Increase in glucose 1,6-bisphosphate levels, activation of phosphofructokinase and phosphoglucomutase, and inhibition of glucose 1,6-bisphosphatase in muscle induced by trifluoperazine

Injection of trifluoperazine (TFP) to rats induced a significant rise in the level of glucose 1,6-bisphosphate (Glc-1,6-P2) in muscle. This increase in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked activation of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. Glucose-1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2) was markedly inhibited following the injection of TFP, which may account for the rise in the Glc-1,6-P2 level. Previous results from this laboratory have revealed that muscle damage or weakness is characterized by a decrease in Glc-1,6-P2 levels, leading to a marked reduction in the activities of phosphoglucomutase and phosphofructokinase (the rate-limiting enzyme in glycolysis). The present results suggest that TFP treatment may have a beneficial effect on the depressed glycolysis in muscle weakness or damage.     

Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle

Two enzymes which possess 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities have been purified from pig skeletal muscle. One of the enzymes corresponds to type M phosphoglycerate mutase. The other enzyme shows properties similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase present in mammalian erythrocytes. The erythrocyte and the muscle enzyme possess the same molecular (56 000) and subunit (27 000) weights. The synthase, phosphatase and mutase activity ratio is similar in both enzymes, and they are affected by the same inhibitor (glycerate 3-P) and activators (glycolate 2-P, pyrophosphate, sulfite and bisulfite).     

Oscillatory synthesis of glucose 1,6-bisphosphate and frequency modulation of glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity, due to autocatalytic activation by fructose-1,6-P2. Glucose-1,6-P2 similarly might activate phosphofructokinase in an autocatalytic manner, because it is produced in a side reaction of phosphofructokinase and in a side reaction of phosphoglucomutase using fructose-1,6-P2. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, glucose-1,6-P2 accumulated in a stepwise, but monotonic, manner to 0.7 microM in 1 h. The stepwise increases occurred during the phases when fructose-1,6-P2 was available, consistent with glucose-1,6-P2 synthesis in the phosphoglucomutase side reaction. Addition of 5-20 microM glucose-1,6-P2 increased the frequency of the oscillations in a dose-dependent manner and progressively shortened the time interval before the first burst of phosphofructokinase activity. Addition of 30 microM glucose-1,6-P2 blocked the oscillations. The peak values of the [ATP]/[ADP] ratio were then eliminated, and the average [ATP]/[ADP] ratio was reduced by half. In the presence of higher, near physiological concentrations of ATP and citrate (which reduce the activation of phosphofructokinase by glucose-1,6-P2), high physiological concentrations of glucose-1,6-P2 (50-100 microM) increased the frequency of the oscillations and did not block them. We conclude that autocatalytic activation of phosphofructokinase by fructose-1,6-P2, but not by glucose-1,6-P2, is the mechanism generating the oscillations in muscle extracts. Glucose-1,6-P2 may nevertheless play a role in facilitating the initiation of the oscillations and in modulating their frequency.     

The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofructokinases

The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-P2 show identical effects on the allosteric kinetic properties of both enzymes. Fructose-2,6-P2 counteracts inhibition by ATP and citrate and decreases the Km for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The Km values of liver and muscle phosphofructokinase for fructose-2,6-P2 in the presence of 1.25 mM ATP are 12 milliunits/ml (or 24 nM) and 5 milliunits/ml (or 10 nM), respectively. At near physiological concentrations of ATP (3 mM) and fructose-6-P (0.2 mM), however, the Km values for fructose-2,6-P2 are increased to 12 microM and 0.8 microM for liver and muscle enzymes, respectively. Thus, fructose-2,6-P2 is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-P2. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-P2, but the rates become linear in the presence of higher concentrations of fructose-2,6-P2. Fructose-2,6-P2 also protects phosphofructokinase against inactivation by heat. Fructose-2,6-P2, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.     

Exogenous ATP antagonizes the actions of phospholipase A2, local anesthetics, Ca2+ ionophore A23187, and lithium on glucose-1,6-bisphosphate levels and the activities of phosphofructokinase and phosphoglucomutase in rat muscle

ATP, added externally to the incubation medium of rat diaphragm muscles, abolished the decrease in the levels of glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, induced by phospholipase A2, local anesthetics, Ca2+ ionophore A23187, or lithium. Concomitantly to the changes in Glc-1,6-P2, the potent activator of phosphofructokinase (the rate-limiting enzyme in glycolysis) and phosphoglucomutase, the activities of these enzymes were reduced by the myotoxic agents and restored by exogenous ATP, when assayed under conditions in which these enzymes are sensitive to regulation by Glc-1,6-P2. These findings suggest that ATP may have broad therapeutic action, as it may stimulate the impaired glycolysis in muscle induced by various drugs and conditions which cause muscle weakness or damage.     

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.     

Fructose 2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity due to autocatalytic activation by fructose-1,6-P2. Fructose-2,6-P2 is an even more potent activator of phosphofructokinase and is competitive with fructose-1,6-P2 in binding and kinetic studies. The possible role and effects of fructose-2,6-P2 on the oscillating system were therefore examined. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, fructose-2,6-P2 slowly accumulated to 50 nM in 1 h. The nearly monotonic rise, in contrast to the 50-fold oscillations in fructose-1,6-P2, indicated no involvement of fructose-2,6-P2 in the oscillatory process. Addition of 0.5 microM fructose-2,6-P2 blocked the oscillations, and there was negligible appearance of glycolytic intermediates from fructose-1,6-P2 to phosphoenolpyruvate, although similar amounts of lactate accumulated. In the presence of 0.2 microM fructose-2,6-P2, there were small, transient accumulations of fructose-1,6-P2, suggesting aborted activations of phosphofructokinase. Oscillations were not blocked by 0.1 microM fructose-2,6-P2. The average [ATP]/[ADP] ratio in the presence of 0.2 or 0.5 microM fructose-2,6-P2 was half the value in its absence, demonstrating the advantage of the oscillatory behavior in maintaining a high energy state. In the presence of higher, near physiological levels of ATP and citrate, inhibitors which reduce the affinity of phosphofructokinase for fructose-2,6-P2, glycolytic oscillations were not blocked by 1 microM fructose-2,6-P2, its approximate concentration in vivo.     

Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells.

Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs. By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P; these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes.     

Kinetics of the D and I glycogen synthetase forms and their interconversion, in the sea scallop Pecten maximus.

In extracts from the adductor muscle of the shell-fish, Pecten maximus, glycogen synthetase (EC.2.4.1.11) was found. The enzyme occurs predominantly as D form (glucose-6-P dependent for activity). An I form (G-6-P independent) was also present. Kinetics of glycogen synthetase showed that the Ka for G-6-P in the D form was 10 fold higher than in the I form. Both forms of glycogen synthetase were interconverted through reactions catalyzed by phosphatase and kinase enzymes respectively. Glucose-6-P and Mg+2 must be present to stabilize glycogen synthetase and to activate the synthetase D phosphatase, found in the 90,000 X g protein-glycogen complex. The conversion of synthetase D to I was inhibited by F-, glycogen, ATP and UTP. When F- was present the effect of G-6-P on synthetase and phosphatase suggested that conversion involved the existence of more than a single glycogen synthetase phosphatase enzyme. ATP and Mg+2 were necessary for the conversion of synthetase I to D, and the conversion was stimulated by cAMP.     

Functional characterization of the enzymes with 2,3-bisphosphoglycerate phosphatase activity from pig skeletal muscle

In pig skeletal muscle exist four enzymes with 2,3-bisphosphoglycerate phosphatase activity. Two of them (forms I-A and I-C) are multi-functional enzymes which, in addition to the phosphatase activity, possess 2,3-bisphosphoglycerate synthase and phosphoglycerate mutase activities. The other two enzyme forms (II-A and II-B) only show the phosphatase activity. The four enzymes differ in substrate specificity. Form I-C is highly specific for glycerate 2,3-P2; form I-A also hydrolyzes the monophosphoglycerates and forms II-A and II-B are specific for phosphoester bonds adjacent to a C-1 carboxylic group. The enzymes possess similar Km, Kcat and optimum pH value, but they are differently inhibited by the reaction products. They are also differently affected by glycolate-2-P (their main activator) and by other modifiers. Probably form I-A, which corresponds to M-type phosphoglycerate mutase, is the main enzyme implicated in the breakdown of glycerate 2,3-P2 in pig muscle.     

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.     

Changes in the levels of glucose 1,6-diphosphate and cyclic GMP, and in the activities of phosphofructokinase and phosphoglucomutase induced by serotonin in muscle

Injection of serotonin (5-hydroxytryptamine) induced a marked decrease in the level of glucose 1,6-diphosphate (Glc-1,6-P2) in the rat tibialis anterior muscle. Concomitant to the decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, the activities of both these enzymes were markedly reduced by serotonin. The level of Glc-1,6-P2 and the activities of phosphofructokinase and phosphoglucomutase increased with age in the tibialis anterior muscle and the effect of serotonin was more pronounced in the older animals. Serotonin also induced a significant increase in the level of cyclic GMP in muscle. The serotonin-induced changes in the normal muscle mimic the changes in carbohydrate metabolism we found previously in muscular dystrophy.     

Trifluoperazine abolishes the actions of bradykinin on glucose 1,6-bisphosphate levels and on the activities of glucose 1,6-bisphosphatase, phosphofructokinase and phosphoglucomutase

Injection of trifluoperazine abolished the bradykinin-induced decrease in intracellular concentration of glucose 1,6-bisphosphate (Glc-1,6-P2) in rat tibialis anterior muscle and skin. These changes in Glc-1,6-P2 levels may be attributed to the changes in the activity of glucose 1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2), which was markedly enhanced by bradykinin and reversed by trifluoperazine. Concomitantly to the changes in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, the activities of these enzymes were reduced by bradykinin and restored by trifluoperazine. These findings suggest that trifluoperazine treatment may have a beneficial effect on the depressed glycolysis induced by bradykinin in tissue damage.     

Metabolism of glycerate-2,3-P2--II. Enzymes involved in the glycerate-2,3-P2 metabolism in chicken skeletal muscle

1. Four enzyme fractions which may be involved in the synthesis and breakdown of glycerate-2,3-P2 have been isolated from extracted skeletal muscle by gel-filtration and ion-exchange chromatography. 2. One of the fractions, corresponding to the glycerate-2,3-P2 dependent phosphoglycerate mutase, has been purified to homogeneity. In addition to the main enzymatic activity, it shows intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity stimulable by glycolate-2-P. Its synthase activity represents about 10% of the total synthase activity of the tissue, and its phosphatase activity corresponds to about 60% of the total phosphatase activity. 3. Two of the fractions have glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and phosphoglycerate mutase activities in a ratio similar to that of the glycerate-2,3-P2 synthase described in mammalian skeletal muscle. Their synthase activity corresponds to about 90% of the total synthase activity, and their phosphatase activity represents about 1% of the total phosphatase activity of the tissue. 4. The fourth fraction shows only glycerate-2,3-P2 phosphatase activity and represents about 40% of the total activity of the tissue. 5. It is suggested that in chicken skeletal muscle the metabolism of the glycerate-2,3-P2 is regulated in a way similar to that described in mammalian skeletal muscle.     

The NADP-linked glyceraldehyde-3-phosphate dehydrogenases of Anabaena variabilis and Synechocystis PCC 6803, which lack one of the cysteines found in the higher plant enzyme,are not reductively activated

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29–35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.     

Isozymes of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle

The distribution of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle tissues was examined. With DEAE-cellulose chromatography, two peaks (I and II) of Fru 6-P,2-kinase activity were detected in all tissue extracts. Peak I was the predominant form both in rat and bovine heart tissue, while peak II was the major form in liver and skeletal muscle. Antibodies to heart enzyme reacted specifically with peak I, and antibodies to liver enzyme reacted with peak II from both liver and skeletal muscle. All the isozymes were bifunctional. All the tissues examined contained other isozymes in minor amounts.     

Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C(4) Dicarboxylic Acid Pathway of Photosynthesis

The activities of certain enzymes related to the carbon assimilation pathway in whole leaves, mesophyll cell extracts, and bundle sheath extracts of the C₄ plant Panicum miliaceum have been measured and compared on a chlorophyll basis. Enzymes of the C₄ dicarboxylic acid pathway—phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase—were localized in mesophyll cells. Carbonic anhydrase was also localized in mesophyll cell extracts. Ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, and ribulose diphosphate carboxylase—enzymes of the reductive pentose phosphate pathway—were predominantly localized in bundle sheath extracts. High activities of aspartate and alanine transaminases and glyceraldehyde-3-P dehydrogenase were found about equally distributed between the photosynthetic cell types. P. miliaceum had low malic enzyme activity in both mesophyll and bundle sheath extracts.     

Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase

Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucose-dependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.     

Fructose-1,6-biphosphatase of the small intestine. Purification and comparison with liver and muscle fructose-1,6-bisphosphatases.

The occurrence of specific fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] (Fru-1,6-P2ase) in the small intestine was confirmed. 1. Fru-1,6-P2ase was isolated from mouse small intestine by a simple method. The isolated enzyme preparation was an electrophoretically homogeneous protein. 2. The molecular weight and subunit molecular weight were 140,000 and 38,000, respectively. 3. The intestinal enzyme was electrophoretically distinct from the liver enzyme. 4. The kinetic properties of the purified intestinal enzyme were compared with those of the mouse liver and muscle enzymes. 5. Mouse intestinal and muscle Fru-1,6-P2ases hydrolyzed ribulose-1,5-bisphosphate in addition to fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate.     

Levels of glycerate 2,3-P2, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities in rat tissues. A method to quantify blood contamination of tissue extracts

The levels of glycerate 2,3-P2 and of 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities have been determined in isolated rat hepatocytes and adipocytes and in perfused rat tissues to discard blood contamination. The values obtained are much lower than those previously reported, ranging 0.50-40 nmol/g tissue. No relationship appears to exist between glycerate 2,3-P2 concentration and the levels of the enzymatic activities involved in glycerate 2,3-P2 metabolism. Assay of glycerate 2,3-P2 in tissue extracts constitute a very useful way to quantify blood contamination.