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PubMed1990

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

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 microM. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 microM. 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.

Animals↗
PubMed1996

Physiology of pyruvate metabolism in Lactococcus lactis.

Lactococcus lactis, a homofermentative lactic acid bacterium, has been studied extensively over several decades to obtain sometimes conflicting concepts relating to the growth behaviour. In this review some of the data will be examined with respect to pyruvate metabolism. It will be demonstrated that the metabolic transformation of pyruvate can be predicted if the growth-limiting constraints are adequately established. In general lactate remains the major product under conditions in which sugar metabolism via a homolactic fermentation can satisfy the energy requirements necessary to assimilate anabolic substrates from the medium. In contrast, alternative pathways are involved when this energy supply becomes limiting or when the normal pathways can no longer maintain balanced carbon flux. Pyruvate occupies an important position within the metabolic network of L. lactis and the control of pyruvate distribution within the various pathways is subject to co-ordinated regulation by both gene expression mechanisms and allosteric modulation of enzyme activity.

Adenosine Triphosphate↗
PubMed1978

The pathway of formation of acetate and succinate from pyruvate by Bacteroides succinogenes.

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO2. Active CO2 exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP- specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.

Acetates↗
PubMed1988

Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans.

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.

Aspergillus nidulans↗
PubMed1988

Structure of the Aspergillus nidulans pyruvate kinase gene.

The complete nucleotide sequence of the Aspergillus nidulans pyruvate kinase gene, including its flanking sequences, is presented. The gene has a 1,578 bp coding sequence that encodes a protein of 526 amino acids; the latter is strongly homologous to the pyruvate kinases found in Saccharomyces cerevisiae (66%) and mammals (53%). The gene is interrupted by seven introns, three of which are in a conserved position compared to those present in the mammalian pyruvate kinase genes sequenced thus far. A fourth intron within the mononucleotide binding fold domain is in a conserved position with respect to the position of an intron within the NAD+ binding region of maize ADH I. The transcription start site has been determined; a major site of transcription was found 80 bp before the translation initiation codon. The promoter region of the A. nidulans pyruvate kinase gene contains no direct homologies with the TATA or CCAAT sequences in the expected region (30-70 bp) before the transcription initiation site. However, extended CT-enriched regions are found in the promotor region, similar to what has been observed in genes that are highly expressed in Saccharomyces cerevisiae and filamentous fungi.

Amino Acid Sequence↗
PubMed1988

Cell culture studies on patients with mitochondrial diseases: molecular defects in pyruvate dehydrogenase.

There is a group of inborn errors of metabolism that result in the condition of chronic lacticacidemia of childhood. Nearly all of the defects that can be identified occur in mitochondrial proteins, and can be demonstrated in cultured skin fibroblasts from the patients concerned. One approach to the diagnosis of these defects involves a simple incubation of the fibroblast culture with glucose-containing medium followed by the measurement of accumulated lactate and pyruvate. The total amounts of lactate and pyruvate and the ratio between them is different in cells from patients with defects in the pyruvate dehydrogenase complex or the respiratory chain. Measurement of 1-14C-pyruvate oxidation to 14CO2 can also reveal defective oxidative metabolism. Localization of the defects can be achieved using individual assays for the enzymes concerned. The clinical sequelae of the different defects is discussed.

Cells, Cultured↗
PubMed1989

Activities of pyruvate dehydrogenase, enzymes of citric acid cycle, and aminotransferases in the subcellular fractions of cerebral cortex in normal and hyperammonemic rats.

Activity levels of pyruvate dehydrogenase, enzymes of citric acid cycle, aspartate and alanine aminotransferases were estimated in mitochondria, synaptosomes and cytosol isolated from brains of normal rats and those injected with acute and subacute doses of ammonium acetate. In mitochondria isolated from animals treated with acute dose of ammonium acetate, there was an elevation in the activities of pyruvate, isocitrate and succinate dehydrogenases while the activities of malate dehydrogenase (malate----oxaloacetate), aspartate and alanine aminotransferases were suppressed. In subacute conditions a similar profile of change was noticed excepting that there was an elevation in the activity of alpha-ketoglutarate dehydrogenase in mitochondria. In the synaptosomes isolated from animals administered with acute dose of ammonium acetate, there was an increase in the activities of pyruvate, isocitrate, alpha-ketoglutarate and succinate dehydrogenases while the changes in the activities of malate dehydrogenase, aspartate and alanine amino transferases were suppressed. In the subacute toxicity similar changes were observed in this fraction except that the activity of malate dehydrogenase (oxaloacetate----malate) was enhanced. In the cytosol, pyruvate dehydrogenase and other enzymes of citric acid cycle except malate dehydrogenase were enhanced in both acute and subacute ammonia toxicity though their activities are lesser than that of mitochondria. In this fraction malate dehydrogenase (oxaloacetate----malate) was enhanced while activities of malate dehydrogenase (malate----oxaloacetate), aspartate and alanine aminotransferases were suppressed in both the conditions. Based on these results it is concluded that the decreased activities of malate dehydrogenase (malate----oxaloacetate) in mitochondria and of aspartate aminotransferase in mitochondria and cytosol may be responsible for the disruption of malate-aspartate shuttle in hyperammonemic state.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗
PubMed1988

Pyruvate dehydrogenase complex is inhibited in calcium-loaded cerebrocortical mitochondria.

An impairment of mitochondrial functions as a result of Ca-loading may be one of the significant events that lead to neuronal death after an ischemic insult. To assess the metabolic consequences of excess Ca on brain mitochondria, pyruvate oxidation was studied in isolated cerebrocortical mitochondria loaded with Ca in vitro. The flux of pyruvate dehydrogenase complex (PDHC) [( 1-14C]pyruvate decarboxylation) was inhibited as the mitochondria accumulated excess Ca under the conditions tested: the inhibition in state 3 (i.e., in the presence of added ADP) was greater than in state 4 (i.e., in the absence of added adenine nucleotides). In state 4, the inhibition of the PDHC flux was accompanied by a similar reduction of the in situ activity of PDHC, indicating a change in PDHC phosphorylation. In state 3, the inhibition of the PDHC flux was greater than the corresponding decrease of the in situ PDHC activity. Thus, mechanisms other than the phosphorylation of PDHC might also contribute to the inhibition of pyruvate oxidation. Measurement of PDHC enzymatic activity in vitro indicated that PDHC, similar to alpha-ketoglutarate dehydrogenase complex, was inhibited by millimolar levels of Ca. This observation suggests that PDHC may also be inhibited non-covalently in Ca-loaded mitochondria in a manner similar to that of alpha-ketoglutarate dehydrogenase complex.

Animals↗
PubMed1986

The activity of the pyruvate dehydrogenase complex in heart muscle in the previously obese mouse model.

Obese gold thioglucose injected mice were reduced to lean control weight by food restriction. When pair fed with lean controls these animals then gained weight (were metabolically more efficient). Serum glucose was also elevated in this group (14.5 +/- 0.4 (14) vs 12.1 +/- 0.3 mmol/L, p less than 0.001). If previously obese animals were weight maintained with lean controls (by mild food restriction), serum glucose remained at control levels. The activity of the pyruvate dehydrogenase complex in heart muscle was decreased in both obese and pair fed previously obese, whilst it was similar to that of lean controls in the weight maintained previously obese and in obese mice actually dieted. In all obese and previously obese animals serum insulin was elevated. In hearts from control animals subjected to mild food restriction the pyruvate dehydrogenase complex was activated (11.53 +/- 1.80 (5) vs 3.34 +/- 0.62 (9) U/g dry weight), despite a reduced serum insulin level (42 +/- 2 vs 74 +/- 10 microU/ml, p less than 0.01). These diverse changes in the proportion of the pyruvate dehydrogenase complex in the active form and insulin levels argue for a persistent alteration in the sensitivity of the pyruvate dehydrogenase complex to insulin in obesity, as well as indicating that glucose metabolism in obese animals is altered by both body weight and diet amount.

Animals↗
PubMed1984

Apparent identity of alpha-subunit of pyruvate dehydrogenase and the protein phosphorylated in the presence of glutamate in P2-fractions of rat cerebral cortex.

Addition of L-glutamate or several citric acid cycle intermediates stimulated the phosphorylation of a protein with apparent molecular weight of 43,000 ( P43 ) in P2-fractions from rat cerebral cortex, and this phosphorylation was inhibited by dichloroacetic acid, a specific inhibitor of pyruvate dehydrogenase kinase. Comparison of several molecular properties of phosphorylated P43 and the phosphorylated alpha-subunit of pyruvate dehydrogenase indicated that both proteins are extracted by a similar procedure and have an identical apparent molecular weight and isoelectric point. Furthermore, digestion of both phosphorylated proteins by several different proteases in the presence of SDS yielded a similar pattern of phosphorylated peptides indicating that these proteins have a considerable sequence homology. Thus, various pieces of evidence indicate that P43 and the alpha-chain of pyruvate dehydrogenase are very similar if not identical. The possible implication of a glutamate stimulated phosphorylation of pyruvate dehydrogenase for long term potentiation and epilepsy is discussed.

Adenosine Diphosphate↗
PubMed1978

Blood lactate and pyruvate levels in diabetic patients treated with biguanides with and without sulphonylureas.

Blood lactate and pyruvate were measured two to three hours after breakfast in 153 patients with maturity-onset (type 2) diabetes. Blood lactate was raised in 56% of all patients taking phenformin and in 35% of all patients taking metformin, while 75% and 35% respectively had raised pyruvate. Mean blood lactate level was raised at 2.2 mmol/1 (range 0.2-4.4 mmol/1) in the patients on phenformin and 1.7 mmol/1 (range 0.2-4.3 mmol/1) in those on metformin, with mean blood pyruvate levels of 0.122 mmol/1 (range 0.050-0.215 mmol/1) and 0.111 mmol/1 (range 0.038-0.259 mmol/1) respectively. In patients treated with a sulphonylurea drug alone mean blood lactate and pyruvate levels were not increased and were no different in those receiving sulphonylurea therapy in addition to a biguanide from those receiving a biguanide alone. Blood lactate did not correlate with any clinical or biochemical measurement in any of the treatment groups except for a correlation with serum creatinine in those patients taking metformin alone. The finding of raised blood lactate levels in diabetic patients treated with metformin suggests that this drug should be prescribed with caution.

Adolescent↗
PubMed1980

Pyruvate blocks expression of sensitivity to antimycin A and chloramphenicol.

Selectivity in Chinese hamster cells with antimycin A and chloramphenicol depends on a metabolic balance which can be modulated by varying the level of exogenous pyruvate. The effects of both inhibitors are most clearly seen in pyruvate-free nutrients. Addition of 1 mM pyruvate in plating assays shifts dose-response curves for antimycin A or chloramphenicol to higher concentration levels and reduces the differential in response between sensitive and resistant cells. In mass populations, growth inhibition by antimycin A is reduced by adding pyruvate, and growth curves for sensitive and resistant cells tend to converge. These observations show that responses to antimitochondrial drugs can be conditioned by extrinsic factors and indicate the need for further definition of selective systems.

Animals↗
PubMed1980

Studies on pyruvate carboxylase from cultured human fibroblasts and amniotic fluid cells.

The properties of pyruvate carboxylase in cultured human fibroblasts were investigated. A pH optimum around pH 7.6 was found in Tris buffer at 37 degrees C. The apparent Km for pyruvate and bicarbonate were 0.22 mmol/l and 2.1 mmol/l respectively. The activity of the crude homogenate was most stable at room temperature. The major end product was identified as citric acid during the assay conditions used. During growth the specific activity increased from 0.5 to 2 nmol/min per mg protein. The activity of pyruvate carboxylase in the crude homogenate from cultured human fibroblasts was 0.76 +/- 0.12 nmol/min per mg protein, while the activity in cultured amniotic fluid cells was 0.66 +/- 0.17 nmol/min per mg protein, suggesting the possibility of prenatal diagnosis of pyruvate carboxylase deficiency.

Amniotic Fluid↗
PubMed1996

Metabolic fuel utilization and pyruvate oxidation during the postnatal period.

The transplacental supply of nutrients is interrupted at birth, which diverts maternal metabolism to lactation. After birth, energy homeostasis is rapidly regained through milk nutrients which supply the newborn with the fatty acids and ketone bodies required for neonatal development. However, immediately after birth and before the onset of suckling there is a time lapse in which the newborn undergoes a unique kind of starvation. During this period glucose is scarce and ketone bodies are not available owing to the delay in ketogenesis. Under these circumstances, the newborn is supplied with another metabolic fuel, lactate, which is utilized as a source of energy and carbon skeletons. Neonatal rat lung, heart, liver and brain utilize lactate for energy production and lipogenesis. Lactate is also utilized by the brain of human babies with type I glycogenosis. Both rat neurons and astrocytes in primary culture actively use lactate as an oxidizable substrate and as a precursor of phospholipids and sterols. Lactate oxidation is enhanced by dichloroacetate, an inhibitor of the pyruvate dehydrogenase kinase in neurons but not in astrocytes, suggesting that the pyruvate dehydrogenase is regulated differently in each type of cell. Despite the low activity of this enzyme in newborn brain, pyruvate decarboxylation is the main fate of glucose in both neurons and astrocytes. The occurrence of a yeast-like pyruvate decarboxylase activity in neonatal brain may explain these results.

Animals↗
PubMed1982

Normal 2-aminobutyrate oxidation and increased valine oxidation in fibroblasts deficient in pyruvate dehydrogenase.

Human skin fibroblasts deficient in pyruvate dehydrogenase and five normal control strains were incubated with one of the following labelled substrates: DL-[1-14C]-2-amino-n-butyric acid, DL-[3-14C]-2-amino-n-butyric acid, L-[1-14C]leucine, L-[1-14C]valine, L-[1-14C]alanine, and [1-14C]pyruvate. The rate of 14CO2-production in the deficient cells was normal from 2-aminobutyrate and leucine, increased from valine, and decreased from alanine and pyruvate. These results indicated that in human skin fibroblasts the decarboxylation of 2-oxobutyrate is catalysed by an enzyme system different from the pyruvate dehydrogenase complex.

Aminobutyrates↗
PubMed1981

Pyruvate kinase: activation by and catalytic role of the monovalent and divalent cations.

This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K+ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact, E. coli synthesize, depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4-8 A of the divalent cation activator and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg+2 is optimal for the physiological reaction, however, Co+2, Mn+2, and Ni+2 also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.

Animals↗
PubMed1983

Anaesthesia and pyruvate dehydrogenase deficiency.

A ten-month-old infant with pyruvate dehydrogenase deficiency received anaesthesia on two occasions, once for a laparotomy and once for a tracheostomy. During both anaesthetics (different techniques) she developed an increase in arterial lactate levels and a metabolic acidosis. Pyruvate dehydrogenase deficiency results in the inability to metabolize pyruvate with resultant accumulation of pyruvate and lactate. Inhibition of gluconeogenesis, which may be produced by halothane and thiopentone, will also increase lactate levels. Other causes of increased lactate levels are hypocarbia and high carbohydrate intake. In this patient hypocarbia may have produced increased lactate levels and increased the metabolic acidosis. Recommendations include avoidance of halogenated anaesthetics, avoidance of lactate containing solutions, maintenance of normocarbia, and stress-free anaesthesia.

Acidosis↗
PubMed1997

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6.

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an alphabetagammadelta-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80 degrees C. The pH optimum was 7.6-7.8. The apparent Km values for pyruvate and coenzyme A at 70 degrees C were 3.45 mM and 54 microM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t50%) at 70 degrees C was approximately 8 h.

Amino Acid Sequence↗