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PubMed1998

Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1beta-, E1alpha-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg2+. Michaelis constants for the substrates were 47, 3, 77, and 1 &mgr;m for pyruvate, coenzyme A (CoA), NAD+, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD+ and CoA, and the inhibition constants were 15 and 47 &mgr;m, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg2+.

Journal Article↗

PubMed1967

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips.

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-(14)C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a (14)C-product as IAA was by (a) co-chromatography of authentic IAA and (14)C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of alpha-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall congruent with etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.

Journal Article↗

PubMed1969

beta-Alanine as an Ethylene Precursor. Investigations Towards Preparation, and Properties, of a Soluble Enzyme System From a Subcellular Particulate Fraction of Bean Cotyledons.

A method is described for the preparation, from a subcellular particulate fraction of wax bean cotyledons, of a soluble enzyme system that is capable of converting beta-alanine to ethylene. In the presence of ATP, CoA, thiamine pyrophosphate, MgSO(4), and pyridoxal phosphate, ethylene production is maximum at a 0.5 mm concentration of beta-alanine. The system exhibits a pH optimum at 7.0 but when the pH is raised above 8, evolution of the volatile again increases and continues to do so up to pH 12. The enzyme system is stimulated by either NADPH or NADH; the concentration of NADPH necessary to obtain maximum activity is twice that of NADH. The requirement for a reducing agent is in agreement with the proposal that malonate semialdehyde, formed by an aminotransferase reaction from beta-alanine, is reduced to beta-hydroxypropionate. Both malonate semialdehyde and beta-hydroxypropionate are better stimulators of production of the volatile in the soluble system than is beta-alanine, and beta-hydroxypropionate is a better stimulator than malonate semialdehyde. This system is also able to incorporate tritium from tritiated water into ethylene; this supports the proposal that ethylene is formed by the decarboxylation of acrylate, the latter being formed from beta-hydroxypropionate.Experiments with both cold and labeled malonate suggest that this compound stimulates ethylene production by acting as an end product inhibitor that prevents the loss of malonate semialdehyde from the pathway. Malonate does not appear to serve as a precursor.Addition of cytoplasmic enzymes to the ;soluble system' (prepared from particulate enzymes) results in a considerable boost in ethylene production, but the specific activity (mmu1 / mg protein) is lowered from that of the particulate enzymes alone.

Journal Article↗

PubMed1979

Helminthosporium maydis Race T Toxin Induces Leakage of NAD from T Cytoplasm Corn Mitochondria.

The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD(+)-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD(+). Added NAD(+) partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD(+)-linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD(+) and other coenzymes.

Journal Article↗

PubMed1983

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber.

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O(2) consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD(+) and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD(+) ratio was high, alpha-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD(+) ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and alpha-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD(+) ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD(+)-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.

Journal Article↗

PubMed1987

Some kinetic and regulatory properties of the pea mitochondrial pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea (Pisum sativum L., cv Little Marvel) leaf mitochondria. The pH optimum for the overall reaction was 7.6. The divalent cation requirement was best satisfied by Mg(2+). Reaction velocity was maximal at 40 degrees C. Pyruvate was a better substrate than 2-oxo-butyrate; other 2-oxo-acids were not substrates. Michaelis constants for substrates were; pyruvate, 57 micromolar; NAD, 122 micromolar; Coenzyme-A, 5 micromolar; Mg(2+), 0.36 millimolar; Mg-thiamine pyrophosphate, 80 nanomolar. The products, NADH and acetyl-Coenzyme-A, were linear competitive inhibitors with respect to NAD and Coenzyme A. Inhibition constants were 18 and 10 micromolar, respectively. Glyoxylate inhibited complex activity only in the absence of thiol reagents. Glyoxylate inhibition was competitive with respect to pyruvate with an inhibition constant of 51 micromolar. Among mitochondrial metabolites examined as potential effectors, only ADP with an inhibition constant of 0.57 millimolar could be of physiological significance.

Journal Article↗

PubMed1988

Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints.

The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.

Journal Article↗

PubMed1988

Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium.

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC.

Journal Article↗

PubMed1989

Regulation of pea mitochondrial pyruvate dehydrogenase complex : does photorespiratory ammonium influence mitochondrial carbon metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na(+). NH(4) (+), at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K(+) in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na(+) was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH(4) (+) is discussed in relation to a possible regulatory interaction between photorespiratory NH(4) (+) production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.

Journal Article↗

PubMed1990

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide.

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.

Journal Article↗

PubMed1985

A distinct variant of intermediate maple syrup urine disease.

Branched chain alpha-ketoacid dehydrogenase (BCKAD) deficiency, or maple syrup urine disease (MSUD), can be categorized as classical, intermediate, intermittent or thiamine responsive, based on generally concordant in vitro BCKAD activity and severity of phenotype. We present clinical and enzymatic data on a boy with intermediate maple syrup urine disease, and suggest that he represents a novel category of mutation. He presented at age 10 months in ketoacidotic coma, with a history of irritability, poor feeding and growth and developmental delay. Branched chain amino acid restriction effected normal growth and developmental parameters by age 42 months. In contrast to previous patients with intermediate MSUD, his fibroblasts and fibroblast extracts failed to decarboxylate [1-14C]-alpha-ketoisovalerate (KIV). The defect is not in mitochondrial transport of substrate, but rather in the catalytic activity of the E1 component of the BCKAD. Disrupted cells of the proband exhibited negligible BCKAD activity over a wide range of keto acid substrate concentrations, irrespective of the presence of added thiamine pyrophosphate (TPP). These results differ from the sigmoidal kinetics observed using classical MSUD extracts, and the hyperbolic kinetics with control preparations under the same assay conditions. We propose that the structurally altered enzyme possesses reduced but not negligible activity in vivo, and exists as an unstable complex in vitro under assay conditions used, even in the presence of added TPP.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1975

The pyruvate-dehydrogenase complex from Azotobacter vinelandii.

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.

Acetyltransferases↗

PubMed2007

Blood vitamin concentrations in privately owned dogs fed non-standardized commercial diets and after intake of diets with specified vitamin concentrations.

The objective was to investigate in a survey study the blood vitamin concentrations in healthy dogs fed non-specified commercial complete diets and in an intervention study to determine the effects of defined dietary vitamin intakes on blood vitamin levels and hair and skin condition. Sixty-four privately owned dogs, aged from 1 to 8 years, without history of skin or coat problems were included. All animals were fed commercial complete diets with uncertain vitamin concentrations before enrolment. The animals were assigned, according to weight and gender, to four groups with graded vitamin intakes. The blood vitamin levels and skin and coat quality of the dogs were investigated at days 0 and day 122. Coat and hair condition was not influenced by the experimental diets. The retinol concentrations were reduced at the end of the experiment compared with the baseline levels, retinyl esters were not influenced. 25-Hydroxycholecalciferol decreased in all groups, alpha-tocopherol was constant or tended to decrease. Ascorbic acid, thiamine pyrophosphate and riboflavin concentrations were not affected by treatment, flavin adenine dinucleotide and pyridoxal-5'-phosphate were partially reduced on day 122. Cobalamin, pantothenate and biotin concentrations increased with higher dietary intakes, folate levels in tendency. In conclusion, this study gives a survey of blood vitamin concentrations in healthy dogs and provides a data base for the evaluation of the vitamin status in health and disease.

Animal Feed↗

PubMed1980

Thiamin deficiency in the lamb: changes in thiamin phosphate esters in the brain.

Concentration of thiamin (unphosphorylated), thiamin monophosphate (TMP), thiamin diphosphate (TDP), and thiamin triphosphate (TTP) were measured in three regions of the brain of seven pairs of lambs. The lambs were maintained on a thiamin-free synthetic diet for 2, 3, or 4 weeks. Controls were pair-fed and supplemented with thiamin. The three brain regions were: (1) dorso-lateral aspect of the cortex [common site for lesions of polioencephalomalacia (PEM)]; (2) pyriform lobe of the cortex (no PEM lesions are found here); (3) white matter of the internal capsule (no PEM lesions found here). The concentration of TTP ina ll three sections of brain was maintained at control values for up to 4 weeks on the thiamin-deficient diet. TDP concentration decreased to 22% of control values in both regions of grey matter after 4 weeks on the diet. Unphosphorylated thiamin and TMP decreased to a smaller extent than TDP.

Animals↗

PubMed1982

Studies on the pyruvate dehydrogenase complex in brain with the arylamine acetyltransferase-coupled assay.

A spectrophotometric assay for the brain pyruvate dehydrogenase complex (PDHC) with arylamine acetyltransferase (ArAT; EC 2.3.1.5) to follow the production of acetyl-CoA has been standardized. Activity was proportional to time and protein. It depended completely on added pyruvate, CoA, NAD, and MgCl2, and partially on thiamine pyrophosphate. Triton X-100, and a sulfhydryl compound. The activities are the highest in the literature for brain PDHC (50 nmol/min/mg protein) and equal to maximum recorded rates of pyruvate flux for brain in vivo. Activities as low as 0.6 nmol/min could be measured. Use of ArAT at different purities (I--2-fold and II--55-fold) allowed convenient measurement of total PDHC (ArAT-I) and of the active form of PDHC (ArAT-II). The proportion of PDHC in the active form was 50% in mouse brain, 30% in brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4 degrees C or at -20 degrees C for 3 days or at +20 degrees C for 24 h. The relative specific activity of PDHC in cytoplasmic or synaptoplasmic fractions was less than that of two other mitochondrial enzymes, fumarase (EC 4.2.1.2) and monoamine oxidase (EC 1.4.3.4), which argues strongly against the hypothesis of a cytoplasmic PDHC in cholinergic nerve endings.

Acetyltransferases↗

PubMed1984

Phosphate derivatives of thiamine and Na+ channel in conducting membranes.

The results show that thiamine derivatives are copurified with the specific proteins forming the Na+ channel in conducting membranes. Therefore, thiamine derivatives could well play a specific role in the molecular aspects of bioelectrogenesis , an interpretation that could help explain the neurological symptoms observed in human pathology as well as in animals experimentally rendered deficient in vitamin B1.

Animals↗

PubMed1984

Properties and regional distribution of pyruvate dehydrogenase kinase in rat brain.

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.

Adenosine Triphosphate↗

PubMed1986

Brain alpha-ketoglutarate dehydrogenase complex: kinetic properties, regional distribution, and effects of inhibitors.

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.

Ammonia↗