Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PYRUVATES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed1983

Normal activities of hepatic pyruvate dehydrogenase and pyruvate carboxylase in Leigh's syndrome.

A case of Leigh's syndrome (subacute necrotizing encephalomyelopathy, SNE), proven by autopsy, was reported. The persistent elevation of pyruvate and lactate in blood and hyperalanemia suggested an impairment of pyruvate oxidation, but the enzyme activities of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) in liver tissues of the patient revealed normal. It is postulated that Leigh's syndrome and both enzyme deficiencies are distinct entities.

Brain↗
PubMed1975

Membrane-localized pyruvate kinase of red blood cells in hemolytic anemia associated with pyruvate kinase deficiency.

Pyruvate kinase activity of red blood cell membranes, which is normally masked, has been determined after mechanical disruption of the membranes in normal individuals and in three homozygous patients with pyruvate kinase deficiency. Although patients 1 and 2, who were siblings, had relatively high enzyme activities in their hemolysates, they had the severest form of the disorder. The activities of their membrane fragments were decreased to seven per cent of fragments of normal membranes. Patient 3 had a mild form of hemolytic anemia despite a low enzyme activity of his hemolysates. The membrane fragments of this patient contained 28 per cent of the pyruvate kinase activity of normal fragments. The data suggest a relationship between the amount of membrane-localized pyruvate kinase and the severity of the clinical disorder. The reduced production of ATP by the enzyme portion localized within the membrane may cause an impairment of membrane functions in pyruvate kinase deficiency.

Aged↗
PubMed1983

Purification and properties of pyruvate kinase from Streptococcus sanguis and activator specificity of pyruvate kinase from oral streptococci.

It was found that pyruvate kinases with two different regulatory characteristics were distributed among oral streptococci. The pyruvate kinases of Streptococcus mutans, Streptococcus salivarius, and Streptococcus bovis were activated by glucose 6-phosphate, whereas the enzymes of both Streptococcus sanguis and Streptococcus mitis were activated by fructose 1,6-bisphosphate. Pyruvate kinase (EC 2.7.1.40) from S. sanguis NCTC 10904 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 250,000 to 260,000 and consisted of four identical subunits. Whereas the pyruvate kinase from S. mutans was completely dependent on glucose 6-phosphate (K. Abbe and T. Yamada, J. Bacteriol. 149:299-305, 1982), the enzyme from S. sanguis was activated by fructose 1,6-bisphosphate. In the presence of 0.5 mM fructose 1,6-bisphosphate, the saturation curves for the substrates, phosphoenolpyruvate and ADP, were hyperbolic, and the Km values were 0.13 and 0.30 mM, respectively. Without fructose 1,6-bisphosphate, however, saturation curves for both substrates were sigmoidal. GDP, IDP, and UDP could replace ADP. Like the enzyme from S. mutans, the enzyme from S. sanguis required a divalent cation, Mg2+ or Mn2+, and a monovalent cation, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for fructose 1,6-bisphosphate increased. The remarkable fluctuation of intracellular levels of fructose 1,6-bisphosphate and phosphoenolpyruvate observed in the cells growing under glucose limitation and nitrogen limitation implies that the intracellular concentration of fructose 1,6-bisphosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. sanguis in vivo.

Cations↗
PubMed1978

Homogeneous pyruvate kinase isolated from yeast by two different methods is indistinguishable from pyruvate kinase in cell-free extract.

In this report, we have compared homogeneous yeast (Saccharomyces cerevisiae) pyruvate kinase to enzyme from cell-free extracts in several different ways: 1) isoelectric focusing of cell-free extracts indicates one peak of pyruvate kinase activity whose isoelectric point is the same as that of the pure enzyme; 2) antibody prepared to the pure enzyme produces a single, fused precipitin line against enzyme in the cell-free extract and pure enzyme; 3) immunoelectrophoresis of cell-free extract produces one precipitin arc which has the same mobility as that of the pure enzyme; and 4) immunoprecipitation of the pure enzyme from cell-free extract with subsequent solubilization in 1% sodium dodecyl sulfate and electrophoresis on sodium dodecyl sulfate-polyacrylamide gels produces a single protein band attributable to pyruvate kinase which co-migrates with the purified enzyme. Within the limits of the sensitivity of the methods employed, we conclude that the homogeneous pyruvate kinase prepared from yeast lysed either by Manton-Gaulin homogenization (Aust, A., Yun, S.-L., and Suelter, C. (1975) Methods Enzymol. 42, 176-182) or by toluolysis (Yun, S.-L., Aust, A.E., and Suelter, C.H. (1977) J. Biol. Chem. 251, 124-128) is identical with pyruvate kinase in cell-free extract.

Immunodiffusion↗
PubMed1983

Molecular cloning of DNA complementary to rat L-type pyruvate kinase mRNA. Nutritional and hormonal regulation of L-type pyruvate kinase mRNA concentration.

Rat liver L-type pyruvate kinase mRNA was enriched from total polysomes by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to L-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-selected translation, and dot blot hybridization. A partial restriction endonuclease map of cDNA inserts was constructed covering about 1.86 kilobase pairs. The cDNA insert of recombinant plasmid pLPK-14 was used as a hybridization probe to quantitate L-type pyruvate kinase mRNA in rat liver after various treatments. The level of hybridizable L-type enzyme mRNA was markedly increased by a high carbohydrate diet. Diabetes greatly reduced the mRNA level in the liver of rats maintained on a high carbohydrate diet, but insulin administration resulted in restoration of the mRNA level to normal within 24 h. These changes were approximately proportional to the changes in the level of translatable L-type pyruvate kinase mRNA. Thus, we conclude that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.

Animals↗
PubMed1982

Effect of pyruvate decarboxylase activity and of pyruvate concentration on the production of 1-hydroxy-1-phenylpropanone in Saccharomyces carlsbergensis.

The assumption that the pyruvate decarboxylase activity of Saccharomyces carlsbergensis is the main limiting factor determining the formation rate and the total amount of d(--)-l-hydroxy-l-phenyl-propanone (phenylacetylcarbinol, PAC) produced was not confirmed. An increase of about 30% of the total amount of the PAC produced was obtained when 8.5% sodium pyruvate was gradually added. The total PAC production is probably influenced both by the pyruvate decarboxylase activity and the pyruvate concentration in the cells, the latter being actually the determining rate-limiting factor.

Acetone↗
PubMedDate not supplied

Enzyme electrode composed of the pyruvate oxidase from Pediococcus species coupled to an oxygen electrode for measurements of pyruvate in biological media.

Pyruvate oxidase from Pediococcus species was immobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto the tip of an oxygen electrode. The enzyme electrode was specific for pyruvate measurements. This electrode was sensitive to 0.1 mM and could be used up to a final pyruvate concentration of 2 mM. At each step of the enzymatic film preparation and assay 0.7 mM thiamine pyrophosphate, 10 microM flavin adenine dinucleotide, 5 mM Mg2+ and 10 mM phosphate buffer were necessary. A computerized probe allowed successive measurements every 3 min for more than 20 h with the same enzymatic film. The reproducibility for the same pyruvate concentration was 2% during 400 assays without special optimization. This enzyme electrode has many applications in basic (metabolism, enzymology) and applied (blood, yoghurt) research. Results obtained from assays carried out in yoghurt are presented.

Electrodes↗
PubMed1991

DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains.

A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

Amino Acid Sequence↗
PubMed2003

Characterization of the isozymes of pyruvate dehydrogenase phosphatase: implications for the regulation of pyruvate dehydrogenase activity.

The activity of mammalian pyruvate dehydrogenase complex (PDC) is regulated by a phosphorylation/dephosphorylation cycle. Dephosphorylation accompanied by activation is carried out by two genetically different isozymes of pyruvate dehydrogenase phosphatase, PDP1c and PDP2c. Here, we report data showing that PDP1c and PDP2c display marked biochemical differences. The activity of PDP1c strongly depends upon the simultaneous presence of calcium ions and the E2 component of PDC. In contrast, the activity of PDP2c displays little, if any, dependence upon either calcium ions or E2. Furthermore, PDP2c does not appreciably bind to PDC under the conditions when PDP1c exists predominantly in the PDC-bound state. The stimulatory effect of E2 on PDP1c can be partially mimicked by a monomeric construct consisting of the inner lipoyl-bearing domain and the E1-binding domain of E2 component. This strongly suggests that the E2-mediated activation of PDP1c largely reflects the effects of co-localization and mutual orientation of PDP1c and E1 component facilitated by their binding to E2. Both PDP1c and PDP2c can efficiently dephosphorylate all three phosphorylation sites located on the alpha chain of the E1 component. For PDC phosphorylated at a single site, the relative rates of dephosphorylation of individual sites are: 2>site 3>site 1. Phosphorylation of sites 2 or 3 in addition to site 1 does not have a significant effect on the rates of dephosphorylation of individual sites by PDP1c, suggesting a random mechanism of dephosphorylation. In contrast, there is a significant decrease in the overall rate of dephosphorylation of pyruvate dehydrogenase by PDP2c under these conditions. This indicates that the mechanism of dephosphorylation of PDC phosphorylated at multiple sites by PDP2c is not purely random. These marked differences in the site-specificity displayed by PDP1c and PDP2c should be particularly important under conditions such as starvation and diabetes, which are associated with a great increase in phosphorylation of sites 2 and 3 of pyruvate dehydrogenase.

Acetylation↗
PubMed1998

Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth.

Neisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two-hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteria-host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified five different OIPs, designated OIP1-OIP5, two of which are homologous to human proteins--thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two-hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.

Amino Acid Sequence↗
PubMed2000

Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis.

The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains. The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.

Amino Acid Sequence↗
PubMed2001

Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase.

Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.

Acetylation↗
PubMed1998

Effects of pyruvate decarboxylase overproduction on flux distribution at the pyruvate branch point in Saccharomyces cerevisiae.

A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN. PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of the PDC1 plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h-1, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h-1 in the reference strains to 0.23 h-1 in the Pdc-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain. Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h-1), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.

Aerobiosis↗
PubMed2006

Oxidative damage to lens in culture: reversibility by pyruvate and ethyl pyruvate.

It is generally believed that prophylactic intake of antioxidants is beneficial in delaying the onset of some aging manifestations such as cataract. However, whether such a supplementation will also be effective if the pathophysiological process has already set in remains a largely open question. We examined this possibility with lens changes leading to cataract formation, since cataract genesis is intimately related to a continued generation of reactive oxygen species (ROS) in the aqueous humor. Since the formation of cataract is a well-defined progressive disease, starting with an early refractive change and leading to gradual enhancement of opacification, we hypothesized that even a belated start with an appropriate anti-oxidant could halt the pathological process and delay cataract maturation and vision impairment. Using lens cultures, we tested this hypothesis with pyruvate, known to be an effective and highly potent ROS scavenger. Adding pyruvate to the culture medium after lenses had sustained a 50% damage was significantly effective in preventing progress. This was apparent by better maintenance of the active rubidium transport activity in these lenses compared to controls without pyruvate treatment. Glutathione levels were also higher in the pyruvate group.

Animals↗
PubMed1986

Deficiency of the pyruvate dehydrogenase component in pyruvate dehydrogenase complex-deficient human fibroblasts. Immunological identification.

A previously reported deficiency of "total" pyruvate dehydrogenase complex activity is further characterized. Dihydrolipoyl transacetylase (E2) and lipoamide dehydrogenase (E3) activities in the patient's fibroblasts were normal. Pyruvate dehydrogenase activity (E1) was 33% of that in fibroblasts from an age-matched control. The amounts of each of the components of pyruvate dehydrogenase complex were analyzed using an immunoblot technique and specific antibodies. Levels of components E2 and E3 were the same in fibroblasts from the patient and control, confirming the activity measurements. However, the levels of E1 alpha and E1 beta were reduced markedly in fibroblasts from the patient. Thus, impairment in the pyruvate dehydrogenase complex activity was due to a reduction in the amount of the E1 component of the complex.

Acetyltransferases↗
PubMed2003

In vivo pyruvate detected by MR spectroscopy in neonatal pyruvate dehydrogenase deficiency.

We present a unique finding of an elevated level of pyruvate at 2.37 ppm revealed by in vivo MR spectroscopy of a female neonate. Low fibroblast pyruvate dehydrogenase (PDH) complex activity subsequently confirmed a diagnosis of PDH deficiency. Abnormalities of brain development consistent with PDH deficiency were also evident on fetal and postnatal MR images. To our knowledge, this is the first report of pyruvate being shown in vivo in a child and the first report of MR spectroscopy aiding in the diagnosis of inborn error in pyruvate metabolism before confirmation by conventional enzymatic testing. This finding has potential implications for earlier diagnosis in patients with defects in mitochondrial metabolism.

Abnormalities, Multiple↗
PubMedDate not supplied

[The effect of phosphorylation on oxidative and CoA- and NAD+-independent turnover of pyruvate by pyruvate dehydrogenase complex in the brain].

It was shown that in the presence of ATP and Mg2+ the phosphorylation of the partially purified pyruvate dehydrogenase complex and the enzyme in isolated brain mitochondria inhibited the oxidative activity of the pyruvate dehydrogenase complex. The phosphorylation did no affect essentially the nonoxidative decarboxylation of pyruvate to form CO2 and acetaldehyde. In native mitochondria from the bovine brain the nonoxidative activity of the pyruvate dehydrogenase complex reached about 10% as compared to the oxidative activity of enzyme.

Animals↗
PubMed1996

Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex.

The most common defect associated with deficiency of the pyruvate dehydrogenase (PDH) complex occurs in the E1 component, specifically due to mutations in the X-linked E1 alpha gene. Clinical sequelae of these mutations, which range from severe neonatal lactic acidosis to carbohydrate-sensitive ataxia, can be different in males and females depending on the nature of the mutation and, in the case of females, on the X-inactivation pattern in different tissues. Males have a high representation of missense mutations among the patient cohort, while females are much more likely to have DNA rearrangements, particularly toward the 3' end of the coding sequence of the gene. Missplicing mutations involving exon 6 deletion have been reported, as has a missense mutation conferring true thiamin-responsiveness of the enzyme and the patient's clinical symptoms. Pyruvate carboxylase deficiency, on the other hand, is a true autosomal recessive disease, though it has high occurrences in particular ethnic groups, especially in Algonkian-speaking Amerindians and in Arabs. In the former group the defect is a simple type in which material cross-reactive to pyruvate carboxylase antibody is present in cultured cells (CRM+ve). In the latter group, cross-reacting material is rarely present (CRM-ve). The CRM+ve patients can survive into teenage years with careful supervision, while the CRM-ve patients have complications due to hyperammonaemia and dysfunction of the urea cycle and rarely survive beyond 3 months of life.

Amino Acid Sequence↗