Showing papers on "Phosphofructokinase activity published in 1982"

Phosphofructokinases from Escherichia coli.

Abstract: Publisher Summary This chapter describes the enzymic assay and purification of two phosphofructokinases in Escherichia. coli K 12. The most studied one, PFK1, is coded by the pfk A gene, while the other enzyme, PFK2, is specified by the pfk B gene. The two enzymes differ in their biochemical and catalytic properties. Phosphofructokinase activity is routinely assayed by following the formation of fructose 1,6-bisphosphate, which is converted to a-glycerophosphate by aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase in the presence of β -NADH. Purification of PFK1 involves preparation of crude extract, affinity chromatography, concentration, and heat denaturation of protein contaminants. Unlike PFK1, PFK2 in crude extracts does not bind to Blue Dextran–Sepharose, while the heat treatment does not affect the biochemical properties of PFK1 or PFK2. ATP is the best phosphoryl donor for both PFK1 and PFK2, and fructose 6-phosphate is the best phosphoryl acceptor. Growth conditions have no influence on the biosynthesis of PFK2 but the relative amount of PFK1 depends on the nature of the carbon source and on the oxygen tension.

Phosphofructokinase from Streptococcus lactis.

Abstract: Publisher Summary This chapter describes an assay method of phosphofructokinase, purification of phosphofructokinase from Streptococcus lactis , and properties of this enzyme. Phosphofructokinase activity can be coupled via the enzymes fructose 1,6-bisphosphate aldolase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase to NADH oxidation. The enzyme activity can then be determined spectrophotometrically by measuring the decrease in absorbance at 340 nm. The purification steps involved are growth of bacteria, preparation of crude extract, ammonium sulfate fractionation, chromatography on Blue Dextran–Sepharose, and crystallization. Streptococcus lactis phosphofructokinase appears to be a tetramer of identical subunits and the native enzyme has a molecular weight (MW) of 144,500. The purified enzyme can be stored at -12 ° C in Tris-glycerol buffer for up to six months with only 10–20% loss in activity. The enzyme has a pH optimum of 7.2–7.8 in a number of different buffers, but in phosphate buffer, both the pH optimum and the maximum velocity are lowered. The chapter indicates that use of the Blue Dextran–Sepharose affinity column is a very effective purification step in the isolation of S. lactis phosphofructokinase giving high yields of homogeneous enzyme.

The control of glucose 1,6-bisphosphate by developmental state and hormonal stimulation in cultured muscle tissue.

Abstract: 1. The concentration of glucose 1,6-bisphosphate, a potent regulator of muscle glucose metabolism, was examined in embryonic muscle cells in culture. 2. The concentration in fused myotubes was twice that in unfused myoblasts. 3. The effect of various hormones and agonists on the glucose 1,6-bisphosphate concentration in both pre- and post-fusion muscle cells was examined. In pre-fusion cells no effect of adrenaline or cyclic AMP was observed, but stimulation by vasopressin, adrenaline + propranolol, ionophore A23187 and dibutyryl cyclic GMP significantly decreased glucose 1,6-bisphosphate. In post-fusion cells similar effects were observed, except that stimulation by adrenaline and by dibutyryl cyclic AMP significantly increased metabolite concentration. 4. All effects increased with time (over a 1 h period), except for that of vasopressin, which was transient. 5. The changes in glucose 1,6-bisphosphate concentration were accompanied by changes in the fructose 1,6-bisphosphate/fructose 6-phosphate ratio, implying an effect on phosphofructokinase activity.

Ultrastructural and enzymatic development of fetal guinea pig heart.

Abstract: The activities of some enzymes of glycolysis, the citric acid cycle, and amino acid metabolism have been measured in the fetal guinea pig heart over the last third of gestation and correlated with heart ultrastructural development. There is little change in glycolytic enzyme activity except for a two- to threefold increase in phosphofructokinase activity. Mitochondrial content and enzyme activities are low in the early fetal heart, and, although content is similar in the late fetus and adult, enzyme activities increase twofold postnatally, indicating fetal heart mitochondria are incompletely developed. The activities of aspartate and particularly alanine aminotransferase are low in the fetal heart. Over the last third of gestation the myofibrillar content of the fetal myocyte increases twofold to the adult value by term. Associated with this is a fourfold rise in myofibrillar and sarcoplasmic reticulum Ca2+-ATPase activity. Na+-K+-ATPase activity is similar in the late fetal and adult heart but one-third lower in the early fetal heart.

Enzyme adaptations in rat skeletal muscle after two intensities of treadmill training.

Abstract: The effects of high intensity, short duration chronic exercise (HSD, N = 9) and low intensity, long duration chronic exercise (LLD, N = 9) on the selected enzyme activities of three muscles of rats were studied. After 12 wk of treadmill training, the LLD group showed 15-23% decreases in lactate dehydrogenase activity in all muscles (P less than 0.05), while succinic oxidase activity increased in the soleus (49%) and the vastus lateralis profundus (42%) (P less than 0.05). No change in creatine kinase activity was found in either group. The HSD group showed no change in lactate dehydrogenase activity; however, phosphofructokinase activity increased (greater than 50%) in both of the fast-twitch muscles (P less than 0.01), while only the vastus lateralis superficialis showed increased succinic oxidase activity (30-40%) (P less than 0.05). Following training no difference was observed in mean heart weights; however, the mean body weight of the sedentary control group (SC, N = 9) was greater than both exercise groups (P less than 0.05). No difference was observed between the exercise groups. The mean heart weight/body weight ratios were significantly different between all groups (P less than 0.01). These results indicated that the HSD group selectively increased its glycolytic power, while the LLD group increased only its oxidative ability. Furthermore, both exercise groups exhibited less gains in body weight than the non-exercise group. This change was independent of any work performed.

Crossover plot study of glycolytic intermediates in the ischemic canine heart.

Abstract: Effects of regional ischemia on myocardial glycolysis were studied by measuring the levels of glycolytic intermediates in the endo- and epicardial layers of the left ventricle in dogs anesthetized with pentobarbital. Regional ischemia was induced by ligating a small branch of the left anterior descending coronary artery. The myocardial tissue samples were removed before and 1.5, 3, 7, or 30min after coronary artery ligation. Based on a crossover plot study of the glycolytic intermediates, it is suggested that the activity of glycogen phosphorylase was accelerated, while that of phosphofructokinase was inhibited in ischemic myocardium samples removed 1.5, 3, 7, and 30min after ligation. When the frozen myocardium was allowed to stand at room temperature for 10min, the crossover plot study revealed acceleration of phosphofructokinase activity. The metabolic response to regional ischemia of the endocardial layers was more marked than that of the epicardial. During ischemia the levels of adenine nucleotides did not change significantly, but those of citrate and hydrogen ions increased significantly. It appears that inhibition of myocardial phosphofructokinase activity during ischemia is partly due to an increase in the levels of citrate and hydrogen ions in the ischemic tissue.

Modulation of chloroplast phosphofructokinase by NADPH : a mechanism for linking light to the regulation of glycolysis.

Abstract: Phosphofructokinase has been partially purified from spinach (Spinacia oleracea) chloroplasts and studied from the standpoint of light/dark regulation. At concentrations reported to occur physiologically, NADPH effected a sharp inhibition of the enzyme by: (a) lowering its affinity (increasing the apparent Km) for both of its substrates, ATP and fructose 6-phosphate; and (b) lowering its Vmax. Inhibition by NADPH was independent of pH and was observed both at pH 7.9 (pH of chloroplast stroma in the light) and pH 7.0 (stromal pH in the dark). The results are consistent with the conclusion that NADPH provides a mechanism for linking light to the modulation of phosphofructokinase activity and thereby to the regulation of glycolysis in chloroplasts.

Effects of pregnancy and lactation on the activities in sheep liver of some enzymes of glucose metabolism

Abstract: The activities of some enzymes that regulate the gluconeogenic-glycolytic pathway were measured in sheep liver during late pregnancy, at three stages of lactation and after the lambs were weaned. Of the gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxylase activities were unchanged throughout pregnancy and lactation and were similar to the values for non-breeding ewes. The activity of fructose diphosphatase increased 2 times and that of pyruvate carboxylase increased 5–7 times during late pregnancy and in early lactation.In the glycolytic sequence phosphofructokinase activity declined by about 50% during lactation. Pyruvate kinase activity declined by about 40% at the end of pregnancy but throughout lactation it was similar to the value for non-breeding ewes. Hexokinase activity was very low in all animals.

5-Hydroxytryptamine, glucose uptake, glycogen utilization and carbon dioxide fixation in Hymenolepsi microstoma (Cestoda)

Abstract: 1. In vitro glucose uptake and glycogen utilization by Hymenolepis microstoma decreased under high oxygen concentrations. 2. 5-Hydroxytryptamine did not stimulate in vitro glucose uptake but did increase glycogen utilizations by H. microstoma. 3. The reduced glucose uptake under high oxygen concentrations (21 and 95%) resulted in a reduction in excretory products. 4. 14CO2-incorporation studies confirmed that, under both 95% O2:5% CO2 and air-minus-CO2 (identical to 21% O2). CO2-fixation by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) was inhibited. 5. The specific activity of hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) was not stimulated by 5-HT. 6. The concentration of ATP required for optimal stimulation of phosphofructokinase activity was 0.67 mM. Activity was further significantly increased by the addition of cAMP and even greater by AMP.

Rapid modulation of adipocyte phosphofructokinase activity by noradrenaline and insulin.

Abstract: 1. Alterations in phosphofructokinase properties can be reproducibly seen in tissue extracts prepared and rapidly assayed after exposure of rat adipocytes to hormones. 2. Noradrenaline, corticotropin or isoprenaline (isoproterenol; beta-adrenergic agonist) decreased the activity measured with high fructose 6-phosphate concentrations (3--6 mM), but increased activity measured with lower concentrations of this substrate (0.3--0.9 mM). Noradrenaline decreased the Vmax. and the concentration of fructose 6-phosphate that gave half the Vmax.. 3. Insulin opposed the actions of noradrenaline and itself increased phosphofructokinase activity. 4. The effect of noradrenaline appeared to be exerted through a beta- rather than an alpha-type of adrenoceptor. 5. The effects of noradrenaline to decrease phosphofructokinase activity at high [fructose 6-phosphate] and to increase activity at low [fructose 6-phosphate] could be rapidly reversed in cells by addition of the beta-blocker propranolol. 6. The effect of noradrenaline seen at low [fructose 6-phosphate] could be abolished by homogenization of cells in buffer containing albumin or reversed by brief incubation of tissue extracts with albumin, suggesting that this effect of the hormone is due to the association of some ligand with the enzyme.

Mechanisms of the protective effect of methohexital on cerebral energy metabolism

Abstract: The purpose of the present investigation was to study the effect of alpha-(+/-)-5-allyl-1-methyl-5-(1-methyl-2-pentinyl) barbituric acid (methohexital, Brevimytal sodium) on brain energy metabolism. The model of the isolated perfused rat brain was used. The high-energy phosphates, some substrates of glycolysis and the intracellular distribution of hexokinase activity were measured in the cortical tissue of the isolated rat brain after 30 min of perfusion, after ischemia or anoxia and after various recovery periods. The EEG was recorded as a parameter of the neuronal activity. The following results were obtained: 1. Ischemia and anoxia accelerated the glycolysis rate which was inhibited by methohexital. 2. The energy metabolism was more rapidly normalized after ischemia or anoxia when methohexital was added to the perfusion medium (0.2 mmol/l). 3. Compared to the corresponding preparation, the spontaneous electrical activity of the isolated brain was maintained in ischemia or anoxia for a longer period and appeared again more rapidly in the recovery periods when methohexital was present. 4. Phosphofructokinase activity was inhibited by methohexital in the recovery period after ischemia or anoxia, respectively. 5. Inhibition of hexokinase activity predominated in the surgical stage of anesthesia when glycolysis rate was not accelerated. Methohexital seemed to inhibit hexokinase activity by solubilizing the mitochondrial bound form.