Showing papers on "Pyruvate kinase published in 1988"
TL;DR: Results provide the first direct demonstration that the exceptionally high level of hexokinase bound to mitochondria of highly glycolytic tumor cells has preferred access to mitochondrially generated ATP, a finding that may have rather profound metabolic significance for such tumors.
326 citations
TL;DR: 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase and were unrelated to inhibition of autophagic proteolysis and were caused by cAMP, which slightly increased in the presence of 3-methyladenine.
Abstract: 3-Methyladenine is generally used as an inhibitor of autophagy [P. O. Seglen & P. B. Gordon (1982) Proc. Natl Acad. Sci. USA 79, 1889-1892]. Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well. 1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by cAMP, which slightly increased in the presence of 3-methyladenine. 2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.
137 citations
TL;DR: Findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.
121 citations
TL;DR: It was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region, and that some sequences involved in this regulation are missing in the cloned fragment.
Abstract: The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI+. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration--55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration. A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth ona glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region. However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.
117 citations
TL;DR: It is concluded that hepatocyte cultures as described in the present study may be a useful, well‐defined system for long‐term metabolic, pharmacologic and toxicologic studies.
114 citations
TL;DR: Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate, whereas DNP has no significant effect on glucose metabolism.
Abstract: Individual blastocysts from cows were cultured for 3 h under 5% CO2 in air, in 4 microliters droplets of Ham's F-10 medium containing D-[5-3H]glucose, D-[1-14C]-glucose, D-[6-14C]glucose, [2-14C]pyruvate, or L-[U-14C]glutamine, and with or without 2,4-dinitrophenol (DNP) or phenazine ethosulphate (PES). The 14CO2 or 3H2O produced were collected by exchange with an outer bath of 400 microliter 25 mM-NaHCO3. All combinations of substrate and treatment (control, DNP or PES) produced measurable quantities of labelled product except for D-[6-14C]glucose in the presence of PES. Untreated and DNP-treated embryos developed normally during a subsequent 48-h culture period in fresh medium, but PES-treated embryos degenerated. Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate. Conversely, DNP has no significant effect on glucose metabolism, indicating that glycolysis is blocked in the bovine blastocyst due to a lack or inhibition of pyruvate kinase. The production of 14CO2 from D-[1-14C]glucose increased significantly in the presence of PES, indicating that the activity of the pentose shunt is less than maximal.
107 citations
TL;DR: It is argued that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvates in both directions along the glycolytic pathway.
Abstract: Enolase or 2-phospho-D-glycerate hydrolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is converted by pyruvate kinase to pyruvate. We describe here the crystallographic determination of the structure of yeast enolase at high resolution (2.25 A) and an analysis of the structural homology between enolase, pyruvate kinase and triose phosphate isomerase. Each of the two subunits of enolase forms two distinctive domains. The larger domain (residues 143–420) is a regular 8-fold β/α-barrel, as first found in triose phosphate isomerase, and later in pyruvate kinase and 11 other functionally different enzymes. An analysis of the molecular geometries of enolase and pyruvate kinase based on the roughly 8-fold symmetry of the barrel showed a structural homology better than expected for proteins related by convergent evolution. We argue that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvate in both directions along the glycolytic pathway. There is structural and sequence evidence that muconate lactonizing enzyme later evolved from enolase.
93 citations
TL;DR: Control of glycolysis during anoxia was investigated in five organs of the freshwater turtle, Pseudemys scripta, after 1 or 5 h of submergence in N2-bubbled water, and changes in the levels of hexose and triose phosphate intermediates of glyCOlysis indicated an activation and inhibition in brain, heart, and skeletal muscles.
Abstract: Control of glycolysis during anoxia was investigated in five organs (heart, brain, liver, and red and white skeletal muscles) of the freshwater turtle, Pseudemys scripta, after 1 or 5 h of submergence in N2-bubbled water. Lactate was produced as the metabolic end product, with distinct organ differences in the amount (net lactate accumulation was 2.4-fold higher in brain than white muscle) and rate (lactate production in liver dropped 16-fold after the 1st h) of lactate accumulation. ATP and total adenylate contents of all organs were reduced (by 15-32%) after 1 h of submergence, but energy charge was maintained; after 5 h, adenylate contents had fully recovered. Changes in the levels of hexose and triose phosphate intermediates of glycolysis indicated an activation of glycolysis within the 1st h of anoxia exposure in brain, heart, and skeletal muscles. By 5 h, however, these were reversed, and a glycolytic rate depression was indicated, consistent with the overall metabolic rate depression accompanying long-term anaerobiosis in the turtle. Crossover analysis indicated glycolytic control at the pyruvate kinase reaction in all organs during both glycolytic activation and metabolic depression; regulatory control at the phosphofructokinase locus was primarily important only during glycolytic activation in heart and red muscle. The same analysis indicated a very rapid glycolytic inhibition in liver occurring within the 1st h of anoxia exposure; this allows glycogenolysis to be directed toward glucose export yielding the fermentative fuel used by other organs during anoxia.
89 citations
TL;DR: Transformation of CECs by Rous sarcoma virus leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate, and kinetic changes were correlated with tyrosine phosphorylation of M2‐PK, but there is no direct evidence that they are caused by post‐translational modification of the enzyme.
80 citations
TL;DR: It appears that the catecholamine-dependent activation of gluconeogenesis may compensate for the acid-base-dependent shortfall in glucose output by the liver.
68 citations
TL;DR: The enhanced generation of reactive oxygen intermediates and increased inactivation of enzymes by microsomes may contribute toward the hepatotoxic effects associated with ethanol consumption.
Abstract: Enzymatic and nonenzymatic mixed-function oxidase systems have been shown to generate an oxidant that catalyzes the inactivation of glutamine synthetase and other metabolic enzymes. Recent studies have shown that microsomes isolated from rats chronically fed ethanol generate reactive oxygen intermediates at elevated rates compared with controls. Microsomes from rats fed ethanol were found to be more effective than control microsomes in catalyzing the inactivation of enzymes added to the incubation system. The enzymes studied were alcohol dehydrogenase, lactic dehydrogenase, and pyruvate kinase. The inactivation process by both types of microsomal preparations was sensitive to catalase and glutathione plus glutathione peroxidase, but was not affected by superoxide dismutase or hydroxyl radical scavengers. Iron was required for the inactivation of the added enzymes; microsomes from the rats fed ethanol remained more effective than control microsomes in catalyzing the inactivation of enzymes in the absence o...
TL;DR: The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo, and evidence is presented which indicates that plastids also contained low activities of enolase and phosphoglycerate mutase.
Abstract: In order to determine whether the enzymes required to convert triose phosphate to acetyl CoA were present in pea (Pisum sativum L.) seed plastids, a rapid, mechanical technique was used to isolate plastids from developing cotyledons. The plastids were intact and the extraplastidial contamination was low. The following glycolytic enzymes, though predominantly cytosolic, were found to be present in plastids: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and pyruvate kinase(EC 2.7.1.40). Evidence is presented which indicates that plastids also contained low activities of enolase (EC 4.2.1.11) and phosphoglycerate mutase (EC 2.7.5.3). Pyruvate dehydrogenase, although predominantly mitochondrial, was also present in plastids. The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo.
TL;DR: Interactions of F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes, supporting the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.
TL;DR: The full-length sequence of human L-type PK cDNA was isolated and determined and activity was detected in the extract of COS cells by the classical PK electrophoresis method.
Abstract: Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes. Among these isozymes, abnormalities of liver (L)-type PK is considered to be associated with hereditary nonspherocytic hemolytic anemia in humans. We isolated and determined the full-length sequence of human L-type PK cDNA. The cDNA contains 1629 base pairs encoding 543 amino acids, 68 base pairs of 5'-noncoding sequence, and 734 base pairs of 3'-noncoding sequence. The similarity between human and rat L-type PK was 86.9% at the nucleotide sequence level and 92.4% at the amino acid sequence level. The full-length L-type PK cDNA was placed under the promoter of simian virus 40 and introduced into monkey COS cells. Human L-type PK activity was detected in the extract of COS cells by the classical PK electrophoresis method.
TL;DR: Transmembrane proton flow was found to be the most sensitive of the functions tested toward ethanol, and it could represent the first target of ethanol action on fermentation.
Abstract: The effect of ethanol on the activities of the key enzymes of the glycolytic pathway and on two membrane functions related with fermentation, the glucose uptake system, and proton extrusion rate are examined. The results indicate that ethanol, up to 2M, does not cause any change of the glucose uptake velocity nor any substantial change in the key glycolytic enzyme activities while the fermentation rate is reduced by about 50%. In a cell extract 3M ethanol as well as incubation of yeast cells with 4M ethanol caused a considerable decrease of pyruvate kinase and hexokinase activities. Phosphofructokinase remained unchanged even at higher ethanol concentrations. Transmembrane proton flow was found to be the most sensitive of the functions tested toward ethanol, and it could represent the first target of ethanol action on fermentation.
TL;DR: In situ hybridization is used to mapped the human liver-type pyruvate kinase gene (PKL) to band q21 of chromosome 1 and it is shown that this gene is important for ATP production in the glycolytic pathway.
Abstract: Pyruvate kinase (PK) is an important enzyme for ATP production in the glycolytic pathway. Deficiency of this enzyme in erythrocytes is characterized by hemolytic anemia. Using in situ hybridization, we have mapped the human liver-type pyruvate kinase gene (PKL) to band q21 of chromosome 1.
TL;DR: Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase.
Abstract: On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.
TL;DR: Cytosolic pyruvate kinase from endosperm of germinating castor beans has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruVate produced/minute per milligram protein.
Abstract: Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.
Journal Article•
TL;DR: The hepatic gluconeogenic/glycolytic pathway undergoes a complex but extremely well-integrated regulation by hormones that accounts in large part for the major role the organ plays in the control of glucose homeostasis.
Abstract: Cyclic AMP plays a major, if not primary, role in the regulation of hepatic gluconeogenesis. The cyclic nucleotide acts on two levels. First, cAMP levels determine the phosphorylation state of key regulatory enzymes including pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Regulation of cAMP levels by glucagon, insulin, and catecholamines accounts in large part for minute-to-minute hormonal control of pathway flux in fed animals and during the transition from fed to starved; second, cAMP plays a key role in regulation of gene transcription of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucokinase, and probably 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Cyclic AMP acts to induce synthesis of mRNA for phosphoenolpyruvate carboxykinase and probably fructose 1, 6-bisphosphatase while it suppresses transcription of the genes for pyruvate kinase and glucokinase. Its role in the regulation of gene transcription of the bifunctional enzyme and 6-phosphofructo 1-kinase remains to be defined. Insulin is the most important hormone for restraining the level of cAMP. Insulin acts to oppose the acute actions of cAMP on enzyme phosphorylation, presumably by activating a phosphodiesterase and thereby lowering cAMP levels. Insulin also opposes the action of hormones (alpha-adrenergic agonists, angiotensin, vasopressin) that act in liver via cAMP-independent phosphorylation. However, in the systems in which this has been studied, the cAMP-independent effects on gluconeogenic/glycolytic pathway flux are small in comparison to cAMP-dependent regulation. Insulin also opposes the action of cAMP on gene transcription by an as yet unknown mechanism. This effect does not appear to involve changes in the level of cAMP because the hormone also acts in cultured cells when added alone or in the presence of dexamethasone. The ability of insulin to lower hepatic cAMP levels and to modulate gene expression are important because restoration of acute regulatory hormone responsiveness to starved or diabetic animals could not occur if insulin were unable to lower cAMP levels and be the dominant factor in modulating the gene expression of these key regulatory enzymes. Clearly, the hepatic gluconeogenic/glycolytic pathway undergoes a complex but extremely well-integrated regulation by hormones that accounts in large part for the major role the organ plays in the control of glucose homeostasis.
TL;DR: Enzymes involved in pyruvate metabolism were assayed in crude extracts of Rhodobacter capsulatus cells grown photosynthetically with different carbon sources, indicating that this anaplerotic enzyme is essential for growth on pyruVate or related compounds as sole added carbon source.
Abstract: Summary: Enzymes involved in pyruvate metabolism were assayed in crude extracts of Rhodobacter capsulatus cells grown photosynthetically with different carbon sources. Pyruvate dehydrogenase, pyruvate carboxylase, pyruvate kinase and phosphoenolpyruvate carboxykinase (ATP) were all present in extracts of cells grown on DL-lactate, whereas phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase and pyruvate, orthophosphate dikinase were undetectable in extracts of cells grown on either DL-lactate, DL-malate or acetate. Malate-grown cells and acetate-grown cells contained lower levels of pyruvate carboxylase and higher levels of pyruvate kinase than did lactate-grown cells, and malate-grown cells also contained lower levels of phosphoenolpyruvate carboxykinase. Pyruvate dehydrogenase activity was undetectable in extracts of acetate-grown cells. Two mutant strains, which were unable to grow on pyruvate, lactate or any other compound metabolized via pyruvate, were found to be deficient in pyruvate carboxylase activity, indicating that this anaplerotic enzyme is essential for growth on pyruvate or related compounds as sole added carbon source. This requirement for pyruvate carboxylase can be bypassed to some extent, however, since the mutants grew on acetate, albeit more slowly than the wild-type and after a long lag period. The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present at high levels in extracts of acetate-grown cells, both of the mutants and of several wild-type strains tested, whereas isocitrate lyase activity was undetectable in three different strains of Rhodobacter sphaeroides. This is consistent with previous suggestions that acetate is assimilated via the glyoxylate cycle in R. capsulatus but not in R. sphaeroides.
TL;DR: The above data suggest that the two variants of PK from the foot muscle of P. caerulea are interconvertible and the direction of the interconversion depends on the duration of anaerobiosis.
Abstract: Pyruvate kinase (PK) from the foot muscle of Patella carulea (L) is present as two variants. These variants are eluted by ion-exchange chromatography from DEAE-cellulose as peak I and peak II at different concentrations of KCl. Studies of their kinetic properties show that both variants of PK exhibit sigmoidal curves with respect to phosphoenolpyuvate (PEP), but the PK in peak I is more sensitive to the activation by PEP and fructose diphosphate (FDP) and less sensitive to inhibition by alanine, ATP, and H+ than the PK in peak II. The proportion of the two PK variants changes during anaerobiosis. The ratio of the number of units of enzyme in peak I to that in peak II increases up to the second hour and then decreases up to the eight hour of anaerobiosis. Studies on the electrophoretic mobility of these two variants on strips of cellulose acetate show that the less active from or peak II PK migrates more quickly toward the anode (Rf = 0.13) than the more active from or peak I PK (Rf = 0.07). Peak II PK, after treatment with alkaline phosphatase, exhibits kinetic properties and electrophoretic mobility quite similar to those of peak I PK. The above data suggest that the two variants of PK from the foot muscle of P. caerulea are interconvertible and the direction of the interconversion depends on the duration of anaerobiosis.
TL;DR: The data provide strong evidence that coordinated glycolytic rate control, as part of an overall metabolic rate depression during anoxia, is mediated via anxia-induced covalent modification of regulatory enzymes.
Abstract: The effects of environmental anoxia (24 h at 7°C in N2/CO bubbled water) on the maximal activities, selected kinetic properties, and isoelectric points of phosphofructokinase and pyruvate kinase were measured in eight tissues of the goldfish,Carassius auratus, in order to evaluate the role of possible covalent modification of enzymes in glycolytic rate control and metabolic depression during facultative anaerobiosis. Both enzymes showed modified kinetic properties as a result of anoxia in liver, kidney, brain, spleen, gill, and heart. Effects of anoxia on properties of pyruvate kinase included reducedV
max, increased S0.5 for phosphoenolpyruvate, increasedK
a for fructose-1,6-bisphosphate, and strongly reduced I50 for alanine; all these effects are consistent with an anoxia-induced phosphorylation of pyruvate kinase to produce a less active enzyme form. Anoxia-induced alterations in phosphofructokinase kinetics included tissue-specific changes in S0.5 for fructose-6-phosphate, Hill coefficient,K
a values for fructose-2,6-bisphosphate, AMP, and NH
4
+
, and I50 values for ATP and citrate, the direction of changes being generally consistent with the production of a less active enzyme form in the anoxic tissue. Enzymes from aerobic versus anoxic skeletal muscle (both red and white) did not differ in kinetic properties but anoxic enzyme forms had significantly different pI values than the corresponding aerobic forms. Enzyme phosphorylation-dephosphorylation as the basis of the anoxia-induced changes in the kinetic properties of PFK and PK was further tested in liver: treatment of the aerobic forms of both enzymes with cAMP dependent protein kinase altered enzyme kinetic properties to those typical of the anoxic enzymes while alkaline phosphatase treatment of the anoxic enzyme forms had the opposite effect. The data provide strong evidence that coordinated glycolytic rate control, as part of an overall metabolic rate depression during anoxia, is mediated via anoxia-induced covalent modification of regulatory enzymes.
TL;DR: The results indicated that the elimination of embryonal dormancy in apple seeds was connected with a change from domination of PPP to domination of glycolysis in sugar catabolism during cold stratification, and a regulatory role of cyanide in removal of dormancy is suggested.
Abstract: The ratio of glycolysis to the pentose phosphate pathway (PPP, C6/C1 ratio), and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and pyruvate kinase (PK, EC 2.7,1.40) were determined in apple seeds (Malus domestica Borb, cv. Antonowka) submitted to cold and warm stratifications. Our results indicated that the elimination of embryonal dormancy in apple seeds was connected with a change from domination of PPP to domination of glycolysis in sugar catabolism during cold stratification. Cyanide pretreatment affected the C6/C1 ratio and the activities of the enzymes under study in such a manner that the maxima of PPP and glycolysis appeared earlier during stratification. We suggest a regulatory role of cyanide in removal of dormancy.
TL;DR: Investigation of the effects of long-term physiological hyperglucagonemia and insulinopenia induced by suckling on expression of the two genes in the liver of normal newborn rats shows that transcription of the L-type pyruvate kinase gene is inhibited at birth and remains low during the whole suckling period, whereas transcription of this gene is maximal in the neonate, and then decreases despite very high levels of plasma glucagon during suckling.
Abstract: Glucagon and its second messenger, cAMP, are known to rapidly block expression of the L-type pyruvate kinase gene and to stimulate expression of phosphoenolpyruvate (PEP) carboxykinase gene in the liver in vivo. The respective roles, however, of hyperglucagonemia, insulinopenia, and carbohydrate deprivation in the inhibition of L-type pyruvate kinase gene expression during fasting are poorly understood. In addition, the long-term effects of physiological hyperglucagonemia on expression of the two genes are not known. In this study, we investigate the effects of long-term physiological hyperglucagonemia and insulinopenia induced by suckling (which provides a high-fat, low-carbohydrate diet) on expression of the two genes in the liver of normal newborn rats. We show that transcription of the L-type pyruvate kinase gene is inhibited at birth and remains low during the whole suckling period, whereas transcription of the PEP carboxykinase gene is maximal in the neonate, and then decreases despite very high levels of plasma glucagon during suckling. In contrast to the adult, however, in which L-type pyruvate kinase gene expression in the liver is blocked by cAMP and stimulated by carbohydrates, the regulation of L-type pyruvate kinase gene expression in the newborn undergoes a developmental maturation: the inhibitory effect of glucagon is never complete in developing rat liver and the stimulatory effect of glucose could not be detected during suckling, due to either hyperglucagonemia, immaturity of the gene regulatory system, or both.
TL;DR: The effects of ligands on the structure of rabbit muscle pyruvate kinase were studied by small angle neutron scattering, and results indicate a "contraction" and "expansion" of the enzyme when it transforms between its active and inactive forms.
TL;DR: Observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle.
Abstract: Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.
TL;DR: All tumour pyruvate kinase isoenzymes were insensitive to normal signal molecules, i.e., to ATP and fructose 1,6-diphosphate, which regulate liver pyruvent kinase activity, and it was, however, noted that the binding of pyruviate kinases to DNA is connected with a diminution in their catalytic activity.
TL;DR: This study removes any necessity to consider the existence of an isomerization equilibrium that is substrate-induced; and provides experimental support for adoption of the Monod model of allostery to interpret enzyme kinetic data for pyruvate kinase.
TL;DR: The results suggest that the ability of turtle brain to survive extended periods of anoxia is the result of metabolic rate depression regulated, at the molecular level, by enzyme inactivation through anoxIA‐induced covalent modification.
TL;DR: Blood samples from 722 unrelated patients with anemia and/or reticulocytosis were submitted to the authors' laboratory for red cell enzyme assay during the past 7 years and 82 cases of 7 different red cell enzymes deficiencies and 2 of unstable hemoglobin were found.