The expression of monocyte chemotactic protein (MCP-1) in human vascular endotheliumin vitro andin vivo

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P₂ in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P₂ in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P₂ levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P₂ due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.Abbreviations fructose-2,6-P₂ D-fructose 2,6-bisphosphate - fructose-6-P D-fructose 6-phosphate - PAGE Polyacrylamide Gel Electrophoresis - PFK 6-phosphofructo-1-kinase - PP_i-PFK Pyrophosphate-dependent Phosphofructokinase, ribose-1,5-P₂, ribose-1,5-bisphosphate - SDS Sodium Dodecyl Sulfate     

Fructose-2,6-bisphosphate in rat mesenteric lymph nodes

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.     

Cardiac expression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase inhibits glycolysis, promotes hypertrophy, impairs myocyte function, and reduces insulin sensitivity

Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiac-specific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P(2)), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P(2). Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

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.     

The stimulation of heart glycolysis by increased workload does not require AMP-activated protein kinase but a wortmannin-sensitive mechanism

Increasing heart workload stimulates glycolysis by enhancing glucose transport and fructose-2,6-bisphosphate (Fru-2,6-P(2)), the latter resulting from 6-phosphofructo-2-kinase (PFK-2) activation. Here, we investigated whether adenosine monophosphate (AMP)-activated protein kinase (AMPK) mediates PFK-2 activation in hearts submitted to increased workload. When heart work was increased, PFK-2 activity, Fru-2,6-P(2) content and glycolysis increased, whereas the AMP:adenosine triphosphate (ATP) and phosphocreatine/creatine (PCr:Cr) ratios, and AMPK activity remained unchanged. Wortmannin, the well-known phosphatidylinositol-3-kinase inhibitor, blocked the activation of protein kinase B and the increase in glycolysis and Fru-2,6-P(2) content induced by increased work. Therefore, the control of heart glycolysis by contraction differs from that in skeletal muscle where AMPK is involved.     

The yeast FBP26 gene codes for a fructose-2,6-bisphosphatase.

Sequencing of an open reading frame 450 bp downstream from the yeast VPS35 gene revealed a putative peptide of 452 amino acids and 52.7 kDa. The predicted amino acid sequence has 45% identity with the 55-kDa subunit of the 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (EC 2.7.1.105/EC 3.1.3.46) from rat liver and 42% identity with 480 amino acids in the center of the recently reported 93.5-kDa subunit of yeast 6-phosphofructo-2-kinase (EC 2.7.1.105). The product of the new yeast gene is similar to the entire sequence of the bifunctional rat liver enzyme and, unlike yeast 6-phosphofructo-2-kinase, has the histidine residue essential for fructose-2,6-bisphosphatase activity. Extracts from a chromosomal null mutant strain, fbp26::HIS3, incubated in the presence of [2-32P]fructose 2,6-P2, lacked in autoradiograms the characteristic 56-kDa labeled band observed in wild-type. The same band was intensified 3-fold over wild-type level with the FBP26 gene introduced on multicopy in the fbp26::HIS3 background. A similar increase was found for fructose-2,6-bisphosphatase activity in the same extracts. The FBP26 gene did not cause detectable increase in 6-phosphofructo-2-kinase activity when introduced on multicopy in a pfk26::LEU2 mutant, indicating that its gene product is predominantly a fructose-2,6-bisphosphatase. Growth on glucose, fructose, galactose, pyruvate, and glycerol/lactate was not impaired in strains carrying the fbp26::HIS3 allele.     

Fructose 2,6-bisphosphate and insulin stimulation of glycolysis in 3T3-L1 adipocytes

1. Insulin is able to stimulate lactate production and to enhance fructose 2,6-bisphosphate (Fru-2,6-P2) content in 3T3-L1 adipocytes. 2. Phorbol 12-myristate 13-acetate is more efficacious than insulin in rising Fru-2,6-P2 content and less effective in the stimulation of glycolysis. 3. 3T3-L1 adipocyte 6-phosphofructo-l-kinase appears to be very sensitive to exogenous Fru-2,6-P2. 4. Insulin treatment does not affect the maximum activity of 6-phosphofructo-1-kinase whereas it markedly increases the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The role of Fru-2,6-P2 in the insulin induced enhancement of glycolytic flux is discussed.     

Cardiac phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase increases glycolysis, hypertrophy, and myocyte resistance to hypoxia

During ischemia and heart failure, there is an increase in cardiac glycolysis. To understand if this is beneficial or detrimental to the heart, we chronically elevated glycolysis by cardiac-specific overexpression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) in transgenic mice. PFK-2 controls the level of fructose-2,6-bisphosphate (Fru-2,6-P2), an important regulator of phosphofructokinase and glycolysis. Transgenic mice had over a threefold elevation in levels of Fru-2,6-P2. Cardiac metabolites upstream of phosphofructokinase were significantly reduced, as would be expected by the activation of phosphofructokinase. In perfused hearts, the transgene caused a significant increase in glycolysis that was less sensitive to inhibition by palmitate. Conversely, oxidation of palmitate was reduced by close to 50%. The elevation in glycolysis made isolated cardiomyocytes highly resistant to contractile inhibition by hypoxia, but in vivo the transgene had no effect on ischemia-reperfusion injury. Transgenic hearts exhibited pathology: the heart weight-to-body weight ratio was increased 17%, cardiomyocyte length was greater, and cardiac fibrosis was increased. However, the transgene did not change insulin sensitivity. These results show that the elevation in glycolysis provides acute benefits against hypoxia, but the chronic increase in glycolysis or reduction in fatty acid oxidation interferes with normal cardiac metabolism, which may be detrimental to the heart.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression is regulated by diet composition and ration size in liver of gilthead sea bream, Sparus aurata

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P(2)ase) gene expression by diet composition and ration size was studied in the liver of gilthead sea bream, Sparus aurata. From five different types of diet supplied to fish, those with either high carbohydrate/low protein or high carbohydrate/low lipid content stimulated 6PF-2-K/Fru-2,6-P(2)ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P(2)) values. The expression of the bifunctional enzyme and Fru-2,6-P(2) levels showed also direct dependence on the quantity of diet supplied. These findings demonstrate for the first time nutritional regulation of 6PF-2-K/Fru-2,6-P(2)ase at mRNA level by diet composition and ration size and suggest that the carnivorous fish S. aurata can adapt its metabolism, by stimulation of liver glycolysis, to partial substitution of protein by carbohydrate in the diet. In addition, the expression of 6PF-2-K/Fru-2,6-P(2)ase can be used as an indicator of nutritional condition.     

Phorbol 12,13-dibutyrate and mitogens increase fructose 2,6-bisphosphate in lymphocytes. Comparison of lymphocyte and rat-liver 6-phosphofructo-2-kinase

The influence of tumour promoters and growth factors on glycolysis and on fructose-2,6-bisphosphate concentration was studied in isolated mouse spleen lymphocytes and in purified B-cells. The intracellular concentration of fructose 2,6-bisphosphate and the rate of lactate release were increased 2-3-fold in spleen lymphocytes exposed to active phorbol esters, mitogenic lectins, interleukin 4 or lipopolysaccharide. The maximal effect was observed after 1 h of exposure. In these cells hexose 6-phosphates increased 2-fold and 6-phosphofructo-2-kinase activity remained unchanged after treatment with phorbol 12,13-dibutyrate or with lectins. Exposure of B-cells to phorbol 12,13-dibutyrate, interleukin 4 or lipopolysaccharide increased the glycolytic flux and the concentration of fructose 2,6-bisphosphate without relation to their mitogenic activity. Lymphocytes and rat liver 6-phosphofructo-2-kinase were partially purified using the same procedure. The lymphocyte enzyme was not inhibited by sn-glycerol 3-phosphate in contrast to the potent inhibition observed in liver. Treatment of both enzymes with the catalytic subunit of the cyclic-AMP-dependent protein kinase failed to inactivate 6-phosphofructo-2-kinase from lymphocytes. These differences suggest that lymphocytes and liver contain different forms of this enzyme.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

Yeast 6-phosphofructo-2-kinase: sequence and mutant.

We have reported yeast 6-phosphofructo-2-kinase (EC 2.7.1.105) as having a ca. 96-kDa subunit size, as well as isolation of its structural gene, PFK26. Sequencing now shows an open reading frame of 827 amino acids and 93.5 kDa. The deduced amino acid sequence has 42% identity with the 55-kDa subunit of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver with extra material at both ends. Although the yeast sequence is especially similar to the liver one in its bisphosphatase domain, the essential His-258 of the liver enzyme is, in yeast, a serine, which may explain the apparent lack of bisphosphatase activity. Also, the yeast enzyme known to be activated via protein kinase A, has a putative phosphorylation site near its C-terminus and lacks the N-terminal phosphorylation sequence involved in inhibition of the liver enzyme. In a chromosomal null mutant strain, pfk26::LEU2, activity was marginal and the protein was not detectable as antigen. The mutant strain grew well on glucose and contained a near-normal level of fructose 2,6-P2. But in its growth on pyruvate, by contrast with the wild-type strain, no fructose 2,6-P2 was detectable, and it did not form after glucose addition in the presence of cycloheximide either. Such resting cells, however, metabolized glucose at the normal high rate. Glucose addition to the pfk26 mutant strain in the absence of cycloheximide, on the other hand, caused a ca. 10% normal rate of fructose 2,6-P2 accumulation, presumably employing a glucose-inducible second enzyme. Using strains also lacking 6-phosphofructo-1-kinase, affinity chromatography revealed the second enzyme as a minor peak amounting to 6% of 6-phosphofructo-2-kinase activity in a PFK26 strain and as the sole peak, in similar amount, in a pfk26 mutant strain.     

Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes

The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6-phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin-independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.     

Fructose 2,6-bisphosphate in isolated foetal hepatocytes

Fru 2,6-P2 was present in isolated foetal hepatocytes at a concentration of 1.6 nmol per g cells. When foetal hepatocytes were exposed to glucagon no changes were observed either in the concentration of Fru 2,6-P2 and lactate release or in the activities of 6-phosphofructo-2-kinase and pyruvate kinase. Incubation of purified 6-phosphofructo-2-kinase with the catalytic subunit of protein kinase did not change the enzyme activity. The inhibition by sn-glycerol 3-phosphate was much lower for the foetal than for adult enzyme. These results suggest that an isoenzyme of 6-phosphofructo-2-kinase in foetal hepatocytes different from that of adult hepatocytes may be present.     

Preparation of monoclonal antibodies against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and their effects on enzyme activities

The effects of the monoclonal antibodies (McAbs) directed against chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) on the structure and function of the enzyme were studied. Using chicken liver 6PF-2-K/Fru-2,5-P2asc as antigen, 7 clones of monoclonal antibodies specifically binding with the antigen were obtained. The epitopes of the antigen recognized by the 6 McAbs localized on the fructose-2,6-bisphosphatase domain of chicken liver 6PF-2-K/Fru-2, 6-P2ase, and the other (H2) are on the 6-phosphofructo-2-kinase domain. All of the 7 McAbs could activate the kinase activity of the bifunctional enzyme by twofold and had a similar effect on the bisphosphatase activity of the bifunctional enzyme which resulted in a fourfold increase of the bisphosphatase activity of the bifunctional enzyme. However, the McAbs did not affect the activity of the separated fructose-2, 6-bisphosphatase domain. The results suggested that the Fru-2, 6-P2ases in the bifunctional enzyme and     

Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver.     

Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase

Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.