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PubMed1980

Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme.

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.

Cyclic AMP↗
PubMed1981

Pyruvate dehydrogenase complex from baker's yeast. 2. Molecular structure, dissociation, and implications for the origin of mitochondria.

1. Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria. 2. The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000. Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria. 3. The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.

Biological Evolution↗
PubMed1982

Glucocorticoid hormones have a permissive role in the phosphorylation of L-type pyruvate kinase by glucagon.

The incorporation of [32P]phosphate into L-type pyruvate kinase in response to glucagon was studied in hepatocytes isolated from control and adrenalectomized rats. In control cells, pyruvate kinase phosphorylation was maximally stimulated by 210% by glucagon. Adrenalectomy reduced both the basal extent of phosphorylation and the response to glucagon to 25% of those measured in control hepatocytes. This impaired pyruvate kinase phosphorylation was associated with altered kinetic properties of L-type pyruvate kinase. Glucagon injected in vivo (1 mg/kg) into adrenalectomized rats did not increase the substrate-co-operativity of L-type pyruvate kinase. These results suggest a molecular basis for the well-recognised glucocorticoid hormone-dependence for the stimulation by glucagon of hepatic gluconeogenesis. Further effects of this defective enzyme regulation on hepatic metabolism are suggested by the observation that adrenalectomy abolished the ability of glucagon to inhibit hepatic fatty acid biosynthesis both in vivo and in isolated hepatocytes.

Adrenalectomy↗
PubMed1983

A comparison of the structure and activity of cat and trout muscle pyruvate kinases.

Pyruvate kinase was purified from cat and trout muscle. The enzymes had similar amino acid compositions and subunit molecular weights. In contrast to the mammalian enzyme, the trout muscle pyruvate kinase was activated by fructose 1,6-bisphosphate. However, unlike the L-type pyruvate kinase from mammalian liver it was not phosphorylated by cyclic-AMP-dependent protein kinase. The purified enzyme from cat muscle was carboxymethylated with iodo[2-14C]acetic acid under conditions that led to the preferential labelling of one especially reactive thiol group. The labelled enzyme was cleaved with CNBr, and the radioactive fragment purified. Amino acid sequence analysis of the reactive-thiol-containing fragment from cat muscle pyruvate kinase showed it had the following sequence: Ile-Gly-Arg-[14C]CmCys-Asn-Arg-Ala-Gly-Lys-Pro-Val-Ile-CmCys-Ala-Thr-Gln- Hse. The corresponding peptide from trout pyruvate kinase had only one difference in its amino acid composition and the sequence around the reactive thiol was identical.

Amino Acids↗
PubMed1984

Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme.

A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin-Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub-unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 +/- 5000. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub-unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl-CoA, 2-oxoglutarate or as the enzyme-avidin complex. L-Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl-CoA. No marked alterations in molecular dimensions are caused by any of these additions. L-Aspartate, but not 2-oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4 degrees C in the presence of 0.5 M KCl. Addition of L-aspartate in low concentrations enhances the effectiveness of inhibition by 2-oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2-oxoglutarate concentrations. Conversely addition of low concentrations of 2-oxoglutarate decreases the concentration of L-aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating L-aspartate concentrations. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub-units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspartic Acid↗
PubMed1988

Structure function studies on the lipoate-acetyltransferase--component-X-core assembly of the ox heart pyruvate dehydrogenase complex.

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues. Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex. Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross-linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations. The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.

Acetyltransferases↗
PubMed1988

Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

Acetyl Coenzyme A↗
PubMed1996

Alcaligenes eutrophus possesses a second pyruvate dehydrogenase (E1).

Two gene loci, which hybridized with pdhA, the structural gene of the E1 component of the Alcaligenes eutrophus pyruvate dehydrogenase complex [Hein, S. & Steinbüchel, A. (1994) J. Bacteriol. 176, 4394-4408], were identified on two nonrelated A. eutrophus chromosomal BamHI fragments by using a pdhA-specific DNA probe. These data indicated that A. eutrophus possesses, beside PdhA, two additional distinct pyruvate dehydrogenases (E1). A 6.8-kbp genomic BamHI fragment of A. eutrophus was cloned, and sequence analysis of a 3.896-kbp region revealed the structural gene pdhE (2.694 kbp) for a second pyruvate dehydrogenase (E1), which was not clustered with structural genes for other components of 2-oxo acid dehydrogenase complexes. The A. eutrophus pdhE gene product (898 amino acid residues) exhibited significant similarities to the E1 components of the pyruvate dehydrogenase complexes of A. eutrophus, Neisseria meningitidis, Escherichia coli and Azotobacter vinelandii, which are also composed of only one type of subunit. Heterologous expression of pdhE in the aceEF deletion mutant E. coli YYC202 was demonstrated by spectrometric detection of enzyme activities and by phenotypic complementation to acetate prototrophy. These complementation studies indicated that the E1 component of the A. eutrophus pyruvate dehydrogenase complex can be replaced by a functionally active pdhE gene product.

Alcaligenes↗
PubMed2006

Routes of pyruvate synthesis in phosphorus-deficient lupin roots and nodules.

Here, nodulated lupins (Lupinus angustifolius (cv Wonga)) were hydroponically grown at low phosphate (LP) or adequate phosphate (HP). Routes of pyruvate synthesis were assessed in phosphorus (P)-starved roots and nodules, because P-starvation can enhance metabolism of phosphoenolpyruvate (PEP) via the nonadenylate-requiring PEP carboxylase (PEPc) route. Since nodules and roots may not experience the same degree of P stress, it was postulated that decreases in metabolic inorganic phosphorus (Pi) of either organ, should favour more pyruvate being synthesized from PEPc-derived malate. Compared with HP roots, the LP roots had a 50% decline in Pi concentrations and 55% higher ADP : ATP ratios. However, LP nodules maintained constant Pi levels and unchanged ADP : ATP ratios, relative to HP nodules. The LP roots had greater PEP metabolism via PEPc and synthesized more pyruvate from PEPc-derived malate. In nodules, P supply did not influence PEPc activities or levels of malate-derived pyruvate. These results indicate that nodules were more efficient than roots in maintaining optimal metabolic Pi and adenylate levels during LP supply. This caused an increase in PEPc-derived pyruvate synthesis in LP roots, but not in LP nodules.

Adenosine Monophosphate↗
PubMed1981

Immunofluorescence and histochemical methods for neural M1 pyruvate kinase localization.

The distribution of pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) in the nervous system has been studied by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. The localization in various parts of rat central nervous system in situ, cerebellar and cerebral cortex, was compared to that found in vitro in cultures of cerebellum, spinal ganglia, cerebral astrocytes, and skin fibroblasts. (1) Pyruvate kinase was found predominantly in the cytoplasm of neuronal cell bodies. (2) Large neurons were better visualized than small ones. (3) No glial localization was clearly demonstrated in situ, although this does not rule out the presence of some M1 pyruvate kinase. (4) Regions expected to be rich in nerve terminals, such as the cerebellar glomeruli or the cerebellar molecular layer, showed intense staining even when the cell bodies themselves were negative. This was expected, owing to the previous demonstration of the presence of M1 pyruvate kinase in nerve ending by subcellular fractionation methods. (5) The localization was similar in situ and in tissue culture, except that nerve processes were better seen in the latter and astrocytes were sometimes stained in vitro. (6) Variation in intensity of staining was observed in similar cell types in the same section or in the same culture. This could represent different metabolic or functional or maturational states.

Animals↗
PubMed1988

Pyruvate dehydrogenase activity in osmotically shocked rat brain mitochondria: stimulation by oxaloacetate.

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.

Animals↗
PubMed2006

Clinical and instrumental evaluation of skin improvement after treatment with a new 50% pyruvic acid peel.

BACKGROUND: Pyruvic acid is an alpha-keto acid that presents keratolytic, antimicrobial, and sebostatic properties as well as the ability to stimulate new collagen production and elastic fibers formation. Because of its low pKa and its small dimension, it penetrates rapidly and deeply through the skin, so far as to be considered a potent chemical peel agent. It has proven its efficacy for the treatment of many dermatological conditions such as acne, superficial scarring, photodamage, and pigmentary disorders. Pyruvic acid application usually induces intense burning, and the postpeeling period is characterized by erythema, desquamation, and, sometimes, crusting. OBJECTIVE: The aim of the study is to assess the efficacy and tolerability of 50% pyruvic acid in a new non-erythematogenic formulation (pyruvic acid 50%, dimethyl isosorbide, propylene glycol, ethyl alcohol, dimethyl sulfone, ethyl lactate, water) for the treatment of photodamage, superficial scarring, and melasma. MATERIALS AND METHODS: Twenty subjects affected by photodamage, superficial scarring, and melasma, but otherwise healthy, entered the study. Four peeling sessions were performed once every 2 weeks. The patients were evaluated clinically and by means of several noninvasive methods in order to monitor the following parameters: hydration, color (erythema and pigmentation), elasticity, skin smoothness, skin roughness, scaliness, and wrinkles. RESULTS: The patients did not report any discomfort either during the peeling session or during the postpeeling period, without any impact on their social life. We did not observe any case of persistent erythema as well as any case of postinflammatory hyperpigmentation. Instrumental evaluations showed a significant reduction in the degree of pigmentation in patients with melasma, a significant increase in skin elasticity, and an improvement of the degree of wrinkling in all the patients. CONCLUSION: This innovative formulation of 50% pyruvic acid peel has been shown to be safe and effective to treat photodamage, melasma, and superficial scarring, allowing the patients to carry out regularly their working life as well as their social life. Furthermore, the results have been evaluated by means of noninvasive devices, which have permitted one to quantify the improvements.

Chemexfoliation↗
PubMed2005

Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle.

Butyrates↗
PubMed1981

Brain pyruvate dehydrogenase: phosphorylation and enzyme activity altered by a training experience.

The active portion of the alpha subunit of pyruvate dehydrogenase in rat frontal cortex was elevated after a training experience. No change in total pyruvate dehydrogenase activity was observed. The phosphorylation in vitro of pyruvate dehydrogenase (band F-2) was also elevated after training. Since activation of pyruvate dehydrogenase requires its dephosphorylation, the following sequence is proposed. Training alters frontal cortex and reduces the phosphate content of pyruvate dehydrogenase in vivo; this leads to enzyme activation; and an increase in back-titration of sites available for phosphorylation in vitro.

Animals↗
PubMed1977

Pyruvate production and excretion by the luminous marine bacteria.

During aerobic growth on glucose, several species of luminous marine bacteria exhibited an imcomplete oxidative catabolism of substrate. Pyruvate, one of the products of glucose metabolism, was excreted into the medium during exponential growth and accounted for up to 50% of the substrate carbon metabolized. When glucose was depleted from the medium, the excreted pyruvate was promptly utilized, demonstrating that the cells are capable of pyruvate catabolism. Pyruvate excretion is not a general phenomenon of carbohydrate metabolism since it does not occur during the utilization of glycerol or maltose. When cells pregrown on glycerol were exposed to glucose, they began to excrete pyruvate, even if protein synthesis was blocked with chloramphenicol. Glucose thus appears to have an effect on the activity of preexisting catabolic enzymes.

Aerobiosis↗
PubMed1990

Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains.

The localization of pyruvate carboxylase (cytosolic or mitochondrial) was studied in nine different Aspergillus species (14 strains). In some species (A. aculeatus, A. flavus, A. foetidus, A. nidulans, A. ochraceus, and A. sojae), the pyruvate carboxylase activity could be detected only in the cytosolic fraction of the cells. Pyruvate carboxylase has been found only in the mitochondrial fraction of two strains of Aspergillus wentii. In Aspergillus oryzae and in five strains of Aspergillus niger, pyruvate carboxylase activity was detected both in the mitochondrial fraction and in the cytosol. There was no quantitative or qualitative correlation between the activities of pyruvate carboxylase in the mitochondrial and cytosolic fractions of the cells and the ability of the various Aspergillus strains to accumulate different organic acids.

Aspergillus↗
PubMed1994

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.

Amino Acid Sequence↗
PubMed1971

Equilibrium constant for conversion of pyruvate to acetyl phosphate and formate.

Preparations of pyruvate formate-lyase were made from Escherichia coli cells. Net reversal of the "phosphoroclastic split" of pyruvate was readily demonstrated with these preparations. Incubation of acetyl phosphate with formate resulted in the accumulation of pyruvate in concentrations up to 0.5 mm. Catalytic amounts of coenzyme A were essential. Pyruvate was also readily formed from acetyl coenzyme A and formate. The equilibrium constant of the reaction (pyruvate(-) + HPO(4) (2-) --> acetyl phosphate(2-) + formate(-)) has been determined to be about 23 at 37 C.

Acylation↗