Distribution of Glucose- 1,6-Bisphosphate and IMP-Activated Glucose Bisphosphatase in Brain and Retina

The activity of glucose-1,6-bisphosphatase and the level of its substrate were measured in 16 gray areas and four fiber areas of mouse brain and 10 layers or sublayers of monkey retina. Because of the low activity of the enzyme and the small sample sizes, it was necessary to develop a method with two different amplification steps (overall amplification about 10⁶). The enzyme ranged in activity 100-fold from a low in monkey retina photoreceptor cells to a high in the pyramidal layer of the hippocampus. However, in gray areas of the brain proper the range was only about fourfold. This, together with its requirement for IMP, suggests that the enzyme has a widespread metabolic function related to states of increased neuronal activity. Glucose-1,6-bisphosphate levels varied from 80 to 960 μmol/kg dry weight in different areas of mouse brain and from 44 to 200 μmol/kg dry weight in different layers of monkey retina. In general, the glucose bisphosphate levels correlated positively with the bisphosphatase activities; however, the three areas with the highest enzyme concentrations did not fit this pattern.     

Phosphoglycerates and Protein Phosphorylation: Identification of a Protein Substrate as Glucose-1,6-Bisphosphate Synthetase

We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate [( 1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [gamma-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP72 preparation.     

Effects of Fructose-1,6-Bisphosphate on Glutamate Release and ATP Loss from Rat Brain Slices During Hypoxia

Abstract: Fructose-1,6-bisphosphate (FBP), an intermediate of glucose metabolism, is neuroprotective in brain hypoxia or ischemia. Because the mechanisms for this protection are not clear, we examined the effects of FBP on two important events in brain ischemia, i.e., loss of ATP and release of the excitatory neurotransmitter glutamate. Glutamate release from cortical brain slices was measured fluorometrically (glutamate dehydrogenase)-catalyzed conversion of glutamate to α-ketoglutarate) during hypoxia (Po₂ 15 mm Hg) or hypoxia plus 100 µ M cyanide. FBP (3.5 m M , with glucose 20 m M ) reduced glutamate release during hypoxia by 55% and during hypoxia/cyanide by 46% ( p < 0.005), and prevented a significant fall in [ATP]. [ATP] was maintained in oxygenated glucose-free conditions with 20 but not 3.5 m M FBP, and fell to <20% of normal with hypoxia. Despite the drop in [ATP], 3.5 or 20 m M FBP without glucose decreased hypoxia-evoked glutamate release. We conclude (1) FBP present without glucose preserves normal [ATP] only when oxygen is available, suggesting limited uptake and metabolism; and (2) FBP decreases hypoxia-evoked glutamate release by processes independent of [ATP]. These results suggest protective actions of FBP that are separate from augmentation of anaerobic energy production, as previously proposed.     

Fructose 2,6-Bisphosphate Changes in Rat Brain During Ischemia

Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6-bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6-bisphosphate content was increased by 20-30% during the early stages of ischemia. Elevation in fructose 1,6-bisphosphate level and lactate formation followed the rise in fructose 2,6-bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.     

Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.     

Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations

Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night.     

Biphasic effect of fructose 2,6-bisphosphate on the liver fructose-1,6-bisphosphatase: mechanistic and physiological implications

Fructose 2,6-bisphosphate has been claimed to be both a substrate analogue and an allosteric inhibitor of fructose-1,6-bisphosphatase. The results reported here show that fructose 2,6-bisphosphate can be both an inhibitor and an activator of the enzyme, depending on the substrate concentration. This biphasic behaviour at saturating concentrations of substrate can only be due to an allosteric effect. In addition to the mechanistic implication it is possible that this finding may have physiological meaning.     

Inhibition of fructose 1,6-bisphosphatase by excess substrate and its reversal by monovalent cations

Purified rabbit liver fructose 1,6-bisphosphatase is maximally active with 2 μM fructose 1,6-bisphosphate. Above this concentration the substrate becomes inhibotory. Inhibition is reversed by $\text{NH}{}_4{}^{\mathrm{+}}$ or by physiological concentrations of K⁺. Substrate inhibition and its modification by monovalent cations may play a role in the regulation of gluconeogenesis at the step catalyzed by fructose 1,6-bisphosphatase.     

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.     

Ischemia and Reperfusion: Effect of Fructose-1,6-Bisphosphate

Several lines of evidence indicating a close relationship among ischemia, concentration of high-energy metabolites and onset of the “oxygen paradox” in reperfused tissues have been published. In this framework, we have recently studied the effects of exogenous fructose-1,6-bisphosphate on energy metabolism and on oxygen free radical damages of isolated rat heart subjected to anoxia and reoxygenation. In comparison with control groups, hearts perfused in the presence of 5mM fructose-1,6-bisphosphate throughout the different perfusion conditions showed higher concentrations of energy metabolites at the end of anoxia, most of which were normalized after reperfusion. Furthermore, in comparison with control hearts, a reduction of tissue malondialdehyde and of lactate dehydrogenase release in the perfusate was observed in fructose-1,6-bisphosphate-perfused hearts. In this article we review most of the available data concerning the ability of fructose-1,6-bisphosphate to protect from ischemia and reperfusion damage outlining those recent findings which contributed both to clarify the pharmacological profile of the drug and to give an insight in its probable mechanism of action.     

Glucose 6-Phosphate and Fructose 1,6-Bisphosphate Can Be Used as ATP-Regenerating Systems by Cerebellum Ca2+-Transport ATPase

Abstract : In this work, it is shown that the Ca²⁺-transport ATPase found in the microsomal fraction of the cerebellum can use both glucose 6-phosphate/hexokinase and fructose 1,6-bisphosphate/phosphofructokinase as ATP-regenerating systems. The vesicles derived from the cerebellum were able to accumulate Ca²⁺ in a medium containing ADP when either glucose 6-phosphate and hexokinase or fructose 1,6-bisphosphate and phosphofructokinase were added to the medium. There was no Ca²⁺ uptake if one of these components was omitted from the medium. The transport of Ca²⁺ was associated with the cleavage of sugar phosphate. The maximal amount of Ca²⁺ accumulated by the vesicles with the fructose 1,6-bisphosphate system was larger than that measured either with glucose 6-phosphate or with a low ATP concentration and phosphoenolpyruvate/pyruvate kinase. The Ca²⁺ uptake supported by glucose 6-phosphate was inhibited by glucose, but not by fructose 6-phosphate. In contrast, the Ca²⁺ uptake supported by fructose 1,6-bisphosphate was inhibited by fructose 6-phosphate, but not by glucose. Thapsigargin, a specific SERCA inhibitor, impaired the transport of Ca²⁺ sustained by either glucose 6-phosphate or fructose 1,6-bisphosphate. It is proposed that the use of glucose 6-phosphate and fructose 1,6-bisphosphate as an ATP-regenerating system by the cerebellum Ca²⁺-ATPase may represent a salvage route used at early stages of ischemia ; this could be used to energize the Ca²⁺ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.     

Modification of cys-128 of pig kidney fructose 1,6-bisphosphatase with different thiol reagents: Size dependent effect on the substrate and fructose-2,6-bisphosphate interaction

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was shown to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Burgos, M. E., Hubert, E., and Slebe, J. C. (1987),J. Biol. Chem. 262, 8451–8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5-dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.     

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 Å, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.     

Fructose 1,6-bisphosphate prevents oxidative stress in the isolated and perfused rat heart

Rat hearts were perfused with the Langendorff technique at constant flux in the presence of the oxidizing agents hydrogen peroxide and diamide. Fructose 1,6-bisphosphate strongly prevented the decline of heart contractility due to the infusion of these oxidizing agents. On the other hand, fructose 1,6-bisphosphate had no effect on the release of total glutathione into the perfusate but prevented the loss of lactate dehydrogenase indicating a protective effect on cell membranes. Comparing the cytosolic and mitochondrial loss of glutathione, fructose 1,6-bisphosphate exerted a beneficial action only on the mitochondrial fraction. Several mechanisms of action have been considered to explain the protective action of frutose 1,6-bisphosphate. In our experimental conditions fructose 1,6-bisphosphate might stimulate its own production giving rise to dihydroxyacetone phosphate, that, after reduction to glycerol 3-phosphate, can permeate the mitochondrial membrane with the final production of energy.     

Glycerol Phosphate Dehydrogenase in Developing Chick Retina and Brain

Abstract: The development of cytoplasmic glycerol phosphate dehydrogenase (GPDH) activity in chick neural retina is compared with that in brain. GPDH converts dihydroxyacetone phosphate to glycerol 3-phosphate, an intermediate in phospholipid synthesis. The enzyme is known to be under corticosteroid control in rat brain and spinal cord (but not muscle or liver) and in primary oligodendrocyte cultures. It has not been previously studied in the eye. In chick brain the GDPH specific activity rises fivefold from the early embryo to the adult, with nearly all the increase occurring between embryonic day 14 and hatching. This time course correlates well with the known maturation of chick adrenal cortex (which produces corticosteroids). On the other hand, in chick retina the GPDH specific activity remains at a low basal level throughout development. Furthermore, adult rat and beef retinas show much lower enzyme activity than do the corresponding brain tissues. GPDH can be induced precociously by hydrocortisone in embryonic chick brain from days 12 through 16, both in the intact embryo and in tissue culture; however, GPDH is not at all inducible in chick retina. The developmental increase in chick brain GPDH can be correlated qualitatively with myelin formation, as shown by luxol fast blue staining, whereas no myelin is seen in retina at any age. Our results are consistent with recent immunocytochemical studies demonstrating that GPDH in rat brain is associated with myelin-producing oligodendroglial cells, absent in retina. In comparison, another glial enzyme, glutamine synthetase (GS), known to be inducible in both chick brain and retina, is localized in brain astrocytes and retinal Müller cells.     

The Differential Effects of GABA-Transaminase Inactivation in the Chick Retina and Brain

Abstract: The inactivation of γ-aminobutyrate (GABA)-transaminase by the highly specific and potent neurotoxin gabaculine leads to different neurochemical consequences in the chick brain as opposed to the chick retina. In the brain, GABA levels continually climb, reaching approximately eightfold increases over control values after 24 h. The elevation in GABA levels leads to a time-dependent and coincident fall in glutamate decarboxylase and cysteine- sulfinatc decarboxylase activities, to approximately 50% of control values. On the other hand, in the retina GABA levels only increase to a plateau level two- to threcfold that of control after inactivation of GABA-transaminase. Further- more, although the glutamate decarboxylase activity decreases to about 50% of control values, cysteinesulfinate decarboxylase activity is not affected. These studies show that the processing of GABA in the retina differs from that in the brain, and that cysteinesulfinate and glutamate decarboxylase activity probably reside in different enzyme molecules in the retina, although they may reside in the same enzyme in the brain.     

Effect of Diabetes on Glycogen Metabolism in Rat Retina

Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.     

Influence of Altered Photoperiods on Serum Melatonin and Its Receptors (MT1 and MT2) in the Brain,Retina, and Ovary in Carp Catla catla

Daily variation in melatonin receptor (MT1 and MT2) density in three specific tissues—brain, retina, and ovary—and its temporal relationship with serum melatonin were evaluated for the first time in a freshwater teleost, the carp Catla catla, under natural as well as altered photoperiods in different reproductive phases of the annual cycle. Cosinor analysis was used to determine rhythmic features of the serum melatonin and receptors (MT1 and MT2) in different tissues. In each photoperiodic group, irrespective of season, the daily minimum serum melatonin level was noted at midday. However, the daily peak value of melatonin varied in relation to both photo-schedules and reproductive phases. Under natural photoperiods (NPs; duration varied with seasons) and short photoperiods (SPs; light [L]:dark [D] 8:16), it occurred in the late dark phase during the preparatory phase, and at midnight in the remaining parts of the annual cycle. On the other hand, in each reproductive phase, compared to corresponding NP carp, the daily melatonin peak under long photoperiods (LPs; L:D 16:8) exhibited a phase delay of ～2–3?h (occurring during the late dark phase). The melatonin levels at each sampling point were highest during the postspawning phase and lowest during the spawning phase, irrespective of the photoperiodic history of the fish. In each tissue, Western blot analysis revealed a band at ～37?kDa and a band at ～36?kDa corresponding to the molecular weights of native MT1 and MT2 receptor proteins, respectively, with the band intensity of MT1 always being higher than that of a 36-kDa protein. The content of both melatonin receptor proteins varied significantly according to the studied tissue (being highest in the retina, intermediate in the brain, and lowest in the ovary), time in the daily cycle (peak at midnight and fall at midday), and reproductive phase in the annual cycle (highest in the spawning phase and lowest in the postspawning phase). Remarkably, no significant effects of altered photoperiod were detected on any rhythm parameters of either MT1 or MT2 in any of the studied tissues. Collectively, the results of the present study suggest a role of photoperiod in determining daily and seasonal profiles of serum melatonin, but not its receptor proteins, on the ovary or on any nongonad tissues in carp. (Author correspondence: dgp_skmaitra@yahoo.co.in)     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.