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PubMed1984

Lactic acidaemia.

Congenital childhood lactic acidaemia is a poorly understood group of genetic diseases. The most common underlying inherited defect encountered in this group is deficiency of the pyruvate dehydrogenase complex. Of 23 cases we have diagnosed, 18 have a deficiency in the first component of the complex, the E1 decarboxylase, while the other five have multiple alpha-keto acid dehydrogenase deficiency due to a defect in lipoamide dehydrogenase. In addition to the lactic acidosis associated with pyruvate decarboxylase deficiency, ten of the cases showed evidence of facial dysmorphism consisting of a narrow head, wide nasal bridge and flared nostrils or gross microcephaly. Two further patients had agenesis of the corpus callosum. Isolated pyruvate carboxylase deficiency was found to present in two different forms, one with lactic acidaemia and mental retardation, the other with lactic acidaemia, hyperammonaemia citrullinaemia and hyperlysinaemia. The former presentation we have shown to be associated with the presence of a biotinylated pyruvate carboxylase protein of the correct subunit molecular weight (125 kd) which has no catalytic activity (CRM + ve). The latter we have shown to be associated with the absence of any recognizable pyruvate carboxylase protein (CRM - ve).

Abnormalities, Multiple↗
PubMed2000

Targeted upregulation of pyruvate dehydrogenase kinase (PDK)-4 in slow-twitch skeletal muscle underlies the stable modification of the regulatory characteristics of PDK induced by high-fat feeding.

In using Western blot analysis with antibodies raised against recombinant pyruvate dehydrogenase kinase (PDK) isoforms PDK2 and PDK4, this study demonstrates selective PDK isoform switching in specific skeletal muscle types in response to high-fat feeding that is associated with altered regulation of PDK activity by pyruvate. The administration of a diet high in saturated fats led to stable (approximately 2-fold) increases in PDK activities in both a typical slow-twitch (soleus [SOL]) muscle and a typical fast-twitch (anterior tibialis [AT]) muscle. Western blot analysis revealed that high-fat feeding significantly increased (approximately 2-fold; P < 0.001) PDK4 protein expression in SOL, with a modest (1.3-fold) increase in PDK2 protein expression. The relative increase in PDK4 protein expression in SOL was associated with a 7.6-fold increase in the pyruvate concentration that was required to elicit a 50% active pyruvate dehydrogenase complex, which indicates a marked decrease in the sensitivity of PDK to inhibition by pyruvate. In AT muscle, high-fat feeding elicited comparable (1.5- to 1.7-fold) increases (P < 0.05) in PDK4 and PDK2 protein expression. Loss of sensitivity of PDK to inhibition by pyruvate was less marked. The data suggest that a positive correlation exists between increases in PDK4 expression and the propensity with which muscles use lipid-derived fuels as respiratory substrates rather than with the degree of insulin resistance induced in skeletal muscles by high-fat feeding. In conclusion, high-fat feeding leads to selective upregulation of PDK4 expression in slow-twitch muscle in response to high-fat feeding in vivo, which is associated with a pronounced loss of sensitivity of PDK activity to acute inhibition by pyruvate. Thus, increased PDK4 expression may underlie the stable modification of the regulatory characteristics of PDK observed in slow-twitch muscle in response to high-fat feeding.

Animals↗
PubMed1996

Different extent of inhibition of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase both containing endogenous thiamine pyrophosphate, by some anticoenzyme analogues.

Inhibitory effects of 4'-oxythiamine pyrophosphate (OTPP) and tetrahydrothiamine pyrophosphate (ThTPP) on the purified bison heart pyruvate dehydrogenase complex (PDC) semisaturated with endogenous thiamine pyrophosphate (TPP) and 2-oxoglutarate dehydrogenase complex (OGDC) saturated about 85% with endogenous TPP, were studied. It has been established that the thiamine derivatives strongly inhibit not only the PDC apoenzyme moiety, but also the PDC holoenzyme moiety. The apparent I50 values for the holoenzyme were 0.006 microM and 0.046 microM for OTPP and ThTPP, respectively. The inhibition of the PDC is reversible. After removal of the anticoenzyme analogues by gel filtration the endogenous TPP within the PDC is retained as it is evidenced by complete recovery of the enzyme activity without added TPP. In contrast with the PDC, OGDC holoenzyme form is weakly inhibited by the anticoenzyme analogues.

Animals↗
PubMed1975

A kinetic study of dihydrolipoyl transacetylase from bovine kidney.

The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase are joined. This report describes studies on the kinetic mechanism of the transacetylase-catalyzed reaction between [1-14C]acetyl-CoA and dihydrolipoamide. This reaction appears to be a model of the physiological reaction, in which the acetyl group is transferred from the S-acetyldihydrolipoyl moiety, bound covalently to the transacetylase, to CoA. The model reaction is not affected by pyruvate dehydrogenase or dihydrolipoyl dehydrogenase, their substrates and products, or by removal of the covalently bound lipoyl moiety. These findings, together with the results of initial velocity, product inhibition, and dead-end inhibition studies, indicate that the model reaction and, apparently, the physiological reaction as well, proceeds via the Random Bi Bi (rapid equilibrium) mechanism. It appears that at the catalytic center of the transacetylase there are two adjacent sites, one that binds CoA and acetyl-CoA and another that binds dihydrolipoamide and S-acetyldihydrolipoamide (or the corresponding forms of the covalently bound lipoyl moiety.

Acetates↗
PubMed1998

Regulation of mammalian pyruvate dehydrogenase alpha subunit gene expression by glucose in HepG2 cells.

We report the effect of glucose on the expression of the gene encoding the pyruvate dehydrogenase (E1) alpha subunit (E1alpha) in human hepatoma (HepG2) cells. Total pyruvate dehydrogenase complex activity as well as the levels of protein and mRNA of the E1alpha subunit were significantly increased in HepG2 cells cultured in medium containing 16.7 mM glucose compared with 1.0 mM glucose for a period of 4 weeks. The level of E1alpha mRNA was elevated approx. 2-fold in HepG2 cells cultured for 24 h in medium containing 16.7 mM glucose compared with 1 mM glucose. This effect was specific to glucose and independent of insulin. Nuclear run-on assays and promoter analysis indicate that the glucose-induced increases in the levels of E1alpha mRNA in HepG2 cells are due to increased transcription of the human E1alpha (PDHA1) gene. Mutational analysis of the E1alpha promoter region has identified two regions, from -78 to -73 bp (CCCCTG) and from -8 to -3 bp (GCGGTG), that are responsible for the effect of glucose on promoter activity; the former exhibits a larger effect. These two sequences represent new variations of the carbohydrate-response element that has been identified in other genes. The stimulation of E1alpha promoter activity by glucose was abolished by okadaic acid at 100 nM but not at 5 nM, suggesting that glucose-mediated regulation of pyruvate dehydrogenase complex E1alpha gene transcription involves a phosphorylation/dephosphorylation mechanism, possibly involving protein phosphatase-1.

Chloramphenicol O-Acetyltransferase↗
PubMed1982

Kinetic studies of mouse brain transketolase.

The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent Km for ribose-5-phosphate was 330 microM, for D-xylulose-5-phosphate was 120 microM, and for thiamine pyrophosphate was 7 microM. However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate-dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.

Animals↗
PubMed1995

A new family of protein kinases--the mitochondrial protein kinases.

Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells. Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest. Dietary protein deficiency increases the activity of branched-chain alpha-ketoacid dehydrogenase kinase associated with the branched-chain alpha-ketoacid dehydrogenase complex. The amount of branched-chain alpha-ketoacid dehydrogenase kinase protein associated with the branched-chain alpha-ketoacid dehydrogenase complex and the message level for branched-chain alpha-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain alpha-ketoacid dehydrogenase kinase. The increase in branched-chain alpha-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain alpha-ketoacid dehydrogenase complex. The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency. Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned. Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue. Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme. In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein. Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex. Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate.

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.

Acetates↗
PubMed2004

Gene expression analysis in mitochondria from chagasic mice: alterations in specific metabolic pathways.

Cardiac hypertrophy and remodelling in chagasic disease might be associated with mitochondrial dysfunction. In the present study, we characterized the cardiac metabolic responses to Trypanosoma cruzi infection and progressive disease severity using a custom-designed mitoarray (mitochondrial function-related gene array). Mitoarrays consisting of known, well-characterized mitochondrial function-related cDNAs were hybridized with 32P-labelled cDNA probes generated from the myocardium of mice during immediate early, acute and chronic phases of infection and disease development. The mitoarray successfully identified novel aspects of the T. cruzi-induced alterations in the expression of the genes related to mitochondrial function and biogenesis that were further confirmed by real-time reverse transcriptase-PCRs. Of note is the up-regulation of transcripts essential for fatty acid metabolism associated with repression of the mRNAs for pyruvate dehydrogenase complex in infected hearts. We observed no statistically significant changes in mRNAs for the enzymes of tricarboxylic acid cycle. These results suggest that fatty acid metabolism compensates the pyruvate dehydrogenase complex deficiencies for the supply of acetyl-CoA for a tricarboxylic acid cycle, and chagasic hearts may not be limited in reduced energy (NADH and FADH2). The observation of a decrease in mRNA level for several subunits of the respiratory chain complexes by mitoarray as well as global genome analysis suggests a limitation in mitochondrial oxidative phosphorylation-mediated ATP-generation capacity as the probable basis for cardiac homoeostasis in chagasic disease.

Animals↗
PubMed2006

High-fat/low-carbohydrate diet reduces insulin-stimulated carbohydrate oxidation but stimulates nonoxidative glucose disposal in humans: An important role for skeletal muscle pyruvate dehydrogenase kinase 4.

AIM: The aim of this report was to study the effect of high-fat (HF)/low-carbohydrate (CHO) diet on regulation of substrate metabolism in humans. METHODS: Ten healthy men consumed either a HF (75% energy as fat) or control (35%) diet for 6 d in random order. On d 7, blood glucose disappearance rate (Rd) was determined before and during a hyperinsulinemic euglycemic clamp. Substrate oxidation was determined by indirect calorimetry. Muscle biopsies were obtained prediet, postdiet, and postclamps. RESULTS: Rd was similar under basal conditions but slightly elevated (approximately 10%, P < 0.05) during the last 30 min of the clamp after the HF diet. HF diet reduced CHO oxidation under basal (by approximately 40%, P < 0.05) and clamp conditions (by approximately 20%, P < 0.05), increased insulin-mediated whole-body nonoxidative glucose disposal (by 30%, P < 0.05) and muscle glycogen storage (by approximately 25%, P < 0.05). Muscle pyruvate dehydrogenase complex activity was blunted under basal and clamp conditions after HF compared with control (P < 0.05) and was accompanied by an approximately 2-fold increase (P < 0.05) in pyruvate dehydrogenase kinase 4 (PDK4) mRNA and protein expression. CONCLUSION: Short-term HF/low-CHO dietary intake did not induce whole-body insulin resistance, but caused a shift in im glucose metabolism from oxidation to glycogen storage. Insulin-stimulated CHO oxidation and muscle pyruvate dehydrogenase complex activity were blunted after the HF diet. Up-regulation of muscle PDK4 expression was an early molecular adaptation to these changes, and we showed for the first time in healthy humans, unlike insulin-resistant individuals, that insulin can suppress PDK4 but not PDK2 gene expression in skeletal muscle.

Adult↗
PubMed1982

Comparison of the metabolic and toxic effects of 2-chloropropionate and dichloroacetate.

The metabolic and toxic effects of 2-chloropropionate and dichloroacetate, activators of the pyruvate dehydrogenase complex, were compared. In 4-hr fasted mice, the oral LD50 values for 2-chloropropionate and dichloroacetate were 15.4 +/- 0.1 and 32.1 +/- 1.1 mmol/kg, respectively. In suckling rats, both compounds effectively lowered blood lactate and glucose levels and increased blood ketone bodies. Although comparable effects were brought about by both compounds on other metabolites, dichloroacetate caused a greater increase in blood ketone bodies. In a prolonged oral toxicity study using male rats, both compounds decreased growth rate and food consumption and caused neurotoxic effects. Both compounds brought about hind limb weakness, slower nerve conduction velocities and decreased diameter of tibial nerves. 2-Chloropropionate treatment caused testicular abnormalities manifested by testicular maturation arrest and degeneration of germ cells. 2-Chloropropionate-treated rats had significantly lower plasma triacylglycerol levels than control or dichloroacetate-treated rats. In mature rats, total serum ketone bodies were increased by dichloroacetate but not significantly elevated by 2-chloropropionate. Although 2-chloropropionate may lack sufficient safety to warrant chronic use in humans, it is a useful research tool for studying the metabolic effects of activation of the pyruvate dehydrogenase complex. Since 2-chloropropionate is not converted to oxalate and is not as ketogenic as dichloroacetate, 2-chloropropionate may be useful clinically in situations requiring only short-term therapy.

Acetates↗
PubMed1992

Enzyme activities of the mitochondrial energy generating system in skeletal muscle tissue of preterm and fullterm neonates.

Quadriceps muscle specimens from autopsy of 28 neonates (gestational age 25-42 weeks) were investigated to determine pyruvate and malate oxidation rates and several enzymes of the mitochondrial oxidative process. In general, the levels of all mitochondrial parameters measured, including carnitine levels, were lower in the neonates who died within the first week of life than those in the control group (age > 5 years). Pyruvate and malate oxidation rates (P < 0.05), activities of pyruvate dehydrogenase complex (P < 0.10) and succinate: cytochrome c oxidoreductase (P < 0.05) increased significantly with gestational age. Pyruvate oxidation rates (P < 0.05) as well as activities of citrate synthase (P < 0.05) and NADH:Q1 oxidoreductase (P < 0.05) were significantly lower in the group of very preterm infants at an age of 1-7 days compared with very preterm infants at an age between 3-8 weeks. We conclude from our study that special reference values are necessary for a correct biochemical diagnosis of mitochondrial encephalomyopathies in the neonatal period. Differences between preterm and fullterm children of the same age (1 week) indicate a maturational process in human muscle tissue during gestation. Comparison of two different age groups within the very preterm neonates point to a postnatal maturation of the mitochondrial energy metabolism, at least in preterm neonates.

Carnitine↗
PubMed2003

Molecular mimicry of mitochondrial and nuclear autoantigens in primary biliary cirrhosis.

BACKGROUND & AIMS: The mechanism for development of primary biliary cirrhosis (PBC) remains enigmatic, but molecular mimicry has been implicated because of well-known cross-reactivity of human mitochondrial autoantigens and equivalent bacterial antigens. Virtually all patients with PBC have antimitochondrial autoantibodies (AMA), but, interestingly, approximately 50% also manifest antinuclear antibodies (ANA). METHODS: To determine whether generation of ANA are due to molecular mimicry of mitochondrial peptides, we established 6 T-cell clones selected by a peptide corresponding to the E2 subunit of mitochondrial pyruvate dehydrogenase complex and analyzed for reactivity to mimicry peptides derived from mitochondrial and nuclear autoantigens, including control sequences. RESULTS: For mitochondrial autoantigens, 1 peptide from the E2 subunit of the pyruvate dehydrogenase complex, 1 peptide from the E2 subunit of the oxo-glutarate dehydrogenase complex, 1 peptide from the E2 subunit of the branched-chain 2-oxoacid dehydrogenase complex, and 1 peptide from the E3-binding protein cross-reacted with these T-cell clones. For the nuclear autoantigens, 5 peptides from gp210 and 1 from Sp100 cross-reacted with these clones. Furthermore, 1 of 3 T-cell clones selected by recombinant gp210 protein reacted with a mimicry peptide corresponding to amino acids 188-201 of gp210, indicating that this part of the protein is a naturally processed immunodominant T-cell epitope. CONCLUSIONS: These results demonstrate molecular mimicry between mitochondrial and nuclear autoantigens in PBC and that a mimicry peptide may become an immunodominant T-cell epitope. These data have significance not only for PBC but also for the production of ANA in other disease processes.

Amino Acid Sequence↗
PubMed1993

Monovalent Cation Activation of Plant Pyruvate Dehydrogenase Kinase.

The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.

Journal Article↗
PubMed1983

Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes.

The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.

Ammonia↗
PubMed1985

Normal pyruvate oxidation in Friedreich ataxia and Charcot-Marie-Tooth disease fibroblasts.

Partial defects in activity of the pyruvate dehydrogenase complex have been described by some investigators in cell lines from Friedreich ataxia and Charcot-Marie-Tooth disease patients. Methylene blue was used to stimulate the rate of pyruvate oxidation in two different assay systems of pyruvate dehydrogenase activity in cultured human fibroblasts to determine if such partial defects, if present, could be detected in a stimulated assay system. Cell lines from normal controls, five patients with Friedreich ataxia, six related persons with Charcot-Marie-Tooth disease patients were studied. Although methylene blue (at a concentration of 25 mumol/l) significantly increased pyruvate oxidation in both assay systems and in all cell lines studied, no significant differences in pyruvate oxidation could be demonstrated between the control cells and either the Friedreich ataxia or Charcot-Marie-Tooth cell lines.

Cell Line↗
PubMed2000

Molecular evidence of a unique lipoamide dehydrogenase in plastids: analysis of plastidic lipoamide dehydrogenase from Arabidopsis thaliana.

Lipoamide dehydrogenase is a subunit of the alpha-ketoacid dehydrogenases and the glycine decarboxylase complex in mitochondria, and the pyruvate dehydrogenase complex in plastids. We report here the unexpected finding of two plastidic isoforms of lipoamide dehydrogenase from Arabidopsis thaliana that are different from the mitochondrial form of the enzyme. The cDNA clones were confirmed by sequence alignment analysis and their location verified by chloroplast import assay. They are single copy genes that appear to be expressed in parallel in different tissues with highest level in developing siliques. Phylogenetic analysis gives further exemplary evidence for the plastidic evolution derived from cyanobacteria.

Amino Acid Sequence↗
PubMed1984

Correlation of enzymatic, metabolic, and behavioral deficits in thiamin deficiency and its reversal.

To clarify the enzymatic mechanisms of brain damage in thiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced 14CO2 and 14C-acetylcholine from [U-14C]glucose at rates similar to controls under resting conditions, their K+-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60-65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30-32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.

Animals↗