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PubMed1983

Essential histidine residues of alpha-ketoglutarate dehydrogenase from pigeon breast muscle.

Modification of histidine residues of alpha-ketoglutarate dehydrogenase results in complete inactivation of the enzyme. The kinetic curves for histidine residues modification and for alpha-ketoglutarate dehydrogenase inactivation are biphasic. The rate constants for both processes show a good coincidence at two phases. The complete inactivation of the enzyme is due to modification of one histidine at each step of the reaction. At saturating concentrations of the substrate, alpha-ketoglutarate, the inactivating effect of the modifier is lowered; the cofactors, thiamine pyrophosphate and Mg2+, exert no protective effect.

Animals↗
PubMed1978

Purification of the "corrinoid" enzyme involved in the synthesis of acetate by Clostridium thermoaceticum.

A corrinoid enzyme has been purified to approximately 80% homogeneity from Clostridium thermoaceticum. It catalyzes the formation of acetate from N5-methyltetrahydrofolate and pyruvate in combination with the required supplementary enzymes which are supplied by an extract that has been treated with propyl iodide. The enzyme was purified by chromatography on a folate affinity column and a DEAE-Bio-Gel column and by ultrafiltration. The molecular weight as determined by sedimentation equilibrium is 158,000 and the sedimentation coefficient is 10.5 S. By gel electrophoresis in sodium dodecyl sulfate, the subunit molecular weight was found to be 40,000, thus, the enzyme may be a tetramer of four similar subunits. The results of electron microscopy confirmed the tetrameric structure. In the absence of sodium dodecyl sulfate, two bands of similar intensity were observed by electrophoresis, but both yielded the 40,000 molecular weight subunit in the presence of sodium dodecyl sulfate. These results indicate the two bands represent either two different molecular weight forms of the enzyme or two differently charged isoenzymes. The enzyme is quite labile being sensitive to dilution, aerobic conditions, and light. Dithiothreitol and glycerol were found to stabilize the enzyme. The cofactor requirements for acetate synthesis have been determined. ATP, thiamin pyrophosphate, S-adenosylmethionine, and Fe2+ were found to be required for maximum activity and the Km values were determined. High concentrations of methyltetrahydrofolate, pyruvate, and S-adenosylmethionine were found to inhibit the synthesis of acetate.

Acetates↗
PubMed1984

Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of L1210 cells by treatment with an N-hydroxysuccinimide ester of [3H]methotrexate.

An N-hydroxysuccinimide ester of [3H]methotrexate has been employed to covalently label a specific binding protein that resides in the plasma membrane of L1210 cells. Incorporation of radioactivity into this protein accounted for 55% of total cellular labeling, was half-maximal at a reagent concentration of 27 nM, and was blocked either by prior exposure to unlabeled reagent or by the addition of excess methotrexate. A role for this protein in methotrexate transport was supported by the observations that: (a) similar concentrations of reagent were required for both labeling of the binding protein and irreversible inhibition of transport; (b) the amount of labeled binding protein was comparable to observed levels of transport protein; (c) protection against labeling was afforded by thiamin pyrophosphate, a potent competitive inhibitor of methotrexate transport; and (d) labeling of the binding protein was not observed in a subline of L1210 cells that has a defect in the ability to transport methotrexate. The binding protein could be solubilized from the membrane by various ionic and non-ionic detergents and the covalent bond between the incorporated [3H]methotrexate and the protein was stable to a variety of conditions, including high concentrations of mercaptoethanol and hydroxylamine and extremes of pH. The labeled protein fractionated as a nearly symmetrical peak on Sephacryl S-300 and it appeared as a single band (Mr = 36,000) after electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate.

Affinity Labels↗
PubMed1982

Thiamin Metabolism in the rat during long-term alcohol administration. 1. Communication: ethanol induced changes at optimal thiamin supply.

The effect of ethanol administration on the phosphorylation of thiamin, and on the transketolase activity (TKA) in erythrocytes, heart and liver, and on the thiamin excretion in urine was investigated in rats optimally supplied with thiamin over a period of 16 weeks. Ethanol ingestion resulted in a diminished concentration of thiamin (T), thiaminmonophosphate (TMP), thiamindiphosphate (TDP) and thiamintriphosphate (TTP) in blood and organs and in a reduced excretion rate of T in urine due to an impaired intestinal absorption. An ethanol induced alteration of the degree of thiamin phosphorylation became evident only after 16 weeks resulting in a decline of T and TMP in blood and of T in heart to undetectable amounts and in an enhancement of the TDP- and TTP-pool. Despite an increasing content of TDP in erythrocytes the TKA was lowered and the alpha-TK was enhanced during the test period suggesting a partially inhibited formation of the active holoenzyme by alcohol in vivo. However, the lowered TKA and the normal alpha-TK in liver are primarily suggestive of an apo-enzyme degeneration influenced by ethanol.

Alcoholism↗
PubMed1982

Application and evaluation of an NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex using chicken liver homogenates.

1. A method for measuring the activity of the pyruvate dehydrogenase complex [EC 1.2.4.1] (Hinman and Blass, 1981) has been adapted for use with crude chicken liver homogenates. 2. It was found necessary to remove cytoplasmic lactate dehydrogenase by centrifugation before releasing the complex from the mitochondria by treatment with sodium deoxycholate. 3. Lipoamide dehydrogenase was preferable to phenazine methosulphate as the electron carrier between the complex and NADH. 4. The assay was not suitable for use at 40 degrees C (approximate body temperature) but was satisfactory at 25 degrees C. 5. The assay exhibited the properties expected of pyruvate dehydrogenase complex i.e. a complete dependence on the presence of NAD, coenzyme A and pyruvate and a partial dependence on MgCl2, dithiothreitol and thiamine pyrophosphate. The pH optimum was between 7.8 and 8.0. 6. The mean activity for a group of normally fed young chickens was 654 mumol/g dry weight h and 88 for birds starved overnight.

Animals↗
PubMedDate not supplied

[Simple method of acetoid determination and its application to the analysis of pyruvate dehydrogenase].

A modification of the method for determining the content of acetoin in biological media is described; the method is based on formation of a stained complex under acyloin heating with the acidified ammonium molybdate water solution. Colorimetric determination of the acetoin amount in the enzymic system containing sodium pyruvate, thiamine pyrophosphate and tris-HCl is shown to be possible. Application of the method to analysis of the catalytic activity of pyruvate dehydrogenase preparations from different sources and with different degree of purification in the reaction of acetoin formation from pyruvate is described.

Acetoin↗
PubMed1981

Kinetics of bisulphite cleavage of the three biological phosphorylated derivatives of thiamine.

Bisulphite/sulphite cleaves thiamine and its biological phosphorylated derivatives into the same pyrimidine moiety and their respective thiazole moiety with pseudo-first order kinetic constants following the order of thiamine greater than thiamine monophosphate greater than thiamine diphosphate greater than thiamine triphosphate over the pH range of 3.9 to 7.0. The activation energy was 14.8 kcal/mol for all four thiamines at pH 5.2 and over the temperature range of 25 to 65 degrees C. The rate differences may be rationalized in terms of differences in the pH-dependent electronic, charge and conformation distributions of the various thiamine species.

Chemical Phenomena↗
PubMed1981

Purification of five components from Clostridium thermoaceticum which catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate. Properties of phosphotransacetylase.

A five-component enzyme system which catalyzes synthesis of acetylphosphate from methyltetrahydrofolate (CH3THF) plus pyruvate has been purified from the homoacetate-fermenting bacterium, Clostridium thermoaceticum. One of the components was identified as the low potential electron carrier, ferredoxin, and the other 4 protein components have been designated F1, F2, F3, and F4. F1, F2, and F4 have been purified to homogeneity and, as estimated by gel filtration, have native molecular weights of 88,100, 58,900, and 255,000, respectively, while the subunit molecular weights obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 20,000, 25,500, and 120,000, respectively. F3 contains 3 to 4 protein bands and has not been characterized with respect to molecular weights. Acetylphosphate synthesis by the purified system is optimal at pH 6.0 and 65 degrees C and requires ATP, CoA, and, to a lesser extent, thiamin pyrophosphate and Fe2+. S-Adenosylmethionine is not required. The F1 component has been identified as phosphotransacetylase and in its absence, the product is acetyl-CoA. Some properties of the phosphotransacetylase are presented. A scheme is given indicating present views of the functions of the individual components.

Acetates↗
PubMed1981

[Dihydroxyacetone synthase from the methanol-utilizing yeast Candida boidinii].

A procedure for purification of dihydroxyacetone synthase catalyzing the formation of dihydroxyacetone and glyceraldehyde 3-phosphate from formaldehyde and xylulose 5-phosphate has been developed. Using ion-exchangers with increasing affinity for dihydroxyacetone synthase, a homogenous preparation of the enzyme with specific activity of 2 u./mg has been obtained. The enzyme is made up of 2 subunits with m. w. of 76,000, contains thiamine pyrophosphate, requires Mg2+ for its activity and differs from yeast transketolase by substrate specificity and some other properties. The role of dihydroxyacetone synthase in metabolism of methanol-utilizing yeasts is discussed.

Aldehyde-Ketone Transferases↗
PubMedDate not supplied

[Biochemical aspects of thiamine transport].

The paper deals with the modern ideas concerning pathways and mechanisms of thiamine and its phosphorylated derivatives transport in the animal cells. The data are analyzed on thiamine and its derivatives resorption by the intestnal wall, their transportation by blood and lymph and also their penetration into cells and subcellular structures. A conclusion is made that these processes occur by mechanism of active and passive transportation and simple diffusion. Realization of one or another mechanism status of the object under study.

Biological Transport↗
PubMed1980

An improved method for the electrophoretic separation and fluorometric determination of thiamine and its phosphates in animal tissues.

An improved method for electrophoretic separation and fluorometric (or radiometric) determination of thiamine and thiamine mono-, di- and tri-phosphates in animal tissues (liver, small intestine, kidney, heart, brain) is described. The amount of total thiamine handled was 1.5-3.5 microgram. The procedure includes: acid excretion of the compounds from the tissue, deproteinization with TCA, purification of the extract on partially deactivated charcoal, elution with 10% n-propanol in 0.1 N formic acid, lyophilization, electrophoresis on gelatinized cellulose acetate strips, elution of the thiaminic bands with 50% ethanol and finally oxidation to thiochromes and reading of eluate fluorescence in a spectrofluorophotometer. The mean internal recovery was 83.3% and the external one 83.5%. Dephosphorylation of the T phosphates was rather scanty and each thiamine compound was comparably recovered (82.0-85.7%), apparently TTP being the worst and T the best recovered compound.

Animals↗
PubMed1995

Nutritional status of elderly female nursing home residents; the effect of supplementation with a physiological dose of water-soluble vitamins.

OBJECTIVE: To assess the nutritional status of female elderly nursing home residents and to study the effect of dietary supplementation with a physiological dose of water-soluble vitamins. DESIGN: Single-blind, randomised, placebo-controlled intervention trial. Daily consumed supplement consisted of fortified fruit juice containing 50 g carbohydrate and 50% of the daily dietary recommendations of water-soluble vitamins. Regular fruit juice was used as placebo. Dietary intake assessment at baseline. Fasting blood sample, anthropometric measurements and medical record at baseline and after 4 and 12 weeks of supplementation. SETTING: Three nursing homes within 40 km of the coordination centre (Department of Human Nutrition, Wageningen Agricultural University, the Netherlands). SUBJECTS: Forty-two female nursing home residents aged 60 years and older, in relatively stable health condition, at least 3 months resident at baseline. In each nursing home, 14 subjects were randomly assigned to the supplement or control group. INTERVENTIONS: Twelve weeks of supplementation. RESULTS: Participants had a poor nutritional status. Dietary supplementation significantly improved the concentrations of thiamin pyrophosphate and pyridoxal 5'-phosphate, increased body weight and decreased serum homocysteine concentration in the supplement group. Plasma vitamin C levels were significantly increased in both supplement and control group. CONCLUSIONS: Even in this heterogeneous population of female nursing home elderly it is possible to improve the nutritional status through dietary intervention with a physiological dose of water-soluble vitamins.

Aged↗
PubMed1995

Primary structure and eubacterial relationships of the pyruvate:ferredoxin oxidoreductase of the amitochondriate eukaryote Trichomonas vaginalis.

In the eukaryotic unicellular organism Trichomonas vaginalis a key step of energy metabolism, the oxidative decarboxylation of pyruvate with the formation of acetyl-CoA, is catalyzed by the iron-sulfur protein pyruvate:ferredoxin oxidoreductase (PFO) and not by the almost-ubiquitous pyruvate dehydrogenase multienzyme complex. This enzyme is localized in the hydrogenosome, an organelle bounded by a double membrane. PFO and its closely related homolog, pyruvate:flavodoxin oxidoreductase, are enzymes found in a number of archaebacteria and eubacteria. The presence of these enzymes in eukaryotes is restricted, however, to a few amitochondriate groups. To gain more insight into the evolutionary relationships of T. vaginalis PFO we determined the primary structure of its two genes (pfoA and pfoB). The deduced amino acid sequences showed 95% positional identity. Motifs implicated in related enzymes in liganding the Fe-S centers and thiamine pyrophosphate were well conserved. The T. vaginalis PFOs were found to be homologous to eubacterial pyruvate:flavodoxin oxidoreductases and showed about 40% amino acid identity to these enzymes over their entire length. Lack of eubacterial PFO sequences precluded a comparison. pfoA and pfoB revealed a greater distance from related enzymes of Archaebacteria. The conceptual translation of the nucleotide sequences predicted an amino-terminal pentapeptide not present in the mature protein. This processed leader sequence was similar to but shorter than leader sequences noted in other hydrogenosomal proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Indolepyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. A new enzyme involved in peptide fermentation.

Pyrococcus furiosus is a strictly anaerobic archaeon that grows optimally at 100 degrees C by a fermentative-type metabolism in which complex peptide mixtures such as yeast extract and Tryptone, and also certain sugars, are oxidized to organic acids, H2 and CO2. Enzymes involved in the utilization of peptides such as proteases, aromatic amino transferases, and glutamate dehydrogenase have been previously purified from this organism. It is shown here that P. furiosus also contains significant cytoplasmic concentrations of a new enzyme termed indolepyruvate ferredoxin oxidoreductase (IOR). This catalyzes the oxidative decarboxylation of aryl pyruvates, which are generated by the transamination of aromatic amino acids, to the corresponding aryl acetyl-CoA. IOR is a tetramer (alpha 2 beta 2) of two identical subunits (66,000 and 23,000 Da) with a molecular weight of 180,000. The enzyme contains one molecule of thiamine pyrophosphate and four [4Fe-4S]2+,1+ and one [3Fe-4S]0,1+ cluster, as determined by iron analyses and EPR spectroscopy. Significant amounts of other metals such as copper and zinc were not detected. IOR was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.5-10.5 (at 80 degrees C). The enzyme was sensitive to inactivation by O2, losing 50% of its activity after exposure to air for 20 min at 23 degrees C, and was quite thermostable, with a half-life of activity at 80 degrees C (under anaerobic conditions) of about 80 min. The Km values (in microM) for indolepyruvate, p-hydroxyphenylpyruvate, phenylpyruvate, CoASH, and P. furiosus ferredoxin, the physiological electron carrier, were 250, 110, 90, 17, and 48, respectively. IOR was inhibited by KCN (apparent Ki = 7.5 mM), but not by CO (1 atm). An enzyme analogous to IOR has not been reported previously. Curiously, it has few properties in common with the pyruvate ferredoxin oxidoreductase of P. furiosus, even though the two enzymes catalyze virtually identical reactions. In fact, of known ketoacid oxidoreductases, the catalytic mechanism of IOR appears to be most similar to that of the pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium Thermotoga maritima.

Amino Acid Sequence↗
PubMedDate not supplied

[Metabolism of vitamin B1 and its di- and triphosphate esters in experimental allergic encephalomyelitis].

Biological significance of thiamin in development of experimental allergic encephalomyelitis has been elucidated. It has been shown that at the late preclinical stage of the disease thiamine metabolism is predominantly directed towards the maintaining of cellular metabolic homeostasis, whereas at the stage of clinical symptoms the anabolic process gives way to catabolic decomposition. Among tested thiamine phosphates the triphosphate ester is the most informative parameter in demyelinizing processes. Thiamine injections to immunized animals accelerate the vitamin phosphorylation depleting the reducing and energy potentials of the cell. Such thiamine antagonist as oxythiamine inhibits phosphatase reactions.

Animals↗
PubMed1993

Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family.

Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism. We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E. coli Gcl and isolation of mutants lacking the enzyme. Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway. Significant sequence identity (26%) was also observed with E. coli pyruvate oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J. E., Jr. (1988) J. Bacteriol. 170, 3937-3945). Consistent with a grouping of Gcl with the AHAS and pyruvate oxidase enzymes. Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD. We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues. Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E. coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit. Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate.

Acetolactate Synthase↗
PubMed1995

An investigation of the kinase activity of aurochs heart pyruvate dehydrogenase complex.

The purified aurochs heart pyruvate dehydrogenase complex (PDC) saturated to approximation 60% with endogenous thiamine pyrophosphate (TPP), was slowly and incompletely inactivated by its kinase in the presence of ATP. Exogenous TPP or ADP, but not pyruvate, strongly inhibited the kinase activity. The kinetic properties of the aurochs heart PDC kinase suggested the occurrence of two active sites each with different affinities for ATP (K'm - 1.7 microM, K''m = 48 microM).

Adenosine Triphosphate↗