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 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 β-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 β-glucose 1,6-bisphosphate from β-glucose 1-phosphate and ATP is described.     

The mechanism by which ethanol decreases the concentration of fructose 2,6-bisphosphate in the liver.

The intragastric administration of ethanol to fed rats caused in their liver, within about 1 h, a 20-fold decrease in the concentration of fructose 2,6-bisphosphate, an activation of fructose 2,6-bisphosphatase, an inactivation of phosphofructo-2-kinase but no change in the concentration of cyclic AMP. Incubation of isolated hepatocytes in the presence of ethanol caused a rapid increase in the concentration of sn-glycerol 3-phosphate and a slower and continuous decrease in the concentration of fructose 2,6-bisphosphate with no change in that of hexose 6-phosphates. There was also a relatively slow activation of fructose 2,6-bisphosphatase and inactivation of phosphofructo-2-kinase. Glycerol and acetaldehyde had effects similar to those of ethanol on the concentration of phosphoric esters in the isolated liver cells. 4-Methylpyrazole cancelled the effect of ethanol but reinforced those of acetaldehyde. High concentrations of glucose or of dihydroxyacetone caused an increase in the concentration of hexose 6-phosphates and counteracted the effect of ethanol to decrease the concentration of fructose 2,6-bisphosphate. As a rule, hexose 6-phosphates had a positive effect and sn-glycerol 3-phosphate had a negative effect on the concentration of fructose 2,6-bisphosphate in the liver, so that, at a given concentration of hexose 6-phosphates, there was an inverse relationship between the concentration of fructose 2,6-bisphosphate and that of sn-glycerol 3-phosphate. These effects could be explained by the ability of sn-glycerol 3-phosphate to inhibit phosphofructo-2-kinase and to counteract the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate. sn-Glycerol 3-phosphate had also the property to accelerate the inactivation of phosphofructo-2-kinase by cyclic AMP-dependent protein kinase whereas fructose 2,6-bisphosphate had the opposite effect. The changes in the activity of phosphofructo-2-kinase and fructose 2,6-bisphosphatase appear therefore to be the result rather than the cause of the decrease in the concentration of fructose 2,6-bisphosphate.     

Effects of glucose and glucagon on the fructose 2,6-bisphosphate content of pancreatic islets and purified pancreatic B-cells. A comparison with isolated hepatocytes.

Glucose caused a sustained and dose-related increase in the fructose 2,6-bisphosphate content of isolated pancreatic islets, as well as of purified pancreatic B-cells. With isolated B-cells, the glucose saturation curve was sigmoidal and superimposable on that obtained with hepatocytes isolated from unfed rats. However, the response to glucose was notably faster in purified B-cells than in isolated hepatocytes. In contrast again with the situation prevailing in the liver, glucagon failed to decrease significantly the concentration of fructose 2,6-bisphosphate in either islets or purified B-cells. It is proposed that, in the process of glucose-stimulated insulin secretion, an early increase in fructose 2,6-bisphosphate formation may, by causing activation of 6-phosphofructo-1-kinase, allow glycolysis to keep pace with the rate of glucose phosphorylation.     

Inactivation of phosphofructokinase 2 by cyclic AMP - dependent protein kinase

Treatment with the catalytic subunit of cyclic AMP-dependent protein kinase induced the following modifications in the kinetic properties of purified phosphofructokinase 2. The affinity for Fru-6-P, the V_max and the stimulatory effect of P_i were decreased; the inhibitory actions of P-enol-pyruvate and citrate were increased; the pH activity curve, measured in the presence of 5 mM Fru-6-P and 5 mM P_i was modified in the respect that the peak of activity normally measured at pH 6.6 was abolished whereas no effect of the treatment was observed at pH 8. Similar changes in the properties of phosphofructokinase 2 were also observed in a crude preparation obtained from hepatocytes incubated with glucagon.     

High residual activity of PMM2 in patients' fibroblasts: possible pitfall in the diagnosis of CDG-Ia (phosphomannomutase deficiency)

Congenital disorders of glycosylation (CDGs) are a rapidly enlarging group of inherited diseases with abnormal N-glycosylation of glycoconjugates. Most patients have CDG-Ia, which is due to a phosphomannomutase (PMM) deficiency. In this article, we report that a significant portion (9 of 54) of patients with CDG-Ia had a rather high residual PMM activity in fibroblasts included in the normal range (means of the controls +/- 2 SD) and amounting to 35%-70% of the mean control value. The clinical diagnosis of CDG-Ia was made difficult by the fact that most (6 of 9) of these patients belong to a subgroup characterized by a phenotype that is milder than classical CDG-Ia. These patients lack some of the symptoms that are suggestive for the diagnosis, such as inverted nipples and abnormal fat deposition, and, as a mean, had higher residual PMM activities in fibroblasts (2.05+/-0.61 mU/mg protein, n=9; vs. controls 5.34+/-1.74 mU/mg protein, n=22), compared with patients with moderate (1.32+/-0.86 mU/mg protein, n=18) or severe (0.63+/-0.56 mU/mg protein, n=27, P<.001) cases. Yet they all showed mild mental retardation, hypotonia, cerebellar hypoplasia, and strabismus. All of them had an abnormal serum transferrin pattern and a significantly reduced PMM activity in leukocytes. Six of the nine patients with mild presentations were compound heterozygotes for the C241S mutation, which is known to reduce PMM activity by only approximately 2-fold. Our results indicate that intermediate PMM values in fibroblasts may mask the diagnosis of CDG-Ia, which is better accomplished by measurement of PMM activity in leukocytes and mutation search in the PMM2 gene. They also indicate that there is some degree of correlation between the residual activity in fibroblasts and the clinical phenotype.     

Rapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.