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PubMed1997

Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1.

Proliferating cells and tumour cells maintain a high glycolytic rate even under aerobic conditions. FTO2B cells, a rat hepatoma cell line, show high activities of glycolytic enzymes. Within a culture period of 48 h the cell number increases 5-fold. Replacement of glucose by pyruvate in the culture medium lowers glycolytic enzyme activity and prevents proliferation. Transfection assays revealed that glucose deprivation dramatically decreases the transcriptional activities of the Sp1-dependent aldolase and pyruvate kinase promoters leading to reduced reporter gene expression. Sp1 binding activity is also inhibited by ocadaic acid, an inhibitor of protein phosphatase 1. Western blot analyses with nuclear extracts from FTO2B cells cultured in the presence or absence of glucose revealed differences in the phosphorylation state of Sp1. From these results we conclude that glucose increases the amount of the dephosphorylated form of Sp1 which has a higher DNA binding activity. As a consequence gene expression of the glycolytic enzymes is increased which is a prerequisite for cell proliferation.

Animals↗
PubMed1998

A new sickle cell disease phenotype associating Hb S trait, severe pyruvate kinase deficiency (PK Conakry), and an alpha2 globin gene variant (Hb Conakry).

A Guinean woman, heterozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3-DPG concentration of twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C-->A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [alpha80(F1) Leu-->Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n(max) value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3-DPG concentration.

Adult↗
PubMed1994

Expression of L- and M2-pyruvate kinases in proliferating oval cells and cholangiocellular lesions developing in the livers of rats fed a methyl-deficient diet.

Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.1% w/w DL-ethionine (CDE) for up to 22 weeks. The expression of the pyruvate kinase isoenzymes L (L-PK) and M2 (M2-PK) was immunohistochemically analyzed in liver slices from rats killed 4, 10, 14 and 22 weeks after starting the treatment. M2-PK was detected in bile duct epithelial cells of untreated rats and in proliferating oval cells, cholangiofibroses and cholangiofibromas of CDE-fed animals. Thus, M2-PK can be viewed as a positive marker of the bile duct epithelial/oval cell compartment. L-PK, a parenchymal cell-specific protein in untreated rat liver, was not present in proliferating oval cells, but was consistently observed in cells that were part of the ductal structures in the cholangiofibroses and cholangiofibromas. Based on their morphology, the L-PK-positive duct cells were undoubtedly part of the bile duct epithelial cell lineage and no L-PK-positive hepatocyte-like cells were observed in the ducts. Hence, this study clearly shows that the mere presence of a liver parenchymal cell marker in cells of the bile duct epithelial/oval cell compartment does not necessarily preclude that these cells are undergoing a differentiation into preneoplastic parenchymal cells, as has previously been suggested.

Animals↗
PubMed2003

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae): effects of development and temperature.

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 degrees C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII-XIV (PL XII-XIV). PK activity reached a pronounced peak in PL V-VI, followed by a further decrease in PL XII-XIV. Temperature reduction produced variation in oxygen consumption rates (QO(2)), ammonia-N excretion and in enzyme activities. Ammonia-N excretion was higher at 20 degrees C in mysis III (M III), PL V-VI and PL XII-XIV, resulting in substantially lower O:N ratios in these stages. QO(2) was increased in protozoea II (PZ II) and mysis I (M I) at 26 degrees C, while no difference in QO(2) was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V-VI and PL XII-XIV at 20 degrees C compared with 26 degrees C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85-0.92 for CS and 1.1-1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 degrees C for both enzymes. Weight-specific CS activity was positively correlated with QO(2) at 20 and 26 degrees C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.

Ammonia↗
PubMed1980

Reduced induction of pyruvate kinase after feeding 2-acetylaminofluorene.

After administration of carcinogens to laboratory animals reduced induction of some enzymes has been found which is believed to be associated with the carcinogenic process. It is suggested that this effect may be used for the testing of chemical substances for carcinogenicity. For this purpose the dietary induction of pyruvate kinase in rat liver was investigated after feeding 2-acetylaminofluorene. The induced activity in the animals fed the carcinogen was found to be significantly lower than in the control, however, the extent of this reduction was too low to be used for testing chemical substances for carcinogenicity.

2-Acetylaminofluorene↗
PubMed2003

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.

Antisense Elements (Genetics)↗
PubMed1988

The apparent target size of rat brain benzodiazepine receptor, acetylcholinesterase, and pyruvate kinase is highly influenced by experimental conditions.

Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme.

Acetylcholinesterase↗
PubMed2005

The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

Bacterial Proteins↗
PubMed1978

Comparative studies of vertebrate and invertebrate pyruvate kinases.

1. Electrophoretic patterns showed that among the vertebrates studied, L isozyme was present in rat and frog liver and also in rat kidney. 2. M2 is a frequent component in vertebrate tissues, giving support to the proposal that M2 is the ancestral form differentiating to other isozymes. 3. The above trend cannot simply apply to invertebrates. 4. Muscle pyruvate kinases from various animals were inhibited by the particular phosphagen present in their muscle. 5. The inhibition may have an important role in the regulation of glycolysis in muscle.

Animals↗
PubMed1991

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase.

Leucoplast pyruvate kinase (PK(p); EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4 degrees C. Purified castor seed PK(p) was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK(p)) immunoglobulin G. Proteolytic activity against PK(p) was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4 degrees C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK(p). The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nalpha-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK(p). This endopeptidase is present throughout all stages of endosperm development. Its PK(p)-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4 degrees C for up to 21 hours, no degradation of PK(p) was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK(p) remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK(p).

Journal Article↗
PubMed1978

Plasma activity of pyruvate kinase and glutamic oxalacetic transaminase as indices of myopathy in the vitamin E deficient rat.

Weanling rats were fed vitamin E deficient diets for 6 to 15 weeks and then given vitamin E orally for 4 days. Plasma obtained 1 day after the last dose was assayed for glutamic oxalacetic transaminase (GOT) and pyruvate kinase activity (PK). Administration of vitamin E resulted in reduction in activity of both enzymes. Plasma levels of alkaline phosphatase, lactic dehydrogenase, and bilirubin were unaffected by vitamin E and there was no histological evidence of liver degeneration. The number of phagocytized muscle fibers was greatly reduced by vitamin E treatment, but a substantial number of necrotic fibers were still present. With more prolonged (8 days) treatment, plasma PK and GOT levels were reduced to levels found in plasma of vitamin E replete animals and few degenerated muscle fibers could be observed. It was concluded that resolution of the necrotizing myopathy in vitamin E deficient rats is a rapid process and that the decreased activity of PK and GOT in plasma is a sensitive indicator of the resolution process. The decrease in plasma enzyme levels is an easily quantitated and reproducible biological response to vitamin E administration. Thus, this approach provides a basis for a sensitive and accurate bioassay for vitamin E activity.

Animals↗
PubMed1993

Cold-sensitive cytosolic 3,5,3'-triiodo-L-thyronine-binding protein and pyruvate kinase from human erythrocytes share similar regulatory properties of hormone binding by glycolytic intermediates.

Similar cold-sensitive properties, values of dissociation constants (Kd = 1 x 10(-10) M), and regulatory effectors were found for the cold-sensitive cytosolic 3,5,3'-triiodo-L-thyronine (L-T3)-binding protein (CTBP) and pyruvate kinase from human erythrocyte. Various metabolites of the blood cell were assayed for their effects on CTBP activity after heat and cold preincubation treatments. Among these compounds, five- and six-carbon phosphorylated sugars were effective in protecting the CTBP activity against cold inactivation, whereas only ATP and dATP blocked activation by heat treatments. The effects of fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, and ATP were obtained at physiological concentrations. Three-carbon phosphorylated intermediates of glycolysis, ADP, AMP, cAMP, and GTP had no effect on cold and heat treatments. The monomer-tetramer interconversion of the enzyme was also regulated by fructose 1,6-bisphosphate and ATP. The association is under the control of fructose 1,6-bisphosphate, whereas the dissociation is under ATP control. This regulation may have physiological relevance since the hormone binds to the tetrameric form of the enzyme at a site other than the active site.

Adenosine Triphosphate↗
PubMed2000

Quantitative detection of tumor M2-pyruvate kinase in plasma of patients with lung cancer in comparison to other lung diseases.

Lung cancer is one of the predominant causes of cancer death. The aim of this project is the development of a screening method in persons with high risk for developing lung cancer, based on the measurement of Tumor M2-pyruvate kinase (Tumor M2-PK). Tumor M2-PK is quantitatively detectable in ethylenediaminetetraacetic acid-plasma with a sensitive enzyme-linked immunosorbent assay. So far, 60 patients with newly diagnosed lung cancer were included. These were compared to 24 patients with acute inflammatory lung diseases, 56 patients with pneumoconiosis, 22 patients with obstructive airway diseases, and 28 healthy persons. Tumor patients and some individuals suffering from severe inflammatory lung diseases had significantly higher Tumor M2-PK concentrations in ethylenediaminetetraacetic acid-plasma than all the other groups. The histologic tumor type had no influence on the plasma levels of Tumor M2-PK. Tumor M2-PK concentrations correlate strongly with the tumor stage, showing significantly increasing concentrations with progressive tumor stages. The present data indicate that Tumor M2-PK could be a valuable tumor marker for the detection of lung cancer.

Adenocarcinoma↗
PubMed1988

Epidermal-growth-factor stimulation of gluconeogenesis in isolated rat hepatocytes involves the inactivation of pyruvate kinase.

Preincubation of rat hepatocytes with EGF (epidermal growth factor) caused a stimulation of gluconeogenesis from alanine. The effect was maximal after preincubation of 20 min, and a half-maximal effect of EGF was obtained at 10 nM. EGF also stimulated gluconeogenesis from lactate and asparagine, but not from glutamine or from proline. Preincubation of hepatocytes with EGF caused a stable inactivation of pyruvate kinase, which may account, at least in part, for the observed effects of EGF on gluconeogenesis.

Animals↗
PubMed1995

Pyruvate kinase deficiency of mice associated with nonspherocytic hemolytic anemia and cure of the anemia by marrow transplantation without host irradiation.

Mutant mice with splenomegaly and nonspherocytic hemolytic anemia were found in an inbred colony of the CBA/N (hereafter CBA) strain maintained in the Japan SLC Haruno farm (Shuchi-gun, Shizuoka, Japan). The activity of pyruvate kinase (PK) in red blood cells (RBCs) of the anemic mutants decreased to 16.2% of normal (+/+) CBA mice. Because the mutant CBA mice showed a remarkable reticulocytosis (41.6%) and because the PK activity of reticulocytes is much higher than that of mature RBCs, the PK activity in mature RBCs of the mutant CBA mice was calculated to be 2.8% that of mature RBCs of CBA-(+/+) mice. Because RBC type PK is encoded by the Pk-1 locus of the mouse (chromosome 3), we designated the mutant locus as Pk-1slc. The anemia and PK deficiency of CBA-Pk-1slc/Pk-1slc mice were cured by bone marrow transplantation (BMT) from CBA-(+/+) mice. Prior irradiation was not necessary for the curative BMT. On the other hand, the BMT from CBA-Pk-1slc/Pk-1slc mice to nonirradiated CBA-(+/+) mice did not result in the decrease of RBCs and the reduction of PK activity. The present results indicate that CBA-Pk-1slc/Pk-1slc mice are a potentially useful animal model for studying pathophysiology of PK deficiency and for developing new therapeutic methods to correct PK deficiency.

Anemia, Hemolytic, Congenital Nonspherocytic↗