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Showing papers on "Pyruvate kinase published in 1989"


Journal ArticleDOI
01 Jul 1989-Yeast
TL;DR: Eight different enzyymes for glycolysis and alcoholic fermentation were overproduced in a common Saccharomyces cerevisiae strain by placing their genes on multicopy vectors by increasing the specific enzyme activities between 3·7 and 13·9‐fold above the wild‐type level.
Abstract: Eight different enzymes for glycolysis and alcoholic fermentation were overproduced in a common Saccharomyces cerevisiae strain by placing their genes on multicopy vectors. The specific enzyme activities were increased between 3.7- and 13.9-fold above the wild-type level. The overproduction of the different glycolytic enzymes had no effect on the rate of ethanol formation, even with those enzymes that catalyse irreversible steps: hexokinase, phosphofructokinase and pyruvate kinase. Also the simultaneous increase in the activities of pairs of enzymes such as pyruvate kinase and phosphofructokinase or pyruvate decarboxylase and alcohol dehydrogenase, did not increase the rate of ethanol production. The levels of key glycolytic metabolites were also normal, compared to the reference strain.

304 citations


Journal ArticleDOI
TL;DR: It is hypothesized that pyrophosphate:fructose 6-ph phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3- phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion.
Abstract: When Brassica nigra leaf petiole suspension cells were subjected to 7 days of inorganic phosphate (Pi) starvation the extractable activity of: (a) pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate phosphatase, and phosphoenolpyruvate carboxylase increased at least fivefold, (b) phosphorylating NAD-glyceraldehyde 3-phosphate dehydrogenase decreased about sixfold, and (c) ATP:fructose 6-phosphate 1-phosphotransferase, 3-phosphoglycerate kinase, pyruvate kinase, or NAD malic enzyme was not altered. Pi deprivation also resulted in significant reductions in extractable levels of Pi, ATP, ADP, fructose 2,6-bisphosphate, and soluble protein, but caused a sixfold elevation in free amino acid concentrations. No change in inorganic pyrophosphate concentration was observed following Pi starvation. It is hypothesized that pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion.

270 citations


Journal ArticleDOI
TL;DR: Hepatocytes isolated from adult fasted rats and cultured in the presence of thyroid hormones, glucocorticoids, and in a serum-free medium conserve the essentials of their differentiated function and hormonal sensitivity for at least 1 week.

160 citations


Journal ArticleDOI
TL;DR: The human hepatoblastoma line Hep G2 modulates gene expression in vitro in response to increasing culture density to provide a cell culture model for the modulation of the liver phenotype which occurs during fetal/adult development or during liver regeneration.
Abstract: The human hepatoblastoma line Hep G2 modulates gene expression in vitro in response to increasing culture density. Two stages of growth can be defined. At low density ( 1×106 cells/cm2) have a doubling time of 193 h, a four fold higher level of albumin production, increased levels of the adult isoenzymes of aldolase and pyruvate kinase and increased asialoglycoprotein receptor. The alteration in albumin and alphafetoprotein synthesis was reflected by changes in the messenger RNA levels and the relative transcription of these two genes. Hep G2 provides a cell culture model for the modulation of the liver phenotype which occurs during fetal/adult development or during liver regeneration.

134 citations


Journal ArticleDOI
TL;DR: The hypothesis that catecholamines regulate glucose availability during hypoxia in the rainbow trout by activating glycogen phosphorylase (GPase) while inhibiting pyruvate kinase (PK) in the liver is tested.
Abstract: This study tests the hypothesis that catecholamines regulate glucose availability during hypoxia in the rainbow trout by activating glycogen phosphorylase (GPase) while inhibiting pyruvate kinase (PK) in the liver. The net result would be an increase in liver glycogenolysis and a reduction of glycolysis and/or enhancement of gluconeogenesis. We used the criteria of Stalmans & Hers (1975) and report much lower resting percent GPase a (active) values (20–30%) than those previously published. Dorsal aortic injections of epinephrine or norepinephrine increased plasma glucose (16–46%), had no effect on liver or muscle glycogen levels, decreased the activity of PK, and increased total and percent GPase a activities. Pre-treatment with the beta-adrenoreceptor antagonist propranolol eliminated these effects. During moderate hypoxia, plasma glucose remained unchanged, while lactate levels increased fourfold. When fish were pre-treated with propranolol, hypoxia depressed plasma glucose levels (−26%), total and percent GPase a, and increased PK activity, suggesting that hypoxia mediated the dephosphorylation of these enzymes. We conclude that catecholamines stimulate hepatic beta-adrenoreceptors during hypoxia and sustain plasma glucose levels by nullifying the deleterious effects of hypoxia on metabolic function. The specific metabolic consequences of these catecholamine-mediated effects are an increase in the activity of the active form of GPase and a reduction in PK activity, which suggests an activation of glycogenolysis and an inhibition of glycolysis and/or activation of gluconeogenesis, respectively.

129 citations


Journal ArticleDOI
TL;DR: It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvating kinase reaction during extended periods of orthophosphate starvation.
Abstract: Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.

111 citations


Journal ArticleDOI
TL;DR: Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis, and each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation, indicating sequence similarity between the twopolypeptides.
Abstract: 1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

108 citations


Journal ArticleDOI
TL;DR: A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated as discussed by the authors, which is a monomer that has approximately 5% the enzymatic activity of the tetrameric pyruvate kinase M2.
Abstract: A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has approximately 5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent Ki values of T3, L-thyroxine, and D-T3 are 30 nM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.

90 citations


Journal ArticleDOI
TL;DR: The in vivo activity of PKc is probably regulated by the relative cytosolic levels of DHAP, Pi, and glutamate; this provides a rationale for the activation of algal cytosolsolic pyruvate kinase which occurs during periods of enhanced ammonia assimilation.

73 citations


Journal ArticleDOI
TL;DR: Different doses of glucagon and glucagon-like peptide (GLP) isolated from coho salmon, Oncorhynchus kisutch were tested in vivo and in vitro on juvenile coho and chinook (O. tshawytscha) salmon.

69 citations


Journal ArticleDOI
TL;DR: The proximal 5'-flanking sequence of the L-PK gene appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by thebinding of another liver- specific factor, LF-A1.
Abstract: A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.

Journal ArticleDOI
TL;DR: Data indicate that an increase in cyclic AMP concentration, sufficiently great to activate A-kinase, is a mechanism that mediates the glucagon-induced increase in [Ca2+]c.
Abstract: The mechanism whereby glucagon causes an increase in the concentration of cytoplasmic free Ca2+, [Ca2+]c, in isolated hepatocytes has been investigated. There have been proposals of cyclic-AMP-dependent and cyclic-AMP-independent mechanisms. In this work, the inactivation of pyruvate kinase was used as an indicator of increases in the activity of cyclic-AMP-dependent protein kinase, A-kinase. [Ca2+]c was measured using the fluorescent probe indo-1. The decrease in activity of pyruvate kinase caused by an increase in [Ca2+]c alone, i.e. mediated by mechanisms not involving cyclic AMP and exemplified by the effect of vasopressin, was of minimal significance under the conditions of the enzyme assay. Studies of the effects of a wide range of glucagon concentrations indicate that any increase in [Ca2+]c caused by glucagon was always associated with a decrease in pyruvate kinase activity. A similar relationship was obtained if glucagon-receptor occupancy was circumvented by using the 8-bromo-derivative of cyclic AMP to activate the A-kinase. It was also found that the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine could potentiate the ability of glucagon to increase [Ca2+]c: no such potentiation was observed when vasopressin was used to raise [Ca2+]c. Together these data indicate that an increase in cyclic AMP concentration, sufficiently great to activate A-kinase, is a mechanism that mediates the glucagon-induced increase in [Ca2+]c.

Journal ArticleDOI
TL;DR: Data indicate that the S. minutum pyruvate kinase isoforms, PK1 and PK2, are not interconvertible forms of the same protein, but probably represent chloroplastic and cytosolic isozymes, respectively.

Journal ArticleDOI
TL;DR: Observations are in good agreement with the role of this enzyme in the two tissues of trout liver and kidney, which was enhanced at all substrate concentrations.

Journal ArticleDOI
TL;DR: The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.
Abstract: Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

Journal ArticleDOI
TL;DR: PEP carboxylase and pyruvate kinase activities appeared to contribute to a complex interaction that regulates the metabolic flow of glycolytic carbon into precursors for both protein and oil biosynthesis during soybean seed development.
Abstract: (…) The correlation of PEP carboxylase activity with protein was generally higher than with oil, suggesting that much of the oxaloacetate (OAA) produced may be used for synthesis of protein precursors. However, the high r values between PEP carboxylase and oil suggested that OAA may also be converted to malate and then to pyruvate (a precursor for oil) via a transhydrogenase system. Therefore, PEP carboxylase and pyruvate kinase activities appeared to contribute to a complex interaction that regulates the metabolic flow of glycolytic carbon into precursors for both protein and oil biosynthesis during soybean seed development

Journal ArticleDOI
TL;DR: The results suggest that the integrated DNA contains all elements necessary for tissue specificity (L' and L) as well as hormonal and nutritional control (L) of expression of the rat transgene.

Journal ArticleDOI
TL;DR: 32P-labeled glucose 6-phosphate and phosphoenolpyruvate were injected into oocytes, fertilized eggs, and early embryos of Xenopus laevis, and the 32P label was followed into glycolytic enzymes and acid-soluble metabolites.

Journal ArticleDOI
TL;DR: Electron spin echo envelope modulation (ESEEM) spectroscopy, with Mn2+ and VO2+ as paramagnetic probes, was used to examine active-site structures at the protein-based divalent cation site of rabbit muscle pyruvate kinase in the presence of substrates, products, and the requisite inorganic cofactors.
Abstract: Electron spin echo envelope modulation (ESEEM) spectroscopy, with Mn2+ and VO2+ as paramagnetic probes, was used to examine active-site structures at the protein-based divalent cation site of rabbit muscle pyruvate kinase in the presence of substrates, products, and the requisite inorganic cofactors. Two different VO.protein complexes were clearly distinguished, which differed with respect to coordination of the active-site lysine to VO2+. Lysine coordination was sensitive to the presence of pyruvate and phosphoenolpyruvate (PEP) and to the nature of the monovalent cation. In the presence of MgATP and oxalate, a 4-MHz 31P contact interaction was observed, which indicates that the ATP is directly coordinated to Mn2+ at the protein-based site. No 31P contact interactions were observed, however, in the presence of PEP. Pyruvate was determined to be a bidentate ligand of VO2+, on the basis of the observation of 2.2- and 5.4-MHz 13C contact interactions between VO2+ and [1-13C]pyruvate and [2-13C]pyruvate, respectively. Magnetic coupling between VO2+ or Mn2+ and 23Na, 39K, and 133Cs was observed, demonstrating the close proximity of the monovalent cation and the protein-based divalent cation.

Journal ArticleDOI
TL;DR: The glycolytic enzymes glycogen phosphorylase, phosphofructokinase (PFK), and pyruvate kinase (PK) were assessed in liver, heart, red muscle, and white muscle of aerobic and 5-h anoxic turtles for changes in total activity and kinetic parameters during anoxia.
Abstract: The glycolytic enzymes glycogen phosphorylase, phosphofructokinase (PFK), and pyruvate kinase (PK) were assessed in liver, heart, red muscle, and white muscle of aerobic and 5-h anoxic turtles (Pseudemys scripta) for changes in total activity and kinetic parameters. Anoxia induced statistically significant changes in these glycolytic enzymes in each of the four organs assayed. Compared with normoxic controls, anoxic liver showed a 3.3-fold increase in glycogen phosphorylase activity, a 1.5-fold increase in the PFK I50 value for citrate (concentration that inhibits initial activity by 50%), a 1.5-fold increase in the PFK Michaelis constant (Km) value for fructose 6-phosphate (P), and an increased maximal activity of PK. Anoxic heart muscle showed a 2.6-fold decrease in glycogen phosphorylase activity and, for PFK, a 1.7-fold decrease in the Km value for ATP and a twofold increase in the I50 value for citrate. In anoxic white muscle, PFK showed a fivefold lower Km value for fructose-6-P and a threefold lower activator concentration producing half-maximal activation (A50) for potassium phosphate than the aerobic enzyme form. Changes in anoxic white muscle PK included a twofold increase in the Km value for ADP and a 1.7-fold decrease in the I50 value for alanine. In red muscle, anoxia affected only the Km value for ATP, which was 50% higher than the value for the aerobic enzyme form. Fructose 2,6-diphosphate (P2) levels also decreased in heart muscle and increased in red and white muscle during anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: It is hypothesized that different hepatic receptors with differing modes of intracellular message transduction are involved in glucagon and GLP actions while targetting identical metabolic routes.
Abstract: This review addresses direct and indirect metabolic actions of hormones co-encoded in the preproglucagon gene of fishes. Emphasis is placed on a critical analysis of the effects of glucagon and glucagon-like peptide (GLP) and the current knowledge of the respective modes of action is reviewed. In mammals GLPs exert no direct metabolic actions. In fish liver, GLP and glucagon act on similar targets of intermediary metabolism by enhancing flux through glycogenolysis, lipolysis and gluconeogenesis. Increases in substrate oxidation are not uniform. Hormonal activation of glycogen phosphorylase and triglyceride lipase and inhibition of pyruvate kinase are implicated in these actions. Hormone-dependent hyperglycemia, depletion of hepatic glycogen and increases in free fatty acids are noticeablein vivo. Glucagon also activates hepatic amino acid uptake and ammonia excretion. Glucagon actions are accompanied by large increases in hepatic cAMP and increased phosphorylation of pyruvate kinase. Metabolic effects measured after GLP administration are associated with minor, if any, increases in cAMP and effects on pyruvate kinase are variable. We hypothesize that different hepatic receptors with differing modes of intracellular message transduction are involved in glucagon and GLP actions while targetting identical metabolic routes. Responses of different species of fish cover a wide spectrum, and variation of response with the circannual cycle of experimental animals makes comparisons of results, even within one species, difficult.

Journal ArticleDOI
TL;DR: Antisera against fish pancreatic peptides proved to be a suitable tool in the studies of hormonal regulation of fish metabolism, especially after the combined administration of aGLU + aGLP.
Abstract: Acute deficiency in pancreatic peptides (insulin, somatostatin-25, glucagon, and glucagon-like peptide) was invoked for 9–12 hr in coho, Oncorhynchus kisutch, and chinook, O. tshawytscha, salmon by administration of specific antisera raised against purified salmon hormones. Insulin-deficient fish were hyperglycemic, had diminished glycogen content in the liver (Plisetskaya et al., '88a, elevated liver triacylglycerol lipase activity, and higher concentration of plasma triiodothyronine (T3) compared to a control group of fish injected with nonspecific rabbit serum. After immunoneutralization of somatostatin-25, fish remained normoglycemic, with higher liver glycogen content, decreased lipase activity, and elevated plasma levels of insulin, while the levels of T3 declined. The induced deficiency in glucagon family peptides led to comparatively smaller changes: liver glycogen content was increased after anti-glucagon-like peptide (aGLP) injection and transient hyperglycemia was apparent following anti-glucagon (aGLU) administration. Circulating levels of insulin remained unaffected for at least 9 hr following aGLU and aGLP treatments. The velocity of pyruvate kinase at 2.5 mM phosphoenolpyruvate (V2.5) was depressed, especially after the combined administration of aGLU + aGLP. The effectiveness of immunoneutralization experiments was greatly dependent on the particular stage of the fish life cycle. Antisera against fish pancreatic peptides proved to be a suitable tool in the studies of hormonal regulation of fish metabolism.

Journal ArticleDOI
TL;DR: It is suggested that stress-susceptible pigs are under a sustained oxidant stress and that a decreased ability to accommodate even a normal free radical load may contribute to the rapid development of the fatal stress response.

Journal ArticleDOI
TL;DR: In this article, Noguchi et al. used phase-modulation measurements of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions to provide insights into the effects of localized sequence change on the global structural and functional behavior of the enzyme.
Abstract: Pyruvate kinase is an important glycolytic enzyme which is expressed differentially as four distinct isozymes whose catalytic activity is regulated in a tissue-specific manner. The kidney isozyme is known to exhibit sigmoidal kinetics, whereas the muscle isozyme exhibits hyperbolic kinetic properties. By integration of the crystallographic [Stuart, D. I., Levine, M., Muirhead, H., & Stammers, D.K. (1979) J. Mol. Biol. 134, 109-142] and primary sequence data [Noguchi, T., Inoue, H., & Tanaka, T. (1986) J. Biol. Chem. 261, 13807], it was shown that the primary sequence for the C alpha 1 and C alpha 2 regions may constitute the allosteric switching site. To provide insights into the effects of the localized sequence change on the global structural and functional behavior of the enzyme, kinetic studies under a wide spectrum of conditions were conducted for both the muscle and kidney isozymes. These conditions include measurements of enzyme activity as a function of substrate concentrations with different concentrations of allosteric inhibitors or activators. These results showed that both isozymes exhibit the same regulatory properties although quantitatively the distribution of active and inactive forms and the various dissociation constants which govern the binding of substrate and allosteric effectors with the enzyme are different. For such a majority of equilibrium constants to be altered, the localized primary sequence change must confer global perturbations which are manifested as differences in the various equilibrium constants. Structural information about these two isozymes was provided by phase-modulation measurement of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities.
Abstract: The separation of red blood cells into reticulocytes and young and old erythrocytes enables investigations of fractions with different contents of reticulocytes. Activities of hexokinase, glucose phosphate isomerase, phosphofructokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase showed a linear relationship to reticulocyte counts. The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities. By linear regression analysis enzyme activities in erythrocytes (AE) and reticulocytes (AR) could be evaluated. The activity of a given enzyme in the reticulocyte exceeded that of the erythrocyte; the quotient AR/AE represents the decline of enzyme activity from the reticulocyte to the erythrocyte stage. This value AR/AE is 16.7 for pyruvate kinase and 9.4 for hexokinase and thus considerably higher than that for the other enzymes investigated (glucose phosphate isomerase: 2.9, phosphofructokinase: 4.3, glucose-6-phosphate dehydrogenase: 4.5). In patients suffering from erythrocyte enzymopathies, the AR/AE for pyruvate kinase was 16.2 and thus almost identical to the normal enzyme. Calibration curves where the enzyme activity is plotted versus the fraction of reticulocytes enable the determination of normal activity of a given erythrocyte enzyme depending on the content of reticulocytes in red blood cell suspensions. Thus an unambiguous diagnosis of enzyme defects irrespective of reticulocyte counts becomes possible.

Journal ArticleDOI
TL;DR: The results are consistent with N Assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch and indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation.
Abstract: In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH(4) (+) addition we found that: (a) N accumulated in glutamine while glutamate and alpha-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH(4) (+) addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH(4) (+) addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.

Journal ArticleDOI
TL;DR: The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non‐preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non-pre preferred codons.

Journal ArticleDOI
TL;DR: The pyruvate kinase from Trypanosoma cruzi epimastigotes was activated by fructose 2,6-diphosphate through a decrease in (S) 0.5 and an increase in Vmax for both substrates, suggesting that the effects are allosteric, and not due to chelation.
Abstract: The pyruvate kinase from Trypanosoma cruzi epimastigotes was activated by fructose 2,6-diphosphate ((A) 0.5 = 0.17 μM), through a decrease in (S) 0.5 and an increase in Vmax for both substrates. The enzyme was 50% inhibited by 0.9 mM ATP or 0.5 mM Pi in the presence of 30 mM MgC12; these inhibitions were completely counteracted by 1.5 μM fructose 2,6-diphosphate. Both facts suggest that the effects are allosteric, and not due to chelation.

Journal ArticleDOI
TL;DR: The observed biphasic kinetics of inactivation are due to the negatively cooperative reaction of 2-BDB-T epsilon A-5'-DP with Tyr147 in the tetramer, suggesting that the reaction occurs in the region of the PEP binding site.

Journal ArticleDOI
TL;DR: Three peptides containing 6-pyridoxyllysine have been isolated from the tryptic digest of the allosteric fructose-1,6-bisphosphate-dependent pyruvate kinase from Escherichia coli, which had been almost completely inactivated with pyridoxal 5'-phosphates.
Abstract: Three peptides containing 6-pyridoxyllysine have been isolated from the tryptic digest of the allosteric fructose-1,6-bisphosphate-dependent pyruvate kinase from Escherichia coli, which had been almost completely inactivated with pyridoxal 5'-phosphate. The labelled peptides have been sequenced. The comparison of their sequences with the primary structure of the cat muscle pyruvate kinase allowed to state that peptide I fits the region spanning residues 423-438 (53% identity), peptide II corresponds to residues 442-457 (44% identity) and peptide III encompasses residues 342-368 (70% identity). These findings are discussed in connection with our previous results on the involvement of the three peptides in the catalytic and regulatory properties of the enzyme (Valentini, G., Speranza, M.L., Iadarola, P., Ferri, G. & Malcovati, M. (1988) Biol. Chem. Hoppe-Seyler 369, 1219-1226) and in connection with their location in the three-dimensional structure of the cat muscle pyruvate kinase (Muirhead, H., Clayden, D.A., Lorimer, C.G., Fothergill-Gilmore, L.A., Schiltz, E. & Schmitt, W. (1986) EMBO J. 5, 475-481).