Glycolytic enzyme activities in liver, heart and gastrocnemius of Sprague-Dawley and Wistar rats: effect of high sucrose diet

1. Pyruvate kinase, phosphofructokinase, and glucophosphate isomerase activities were measured in liver, heart and gastrocnemius of Wistar and Sprague-Dawley rats. 2. Enzyme activities were significantly lower in tissues of Wistar rats except for pyruvate kinase in gastrocnemius. 3. Sensitivities of pyruvate kinase and phosphofructokinase to inhibition by alanine and citrate differed in these two strains except for pyruvate kinase in gastrocnemius. 4. Phosphofructokinase sensitivity to citrate was greater in the three Wistar tissues. 5. Activities of liver enzymes were more responsive to a high sucrose diet in Wistar rats. 6. Heart pyruvate kinase and phosphofructokinase exhibited modest increases in activity with a high sucrose diet.     

Maximum activities and effects of fructose bisphosphate on pyruvate kinase from muscles of vertebrates and invertebrates in relation to the control of glycolysis

1. Comparison of the maximum activities of pyruvate kinase with those of phosphofructokinase in a large number of muscles from invertebrates and vertebrates indicates that, in general, in any individual muscle, the activity of pyruvate kinase is only severalfold higher than that of phosphofructokinase. This is consistent with the suggestion, based on mass-action ratio data, that the pyruvate kinase reaction is non-equilibrium in muscle. However, the range of activities of pyruvate kinase in these muscles is considerably larger than that of phosphofructokinase. This difference almost disappears if the enzyme activities from muscles that are known to possess an anaerobic ;succinate pathway' are excluded. It is suggested that, in these muscles, phosphofructokinase provides glycolytic residues for both pyruvate kinase (i.e. glycolysis) and phosphoenolpyruvate carboxykinase (i.e. the succinate pathway). This is supported by a negative correlation between the activity ratio, pyruvate kinase/phosphofructokinase, and the activities of nucleoside diphosphokinase in these muscles, since high activities of nucleoside diphosphokinase are considered to indicate the presence of the succinate pathway. 2. The effect of fructose bisphosphate on the activities of pyruvate kinase from many different muscles was studied. The stimulatory effect of fructose bisphosphate appears to be lost whenever an efficient system for supply of oxygen to the muscles is developed (e.g. insects, squids, birds and mammals). This suggests that activation of pyruvate kinase is important in the co-ordinated regulation of glycolysis in anaerobic or hypoxic conditions, when the change in glycolytic flux during the transition from rest to activity needs to be large in order to provide sufficient energy for the contractile activity. However, lack of this effect in the anaerobic muscles of the birds and mammals suggests that another metabolic control may exist for avian and mammalian pyruvate kinase in these muscles.     

Age-related changes in activities of hepatic phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in liver and adipose tissue of the swiss albino mouse

The activities of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase were examined in liver as a function of age in Swiss albino mice. The hepatic activity of phosphofructokinase and total pyruvate dehydrogenase peaked in mice between 8 and 12 weeks of age and then decreased to a value that remained stable in mature animals older than 24 weeks of age. Yet, the activity of pyruvate kinase and pyruvate dehydrogenase in the active form in liver remained unchanged in mice up to 12 weeks of age. As mice matured, a progressive increase in the activity of both pyruvate kinase and the active form of pyruvate dehydrogenase in liver was observed while phosphofructokinase was unaltered. The pyruvate dehydrogenase complex, both total activity and the proportion of the enzyme in the active form, in the epididymal fat pad of the mouse showed no consistent age trend. The observed increase in the activity of both pyruvate kinase and the active form of pyruvate dehydrogenase should provide an augmented capacity for the generation of acetyl-CoA units for de novo fatty acid synthesis in livers of mature mice.     

Activation of phosphofructokinase and pyruvate kinase by 6 phosphogluconate in chicken liver (Gallus domesticus): no evidence for a regulatory role

Phosphofructokinase and pyruvate kinase in crude extracts of chick liver were activated by 6 phosphogluconate at subsaturating concentrations of their respective substrates. The apparent Ka for phosphofructokinase was 0.79 mM and for pyruvate kinase 0.44 mM. Total pentose phosphate dehydrogenase activity was low in the livers of both fed and starved chicks. Hepatic 6 phosphogluconate concentration was too low to be measured accurately by a conventional spectrophotometric method. It is concluded that the concentration of 6 phosphogluconate is too low for it to have a role in the regulation of phosphofructokinase and pyruvate kinase activities in chicken liver.     

Effects of chronic hyperinsulinaemia on hepatic enzymes involved in lipogenesis and carbohydrate metabolism in the young rat.

Chronic (6 days) hyperinsulinaemia in young rats produced lower blood glucose concentrations and augmented body- and liver-weight gain. The insulin-treated rats had increased hepatic activities of citrate-cleavage enzyme, 'malic' enzyme and high-substrate (6.6 mM-phosphoenolpyruvate) pyruvate kinase, and decreased glucose 6-phosphatase. There were no changes in activities of phosphoenolpyruvate carboxykinase, phosphofructokinase, low-substrate (1.3 mM-phosphoenolpyruvate) pyruvate kinase, glucokinase and hexokinase.     

2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

METABOLIC CONTROL MECHANISMS IN MAMMALIAN SYSTEMS

Abstract— The regulation by thyroid hormone of the activities of hexokinase (ATP: D-hexose 6-phosphotransferase; EC 2.7.1.1), phosphofructokinase (ATP: D-fructose-6- phosphate 1-phosphotransferase; EC 2.7.1.11) and pyruvate kinase (ATP: pyruvate phosphotransferase; EC 2.7.1.40) has been investigated in the soluble fractions of the cerebral cortex and cerebellum of the rat. Ontogenetic studies on these key glycolytic enzymes demonstrated marked increases in the normal cerebral cortex between 1 day and 1 yr of age; less pronounced increases in enzyme activities were noted in the normal cerebellum. Neonatal thyroidectomy, induced by treatment of 1-day-old rats with 100 μCi of ¹³¹I, ied to an impairment of body and brain growth and inhibited the developmental increases in hexokinase, phosphofructokinase and pyruvate kinase in both the cerebral cortex and cerebellum. Whereas 50 μCi of ¹³¹I had little or no effect on these brain enzymes, 200 μCi of the radioisotope markedly inhibited (35–65 per cent) the developmental increases of the various enzyme activities investigated. When administration of the radioisotope was delayed for 20 days after birth, little or no inhibition of the development of brain glycolytic enzymes was observed. Whereas treatment of normal neonatal animals with L-tri-iodothyronine had no significant effect on the activities of cerebro-cortical and cerebellar glycolytic enzymes, the hormone increased their activities in young cretinous rats. However, when the initiation of tri-iodothyronine treatment was delayed until neonatally thyroidectomized rats had reached adulthood, this hormone failed to produce any appreciable change in enzyme activity. Our results indicate that thyroid hormone exerts an important regulatory influence on the activities of hexokinase, phosphofructokinase and pyruvate kinase in the developing cerebral cortex and cerebellum.     

Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase

In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated. The approach is based on a stirred flow-through reaction chamber. The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase.     

Undifferentiated patterns of key carbohydrate-metabolizing enzymes in injured livers. I. Acute carbon-tetrachloride intoxication of rat.

In acute CCL4 intoxication of rats significantly increased activities of hepatic low-Km hexokinases, glucose-6-phosphate dehydrogenase, phosphofructokinase, aldolase A and pyruvate kinase M2 with concurrently decreased activities of glucokinase, glucose-6-phosphatase, fructose-1,6-diphosphatase, aldolase B and pyruvate kinase L were observed. The resulting enzyme pattern was apparently different from that in dietary induction. Principal component analysis revealed that the degree of enzyme deviation in the injured liver was much greater than that in the regenerating liver after partial hepatectomy and was closer to that in fetal liver or hepatoma tissue.     

Activity and isoenzyme patterns of glycolytic enzymes during perinatal development of rat lung

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.     

Regulatory enzymes of carbohydrate and energy metabolism in the rabbit blastocyst.

The activities of phosphofructokinase, pyruvate kinase, citrate synthase and creatine kinase were determined in blastocysts from rabbits at 144 h post coitum and in similar blastocysts cultured for 24 h with or without oestradiol-17beta (1 microgrm/ml). There was a significant increase in all the enzymes during the 24-h culture period but oestradiol had no effect.     

Studies of regulatory metabolism in Moniezia expansa: general considerations.

The activities of selected enzymes in the branched metabolic pathway to succinate or lactate were determined in cytosol and mitochondrial fractions. The enzymes of lowest activity in the cytosol, and thus possibly regulatory, are phosphofructokinase and pyruvate kinase. Malic enzyme activity could scarcely be detected in either compartment; phosphoenolpyruvate carboxykinase and malate dehydrogenase occur in both. The end products of metabolism are succinate and lactate; under anaerobic conditions lactate production increases whereas succinate production shows a small decrease. The presence of glucose in the medium does not influence the change, but causes an increase in total endproduct accumulation. Levels of metabolic intermediates in worms incubated aerobically and anaerobically are presented, and ‘cross-over’ plots and calculations of apparent equilibrium constants identify hexokinase, phosphofructokinase and pyruvate kinase as regulatory. Under aerobic conditions a large increase in the size of the malate pool is observed suggesting that the depression of lactate production is produced by its inhibitory effect on pyruvate kinase. Adenine nucleotide levels are maintained whether or not the worm is incubated under anaerobic conditions.     

Glycolytic activity of rat aorta after exercise

Activities of glycolytic enzymes in the aorta were investigated in female Wistar rats. There were two groups of rats; one served as the control (sedentary rats), while the other group was forced to run on a treadmill for 10 weeks. In the control animals, the activities of hexokinase, phosphofructokinase and aldolase were relatively lower than those of the other glycolytic enzymes (phosphoglucose isomerase, lactate dehydrogenase and pyruvate kinase). After exercise, the activity of phosphofructokinase increased by 15%, whereas the other enzymatic activities were much the same as in the controls. Within the limits of the experiments, the increased percentage of phosphofructokinase was statistically significant (p less than 0.05). Since phosphofructokinase is a putative rate limiting enzyme, this enzymatic activation may indicate that glycolytic activity in the rat aorta is enhanced during and after running exercise.     

Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis

Abstract The bradyzoite and tachyzoite forms of Toxoplasma gondii , purified from infected animals, were analysed for their activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD⁺- and NADH-linked isocitrate dehydrogenases, and succinic dehydrogenase. Both developmental stages contained high activities of phosphofructokinase (specific for pyrophosphate rather than ATP), pyruvate kinase and lactate dehydrogenase, suggesting that energy metabolism in both forms may centre around a high glycolytic flux linked to lactate production. The markedly higher activity of the latter two enzymes in bradyzoites suggests that lactate production is particularly important in this developmental form. NAD⁺-specific isocitrate dehydrogenase was not detectable in either stage of the parasite (and proved useful as a measure of the purity of the bradyzoite preparation), whereas both parasite forms contained low activities of NADP⁺-linked isocitrate dehydrogenase. The results are consistent with the bradyzoites lacking a functional TCA cycle and respiratory chain and are suggestive of a lack of susceptibility of this developmental stage to atovaquone.     

Metabolic adaptation of the renal carbohydrate metabolism. II

Summary The effects of a high carbohydrate diet on the renal gluconeogenic and glycolytic capacities and on the activities of the main enzymes of the carbohydrate metabolism, fructose 1,6-bisphosphatase, phosphofructokinase and pyruvate kinase have been studied. These parameters have been analysed in two separate and isolated fractions of the renal tubule, the proximal convoluted (PCT) and the distal convoluted (DCT) zones. The results presented in this study show a rapid adaptation capacity of the kidney in response to the high amount of dietary carbohydrate, which are characterized by a decrease in the glucose production and fructose 1,6-bisphosphatase activity in the proximal tubules, and an increase in the glycolytic flux and phosphofructokinase and pyruvate kinase activities in the distal tubules. The changes in these enzyme activities took place only at subsaturating substrate concentrations and not at maximum velocity which suggest that they are probably due to an allosteric and/or covalent modifications and so, they are independent of variations in the cellular levels of the enzymes.     

Alterations in glucose metabolism in chick embryo cells transformed by Rous sarcoma virus. Transformation-specific changes in the activities of key enzymes of the glycolytic and hexose monophosphate shunt pathways

Effects of transformation by Rous sarcoma virus of Schmidt-Ruppin strain on the activities of key enzymes of the glycolytic and the hexose monophosphate shunt pathways in chick-embryo cells were investigated. Activities of hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and glucose-6-P dehydrogenase were increased about twofold in the transformed cells, but that of 6-P-gluconate dehydrogenase remained unaltered. The transformation-mediated increase in the activity of hexokinase was confined entirely to the bound form of the enzyme. Cells infected with a temperature-sensitive mutant (Ts-68) of Schmidt-Ruppin strain of Rous sarcoma virus showed the typical increase in the rate of 2-deoxyglucose uptake and the activities of hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-P dehydrogenase at the permissive temperature (37 °C), but when the infected cells were grown at the nonpermissive temperature (41 °C), the increases in the sugar uptake and activities of these enzymes were abolished. Unlike the regulatory enzymes, lactate dehydrogenase activity was increased at both the permissive and the nonpermissive temperatures.     

Regulation of pyruvate kinase expression and growth in mastocytoma cells : I. Initial observations

The specific activities of pyruvate kinase and phosphofructokinase but not lactate dehydrogenase increase as P-815 mastocytoma cells approach the stationary phase. During this growth period, the rates of uptake of labelled precursors into DNA, RNA and total protein decreases. On the other hand, the pyruvate kinase protein level changes in parallel with activity. Although the K-isozyme is the primary form of pyruvate kinase expressed, some M-type subunit is also present and both forms undergo an increase in specific activity. In addition, pyruvate kinase expression is also elevated by adding cAMP analogues with theophylline, butyrate or conditioned media. This increased level of expression is hypothesized to be a secondary event associated with a differentiation-like-induced expression of the mast cell phenotype.     

Phosphofructokinase and pyruvate kinase in mouse embryonal carcinoma P19 cells in relation to growth and differentiation

Two key enzymes of glycolysis, phosphofructokinase and pyruvate kinase, were studied in embryonal carcinoma cells (P19 EC cells) and three differentiated derivatives in relation to growth rate and differentiation state. The growth rates of P19 EC cells and its differentiated derivatives are positively correlated with both the specific activity of phosphofructokinase and the expression of the L-subunit of this enzyme. The specific activity of pyruvate kinase and its isozyme composition is not correlated with growth rate but seems to be correlated with the differentiation state of these cells. The decrease in specific activity of pyruvate kinase during differentiation of P19 EC cells induced by retinoic acid or dimethylsulfoxide preceded the shift from K- to M-type pyruvate kinase. In contrast to aggregates that were treated with dimethylsulfoxide, the specific activity of pyruvate kinase was reduced after aggregation in the presence of retinoic acid. Only after plating dimethylsulfoxide-treated aggregates again in the presence of dimethylsulfoxide, was a decrease in specific activity obtained. Both retinoic acid and dimethylsulfoxide are able to induce a K- to -M shift of pyruvate kinase.     

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).