokinase-2 and fructose-2,6-bisphosphatase is investigated. Experimentally derived rate laws of the kinase and bisphosphatase activities introduced into the respective differential equations permitted to describe the time evolution of fructose-2,6-bisphosphate to quasi-stationary levels. The two enzyme activities were found to exert strong temperature dependence. The quasi-stationary levels of fructose-2,6-bisphosphate, however, are independent on temperature.     

ATP and fructose-2,6-bisphosphate regulate skeletal muscle 6-phosphofructo-1-kinase by altering its quaternary structure

Recently, it has been demonstrated that fructose-2,6-bisphosphate (F2,6BP) protects skeletal muscle 6-phosphofructo-1-kinase (PFK) from thermal inactivation (50 degrees C) and against the deleterious effects of guanidinium hydrochloride (GdmCl). On the other hand, ATP, when added at its inhibitory concentrations, that is, >1 mM, enhanced either the thermal- or GdmCl-induced inactivation of PFK. Moreover, we concluded that these phenomena were probably due to the stabilization of PFK tetrameric structure by F2,6BP, and the dissociation of this structure into dimers induced by ATP. Aimed at elucidating the effects of F2,6BP and ATP on PFK at the structural and functional levels, the present work correlates the effects of these metabolites on the equilibrium between PFK dimers and tetramers to the regulation promoted on the enzyme catalytic activity. We show that ATP present a dual effect on PFK structure, favoring the formation of tetramer at stimulatory concentrations (up to 1 mM), and dissociating tetramers into dimers at inhibitory concentrations (>1 mM). Furthermore, F2,6BP counteracted this later ATP effect at either the structural or catalytic levels. Additionally, the effects of both F2,6BP or ATP on the equilibrium between PFK tetramers and dimers and on the enzyme activity presented a striking parallelism. Therefore, we concluded that modulation of PFK activity by ATP and F2,6BP is due to the effects of these ligands on PFK quaternary structure, altering the oligomeric equilibrium between PFK tetramers and dimers.     

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.     

Increases in hepatic fructose-2,6-bisphosphate level and fructose-6-phosphate,2-kinase activity in rats with ventromedial lesions of the hypothalamus

To investigate altered fructose-2,6-bisphosphate (fructose-2,6-P2) metabolism, we measured fructose-2,6-P2 levels and fructose-6-phosphate,2-kinase (fructose-6-P,2-kinase) activities in various tissues, including liver, kidney, heart, and skeletal muscle, of ventromedial hypothalamus (VMH)-lesioned rats during feeding and starvation. The plasma insulin level was 6 times or more higher in these rats than in the controls. The fructose-2,6-P2 level in liver was much greater in VMH-lesioned rats than in the controls: 15.1 +/- 2.2 nmol/g tissue versus 7.7 +/- 0.7 in the fed state, 5.3 +/- 1.1 versus 1.6 +/- 0.4 in the starved state. In kidney, heart, and skeletal muscle, fructose-2,6-P2 levels were not different between the two animal groups. The activity of hepatic fructose-6-P,2-kinase remained high after 20 h of starvation in VMH-lesioned rats, whereas it was decreased markedly in the controls. The hepatic concentration of fructose-6-phosphate was also high in VMH-lesioned rats. Both fructose-6-P,2-kinase activity and fructose-6-phosphate concentration in the liver of starved VMH-lesioned rats were comparable to those of control rats in fed conditions. These results indicate that the alteration of fructose-2,6-P2 metabolism is characteristic of liver in VMH-lesioned rats, and that the increase in hepatic fructose-2,6-P2 may activate hepatic glycolysis not only during feeding but also during starvation, leading to the enhanced lipogenesis in these obese rats.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase

Limitation in copper (Cu) leads to pathophysiology in developing brain. Cu deficiency impairs brain mitochondria and results in high brain lactate suggesting augmented anaerobic glycolysis. AMP activated protein kinase (AMPK) is a cellular energy "master-switch" that is thought to augment glycolysis through phosphorylation and activation phosphofructokinase 2 (PFK2) resulting in increases of the glycolytic stimulator fructose-2,6-bisphosphate (F2,6BP). Previously, Cu deficiency has been shown to augment cerebellar AMPK activation. Cerebella of Cu-adequate (Cu+) and Cu-deficient (Cu-) rat pups were assessed to evaluate if AMPK activation in Cu- cerebella functioned to enhance PFK2 activation and increase F2,BP concentration. Higher levels of pAMPK were detected in Cu- cerebella. However, PFK2 activity, mRNA, and protein abundance were not affected by Cu deficiency. Surprisingly, F2,6BP levels were markedly lower in Cu- cerebella. Lower F2,6BP may be due to inhibition of PFK2 by citrate, as citrate concentration was significantly higher in Cu- cerebella. Data suggest AMPK activation in Cu- cerebellum does not augment glycolysis through a PFK2 mechanism. Furthermore, other metabolite data suggest that glycolysis may actually be blunted, since levels of glucose and glucose-6-phosphate were higher in Cu- cerebella than controls.     

Fat body fructose-2,6-bisphosphate content and phosphorylase activity correlate with changes in hemolymph glucose concentration during fasting and re-feeding in larval Manduca sexta

Fasting of second-day fifth instar larval Manduca sexta leads to a rapid decrease in hemolymph glucose concentration from 3.39+/-0.29 to 0.33+/-0.06 mM in 1 h, along with a decrease in the fructose-2,6-bisphosphate content in the fat body (from 5.92+/-0.31 to 2.80+/-0.47 nmol fructose-2,6-bisphosphate/g fat body in 3 h) and activation of fat body glycogen phosphorylase (from 16% to 55-65% phosphorylase a). During re-feeding an increase in the glucose level in the hemolymph was observed (from 0.36+/-0.05 to 3.91+/-0.36 mM in 3 h), along with an increase in the fructose-2,6-bisphosphate level in the fat body (from 2.88+/-0.47 to 6.66+/-0.42 nmol fructose-2,6-bisphosphate/g fat body in 3 h) and inactivation of fat body glycogen phosphorylase (from 56% to 16% phosphorylase a). These data are consistent with the hypothesis that a decrease in hemolymph glucose both activates fat body glycogen phosphorylase and causes a decrease in fat body fructose-2,6-bisphosphate content. Both of these changes would favor conversion of stored glucose to trehalose in the fat body. When second-day larvae were decapitated, the changes in hemolymph glucose and fat body fructose-2,6-bisphosphate were very similar to those observed in fasting whole insects. These data are consistent with a direct role for glucose in controlling carbohydrate metabolism in Manduca sexta.     

Effect of tolbutamide on fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase in rat liver

The effects of tolbutamide on the activities of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were examined using rat hepatocytes. Tolbutamide stimulated fructose-6-phosphate,2-kinase activity and inhibited fructose-2,6-bisphosphatase activity, resulting in an increase of fructose-2,6-bisphosphate level. Changes in the activities of the enzyme by tolbutamide were due to variation in the Km value, but not dependent on alteration of Vmax. Glucagon inhibition of fructose-2,6-bisphosphate formation resulting from an inactivation of fructose-6-phosphate,2-kinase and an activation of fructose-2,6-bisphosphatase was released by tolbutamide. Tolbutamide stimulation of fructose-2,6-bisphosphate formation through regulation of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase may produce enhancement of glycolysis and inhibition of gluconeogenesis in the liver.     

Fructose 2,6-bisphosphate in rat erythrocytes. Inhibition of fructose 2,6-bisphosphate synthesis and measurement by glycerate 2,3-bisphosphate.

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.     

Vanadate inhibits fructose-2,6-bisphosphatase and leads to an inhibition of sucrose synthesis in barley leaves

Vanadate (0.1–1 mM) was supplied to leaves of barley (Hordeum vulgare var. Roland) via the transpiration stream. It led to a selective inhibition of the rate of photosynthesis at high light without altering the initial slope of the light response curve, produced markedly biphasic photosynthesis induction kinetics, and selectively decreased sucrose synthesis compared to starch synthesis. There was a 3-fold increase of the steady state level of the signal metabolite fructose-2,6-bisphosphate in near saturating light. Fructose-2,6-bisphosphate is a potent inhibitor of cytosolic fruc-tose-l,6-bisphosphatase and, in agreement, the fructose-1,6-bisphosphatc level doubled. The increase of fructose-2,6-bisphosphate could not be accounted for by the known regulation of fructose-6-phosphate,2-kinase and fructose 2,6-bisphosphatase by 3-phosphoglycerate and fiuctose-6-phosphate, because these metabolites remained constant or even changed in the opposite direction to that required to generate an increase of fructose-2,6-bisphosphate. Instead, vanadate strongly inhibited the hydrolysis of fructose-2,6-bisphosphate in extracts, producing a half maximal inhibition at 2 \nM and 50 \iM in assays designed to preferentially measure the high-and low-affinity forms of fructose-2,6-bisphosphatase, respectively. Vanadale had no effect on fructosc-6-phosphate,2-kinase activity at these concentrations. Vanadate also led to a deactivation of sucrose phosphate synthase. The results are discussed in relation to the role of fructose-2,6-bisphosphate in regulating sucrose synthesis, and its interaction with the 'coarse' control of sucrose phosphate synthase.     

Regulation of fructose 2,6-bisphosphate metabolism in human fibroblasts

In the present work, the mechanism involved in the regulation of fructose 2,6-bisphosphate (fructose-2,6-P₂) metabolism in human fibroblasts has been studied. Various agents like serum, insulin and adrenaline known to affect glycolysis have been investigated for their ability to influence fructose 2,6-P₂ metabolism in confluent human fibroblasts. Serum appears to be the most potent activator of fructose-2,6-P₂ levels and capable of inducing a marked increase in 6-phosphofructo-2-kinase (ATP: d-fructose-6-phosphate-2-phosphotransferase), EC 2.7.1. 105). To a lesser extent insulin has the same effects. The increase in enzyme activity elicited by serum and insulin does not require de novo protein synthesis since the process is insensitive to cycloheximide. Incubation of fibroblasts in the presence of adrenaline is responsible for a significant rise in fructose-2,6-P₂ levels without affecting 6-phosphofructo-2-kinase. Similar experiments performed on glucose-starved or cytochalasin B-treated cells show that the effects elicited by all the agents are strictly dependent on glucose availability.