Showing papers on "Pyruvate kinase published in 1990"

Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells

Abstract: 1. Maximal activities of some key enzymes of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and glutaminolysis were measured in homogenates from a variety of normal, neoplastic and suppressed cells. 2. The relative activities of hexokinase and 6-phosphofructokinase suggest that, particularly in neoplastic cells, in which the capacity for glucose transport is high, hexokinase could approach saturation in respect to intracellular glucose; consequently, hexokinase and phosphofructokinase could play an important role in the regulation of glycolytic flux in these cells. 3. The activity of pyruvate kinase is considerably higher in tumorigenic cells than in non-tumorigenic cells and higher in metastatic cells than in tumorigenic cells: for non-tumorigenic cells the activities range from 28.4 to 574, for tumorigenic cells from 899 to 1280, and for metastatic cells from 1590 to 1627 nmol/min per mg of protein. 4. The ratio of pyruvate kinase activity to 2 x phosphofructokinase activity is very high in neoplastic cells. The mean is 22.4 for neoplastic cells, whereas for muscle from 60 different animals it is only 3.8. 5. Both citrate synthase and isocitrate dehydrogenase activities are present in non-neoplastic and neoplastic cells, suggesting that the full complement of tricarboxylic-acid-cycle enzymes are present in these latter cells. 6. In neoplastic cells, the activity of glutaminase is similar to or greater than that of hexokinase, which suggests that glutamine may be as important as glucose for energy generation in these cells.

Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite

Abstract: Selected aspects of the metabolism of Plasmodium falciparum are reviewed, but conclusions based on the study of other species of plasmodia are intentionally not included since these may not be applicable. The parasites increase glucose consumption 50-100 fold as compared to uninfected red cells; most of the glucose is metabolized to lactic acid. The parasite contains a complete set of glycolytic enzymes. Some enzymes such a hexokinase, enolase and pyruvate kinase are vastly increased over corresponding levels in uninfected red cells. However, the pathway for synthesizing 2,3-diphosphoglycerate (2,3-DPG) is absent. Parasitized red cells show a decline in the concentration of 2,3-DPG which may function as an inhibitor for certain essential enzyme pathways. Pentose shunt activity is increased in absolute terms, but as a percent of total glucose consumption, there is a decrease during parasite infection of the red cell. The parasite contains a gene for G6PD and can produce a small quantity of parasite-encoded enzyme. It is not clear if the production of this enzyme can be up-regulated in G6PG deficient host red cells. The NADPH normally produced by the pentose shunt can be obtained from other parasite pathways (such as glutamate dehydrogenase). NADPH may subserve additional needs in the infected red cell such as driving diribonucleotide reductase activity--a rate limiting enzyme in DNA synthesis. The role of NADPH in protecting the parasite-red cell system against oxidative stress (via glutathione reduction) remains controversial. Parasitized red cells contain about 10 times more NAD(H) than uninfected red cells, but the NADP(H) content is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts.

Abstract: Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.

Biochemical and histochemical adaptation to sprint training in young athletes

Abstract: The purpose of the present study was to investigate the effects of 8 months of a specific and controlled sprint training programme on three groups of young athletes (two groups of males and one of females). Biopsies of vastus lateralis were taken before and after the period of training. The type percentage and diameter of the fibres, as well as the glycogen content and the activities of the enzymes of glycogen metabolism (glycogen synthase and glycogen phosphorylase), glycolysis (phosphofructokinase, pyruvate kinase, aldolase and lactate dehydrogenase), oxidative metabolism (succinate de-hydrogenase) and creatine kinase and aminotransferases were studied. The results show an increase in the percentage of type I fibres and an increase in the diameter of both fibre types. A significant increase was also observed in glycogen content, and in the activities of glycogen synthase, glycogen phosphorylase, phosphofructokinase, pyruvate kinase, succinate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. We conclude that a long period of sprint training induces a biochemical muscle adaptation to anaerobic exercise. This metabolic adaptation is followed by a morphological adaptation, although this is probably not as specific as the biochemical one.

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

Abstract: The assimilation of NH4+ causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH4+ assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.

Isoenzymes of pyruvate kinase.

Abstract: -. 1 3 . Miernyk. J. A. & Dennis. I>. 1 ( 1982) I’lurit I’hyxiol. 69. 825-828 Journet. E. P. & Douce. R. ( 1 984) (‘. K. Acud. Sci. /’(iris Ser. 111 298, 365-370 Hotha, F. C. & Dennis, D. R. ( 1986) Arch. Bioc,hem. Bioplrvs. 245.96I03 Fothergill. L. A. & Harkins. R. N . ( 1982) /’roc.. K. Soc. London H215, 19-44 White. M. F. & Fothergill-Gilmore. L. A. (1988) FEES Lett. 229,383-387 Winn. S. I., Watson. H. C.. Harkins. R. N. & Fothergill. L. A. ( I 98 I ) l’ldos. Truns. R. Soc. London H 293, I 2 1 1 30 Watson. H. C. (1982) Protein Data Bank Entry: Phosphoglycerare mutase (yeast). Brookhaven, New York Winn. S. I., Watson, H. C.. Fothergill. L. A. & Harkins. R. N. ( 1977) Hiocliem. .Sot. Truns. 5 . 657-659 Sasaki. R., Sugimoto. E. & Chiba. H. ( I 966) Arch. Hiodiom.

Fructose-2,6-bisphosphate, metabolites and 'coarse' control of pyrophosphate: fructose-6-phosphate phosphotransferase during triose-phosphate cycling in heterotrophic cell-suspension cultures of Chenopodium rubrum.

Abstract: Experiments were carried out to determine whether pyrophosphate: fructose-6-phosphate phosphotransferase (PFP) catalyses the rapid recycling of triose phosphates that is found in the cytosol of heterotrophic cell cultures of Chenopodium rubrum L. (W.-D. Hatzfeld, M. Stitt, 1990, Planta, 180, 198–204). Oxygen uptake, carbohydrate turnover, fructose 2,6-bisphosphate (Fru2,6bisP), glycolytic intermediates, adenine and uridine nucleotides, pyrophosphate and the activity of PFP and glycolytic enzymes were monitored for 48 h after subculturing carbohydrate-depleted cells onto glucose. Immediately after transfer there was an increase in the amount of Fru2,6bisP, and of the hexose phosphate. The triose phosphates, fructose-1,6-bisphosphate and inorganic pyrophosphate increased gradually over the next 24 h. This was accompanied by a tripling in the extractable activity of PFP, but not of phosphofructokinase. The activity of fructose-1,6-bisphosphatase was 20–50fold lower than that of PFP. It is calculated that the activity of PFP is high enough to catalyse the observed rate of cycling between the triose phosphates and the hexose phosphates, based on the measured Vmax capacity of the enzyme, the known kinetic properties, and the measured levels of its reactants and Fru2,6bisP. The changes in the levels of Fru2,6bisP were not correlated with the rate of respiration. Instead, the rate of O2 uptake was inversely related to the phosphoenolpyruvate level, showing that pyruvate kinase or phosphoenolpyruvate carboxylase are regulating the use of glucose for respiration. There was also no relation between Fru2,6bisP, and partitioning to sucrose or starch. It is proposed that the main function of the cycle in these cells is to maintain high levels of inorganic pyrophosphate and triose phosphates, which are necessary for the remobilisation of sucrose and for biosynthesis in the plastid, and that ‘coarse’ and ‘fine’ control of PFP play an important role in regulating this cycle.

Inhibition of proton-translocating ATPases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminum.

Abstract: One of the major effects of fluoride on oral bacteria is a reduction in acid tolerance, and presumably also in cariogenicity. The reduction appears to involve transport of protons across the cell membrane by the weak acid HF to dissipate the pH gradient, and also direct inhibition of the F1F0, proton-translocating ATPases of the organisms, especially for Streptococcus mutans. This direct inhibition by fluoride was found to be dependent on aluminum. The dependence on aluminum was indicated by the protection against fluoride inhibition afforded by the Al-chelator deferoxamine and by loss of protection after addition of umolar levels of Al3+, which were not inhibitory for the enzyme in the absence of fluoride. The F1 form of the enzyme dissociated from the cell membrane previously had been found to be resistant to fluoride in comparison with the F1F0 membrane-associated form. However, this difference appeared to depend on less aluminum in the F1 preparation in that the sensitivity of the F1 enzyme to fluoride could be increased by addition of umolar levels of Al3+. The effects of Al on fluoride inhibition were apparent when enzyme activity was assayed in terms of phosphate release from ATP or with an ATP-regenerating system containing phosphoenolpyruvate, pyruvate kinase, NADH and lactic dehydrogenase. Also, Be2+ but not other metal cations, e.g. Co2+, Fe2+, Fe3+, Mn2, Sn2+, and Zn2+, served to sensitize the enzyme to fluoride inhibition. The differences in sensitivities of enzymes isolated from various oral bacteria found previously appeared also to be related to differences in levels of Al. Even the fluoride-resistant enzyme of isolated membranes of Lactobacillus casei ATCC 4646 could be rendered fluoride-sensitive through addition of Al3+. Thus, the F1F0 ATPases of oral bacteria were similar to E1E2 ATPases of eukaryotes in being inhibited by Al-F complexes, and the inhibition presumably involved formation of ADP-Al-F inf3 sup- complexes during catalysis at the active sites of the enzymes.

Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics.

Abstract: Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes Regulation by cAMP dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P 2 ase) The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P 2 ), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P 2 ase The bifunctional enzyme is a homodimer whose activities are regulated by cAMP dependent protein kinase-catalyzed phosphorylation at a single NH 2 -terminal seryl residue/subunit, which results in activation of the Fru-2,6-P 2 ase and inhibition of the PF-1-K reactions Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P 2 levels 6PF-2-K/Fru-2,6-P 2 ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver Pyruvate kinase is regulated by both phosphorylation and allosteric effectors Fru-1,6-P 2 , an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P 2 Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P 2 substrate cycles The Fru-2,6-P 2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P 2 ase gene expression Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector cAMP prevents the effects of glucocorticoids and insulin Although Fru-2,6-P 2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P 2 ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene The muscle form differs from the liver form only at the NH 2 -terminal and does not have a cAMP dependent protein kinase phosphorylation site The hepatic enzyme subunit consists of 470 amino acids The NH 2 -terminal half of the subunit contains the 6PF-2-K domain (residues 1–250), and the COOH-terminal half contains the Fru-2,6-P 2 ase domain (residues 251–470) The bisphosphatase reaction is catalyzed via a phosphohistidine enzyme intermediate This Fru-2,6-P 2 ase domain is evolutionarily related to the phosphoglycerate mutase family of enzymes, which also utilize phosphohistidine in their reaction pathway The 6PF-2-K domain contains a nucleotide binding fold analogous to that found in bacterial 6PF-1-K On the basis of these findings, the bifunctional enzyme was formed by a gene fusion event involving two glycolytic enzyme catalytic units, ie, phosphoglycerate mutase and 6PF-1-K The importance of this system in the regulation of carbohydrate metabolism in many cell types makes it an ideal system to use in molecular approaches to the definitive study of pathway flux and rate-controlling enzymes

Regulation of the gene expression of glucokinase and L-type pyruvate kinase in primary cultures of rat hepatocytes by hormones and carbohydrates.

Abstract: The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyruvate Kinase from the Land Snail Otala Lactea: Regulation by Reversible Phosphorylation During Estivation and Anoxia

Abstract: Pyruvate kinase (PK) from tissues of the desert snail Otala lactea (Muller) undergoes a stable modification of its kinetic properties during estivation or in response to anoxia stress. In foot muscle and mantle, the kinetic changes induced by either state were virtually identical and were consistent with a less active enzyme form in estivation or anoxia: S 0.5 PEP increased, and I 50 values for Mg-ATP and L-alanine decreased, compared to the enzyme in control (aroused) snails. Estivation and anoxia also changed the properties of PK from hepatopancreas; some changes were consistent with a more active enzyme form (S o.5 PEP decreased, I 50 values for Mg-ATP and L-alanine increased) but the enzyme lost all sensitivity to the potent activator fructose-l,6-bisphosphate. A time course of changes in I 50 Mg-ATP for foot PK and S 0.5 PEP for hepatopancreas PK revealed that estivation-induced changes in enzyme properties occurred between 12 and 48 h after snails were deprived of access to food and water, whereas the reversal of these changes occurred within as little as lOmin in foot muscle after arousal was initiated. The molecular basis of the stable modification of PK kinetics appears to be reversible protein phoshorylation. The action of added cyclic-AMP-dependent protein kinase on foot or hepatopancreas PK from control (aroused) snails changed PK kinetic parameters to those characteristic of the enzyme form in estivating animals; the addition of stimulators of endogenous cyclic-GMPdependent protein kinase or protein kinase C had the same effect. Conversely, treatment with added phosphatases reconverted the properties of foot muscle PK from estivating snails to those characteristic of the control enzyme. The data suggest that reversible phosphorylation control over the activity state of regulatory enzymes of glycolysis is one mechanism contributing to the overall metabolic rate depression of the estivating state.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product.

Abstract: The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga.

Effect of microgravity on metabolic enzymes of individual muscle fibers

Abstract: Eleven enzymes were measured in individual fibers of soleus and tibialis anterior (TA) muscles from two flight and two control (synchronous) animals. There were five enzymes of glycogenolytic metabolism: phosphorylase, glucose-6-phosphate isomerase, glycerol-3-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase (group GLY); five of oxidative metabolism: citrate synthase, malate dehydrogenase, beta-hydroxyacyl-CoA dehydrogenase, 3-ketoacid CoA-transferase, and mitochondrial thiolase (group OX); and hexokinase, subserving both groups. Fiber size (dry weight per unit length) was reduced about 35% in both muscles. On a dry weight basis, hexokinase levels were increased 100% or more in flight fibers from both soleus and TA. Group OX enzymes increased 56-193% in TA without significant change in soleus. Group GLY enzymes increased an average of 28% in soleus fibers but underwent, if anything, a modest decrease (20%) in TA fibers. These changes in composition of TA fibers were those anticipated fo...

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells.

Abstract: The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH4+ in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH4+ to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH4+ assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH4+ assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH4+ assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH4+ assimilation.

Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase.

Abstract: The activities of hexokinase (ATP:hexose-6-phosphate transferase, E.C. 2.7.1.1), phosphofructokinase (ATP: fructose-6-phosphate 1-phosphotransferase, E. C. 2.7.1.11) and pyruvate kinase (ATP: pyruvate transferase, E.C. 2.7.1.40), and their kinetic behaviour in two morphological forms of Trypanosoma cruzi (epimastigotes and metacyclic trypomastigotes) have been studied. The kinetic responses of the three enzymes to their respective substrates were normalized to hyperbolic forms on a velocity versus substrate concentration plots. Hexokinase and phosphofructokinase showed a higher activity in epimastigotes than in metacyclics, whereas pyruvate kinase had similar activity in both forms of the parasite. The specific activity of hexokinase from epimastigotes was 102.00 mUnits/mg of protein and the apparent Km value for glucose was 35.4 μM. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 μM. The kinetic parameters (specific activity and Km for fructose 6-phosphate) of phosphofructokinase for epimastigotes were 42.60 mUnits/mg and 0.31 mM and for metacyclics 13.97 mUnits/mg and 0.16 mM, respectively. On the contrary, pyruvate kinase in both forms of T. cruzi did not show significant differences in its kinetic parameters. The specific activity in epimastigotes was 37.00 mUnits/mg and the Km for phosphoenolpyruvate was 0.47 mM, whereas in metacyclics these values were 42.94 mUnits/mg and 0.46 mM, respectively. The results presented in this work, clearly demonstrate a quantitative change in the glycolytic pathway of both culture forms of T. cruzi.

Mechanism of aluminum-induced inhibition of hepatic glycolysis: inactivation of phosphofructokinase.

Abstract: Aluminum, an abundant element in the earth's crust, has been implicated in various pathological disorders and low concentrations of this element have recently been shown to inhibit brain glycolysis. However, despite the fact that aluminum accumulates in high concentrations in the liver, potential effects of this metal on hepatic intermediary metabolism have not been explored. In perfused livers from untreated rats, maximal rates of production of lactate plus pyruvate (glycolysis) were 93 +/- 15 mumols/g/hr. Glycolysis was severely inhibited in livers from aluminum-treated rats (0.5 mg/kg, 6 hr before experiment) with maximal rates of only 23 +/- 4 mumols/g/hr. In contrast, glucose production (glycogenolysis) and hepatic oxygen uptake were not altered significantly by prior treatment with aluminum. In livers from fasted rats, pretreatment with aluminum did not influence gluconeogenesis or production of lactate and pyruvate from fructose (5 mM). This finding indicates that pyruvate kinase is not inhibited by aluminum and implicates phosphofructokinase, hexokinase and/or glucokinase as sites for the inhibitory effect of aluminum on glycolysis. In liver homogenates from untreated rats, increasing concentrations of aluminum did not show any appreciable effect on hexokinase or glucokinase activity but did cause progressive decreases in phosphofructokinase activity. Therefore, aluminum-induced inhibition of liver phosphofructokinase, an important control site in the glycolytic pathway, is most likely responsible for aluminum-induced inhibition of hepatic glycolysis.

Regulation of glycolytic enzymes in the marine invertebrateHalicryptus spinulosus (Priapulida) during environmental anoxia and exposure to hydrogen sulfide

Abstract: A particularly strong reduction of metabolic activity is a precondition for long-term survival ofHalicryptus spinulosus von Siebold under anoxic habitat conditions because of its relatively low fuel reserves (mainly glycogen). The present study analyses the mechanism of this metabolic slow-down. For this purpose the effects of environmental anoxia and exposure to hydrogen sulfide on the activity and selected kinetic properties of glycolytic enzymes [glycogen phosphorylase (GP), pyruvate kinase (PK)] and the concentrations of fructose-2,6-bisphosphate in the body wall ofH. spinulosus were analysed. Anoxia and hydrogen sulfide exposure stimulated modifications of the properties of the enzymes, in both cases due to probable covalent modification of the enzyme proteins. Under both conditions phosphorylase activity was depressed by about 1/3, the result of changes in the percentage of enzyme in the activea-form as well as the total amount of enzyme activity expressed (a +b). Effects of anoxia on the properties of pyruvate kinase included reducedV max , decreasedS0.5 for phospho-enolpyruvate, changes inK a for fructose-1,6-bisphosphate (an initial decrease was followed by a later increase). TheI50 forL-alanine of PK was extremely reduced under anoxia and showed an even greater sensitivity to the presence of hydrogen sulfide. Anoxia stimulated a slight reduction in the content of fructose-2,6-bisphosphate, whereas exposure to hydrogen sulfide caused a dramatic decrease of this allosteric activator of phos-phofructokinase. The study gives evidence that mechanisms of glycolytic rate depression are conserved within a wide variety of vertebrate and invertebrate phyla. With two exceptions (fructose-2,6-bisphosphate levels and alanine inhibition of PK) the responses to hydrogen sulfide were the same as those to anoxia, suggesting that at a metabolic level, the consequences of each stress on energy metabolism are similar.

Purification of Leucoplast Pyruvate Kinase from Developing Castor Bean Endosperm

Abstract: Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L; cv Baker) has been purified 1370-fold to a specific activity of 411 micromoles pyruvate produced per minute per milligram protein Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 575 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed The native molecular mass was approximately 305 kilodaltons Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase The 575- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 575-kilodalton polypeptide In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases

Hydrops fetalis associated with red cell pyruvate kinase deficiency.

Abstract: A hydrops fetalis and multicystic encephalomalacia were diagnosed in a neonate who was one of twins. The co-twin had died 5 weeks prior to delivery. The most likely explantation for both hydrops and multicystic encephalomalacia was fetal anemia caused by a red cell pyruvate kinase deficiency, and aggravated by an intrauterine disseminated intravascular coagulation.

cGMP-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc

Abstract: A cystosolic protein kinase that phosphorylates pyruvate kinase (PK) in vitro has been identified in crude homogenates of heart, radular retractor, and foot muscle from the anoxia-tolerant marine whelk Busycon canaliculatum. Protein kinase action was measured by following changes in PK kinetic parameters: phosphorylated PK has a higher K m value for phosphoenolpyruvate and a lower I50 value for l-alanine. The crude protein kinase readily phosphorylated PK in a Mg2+-and ATP-dependent manner in the absence of any added effector. This activity was not affected by the addition of either cAMP (a stimulator of protein kinase A) or Ca2+ plus phorbol 12-myristate 13-acetate (stimulators of protein kinase C) to the incubation medium. Addition of cGMP to the homogenate, however, increased the rate of PK phosphorylation giving a 3–4-fold increase in the rate of change in PK kinetic parameters that was readily apparent after 5h. Complete time-courses of changes in PK kinetic parameters in the presence and absence of cGMP showed that cGMP increased the rate, but not the final extent, of PK phosphorylation. These results indicate that PK inactivation by enzyme phosphorylation in response to anoxia in whelk tissues may be mediated by a cyclic GMP stimulated protein kinase in response to changing levels of cGMP. This conclusion was further supported by data indicating that the total activity of protein kinase was the same in both anoxic and aerobic animals, and that the total PK phosphatase activity was also constant. Changes in PK phosphorylation during anoxia are not, therefore, the result of changes in the total amount of protein kinase or phosphatase.