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At least 523 records · Page 29Linked to original sources
PubMed1985

Spectroscopic studies of pyruvate oxidase flavoprotein from Escherichia coli trapped in the lipid-activated form by cross-linking.

Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2. The maximal turnover of the enzyme, measured using a ferricyanide reductase assay, is increased 20-to 30-fold by either of two methods. Proteolysis in the presence of the substrate (pyruvate) and cofactor (Mg2+-thiamin pyrophosphate) results in cleavage at a single locus near the carboxyl terminus and concomitant activation. Phospholipids and detergents can bind to the enzyme and result in a similar activation, which is presumed to be physiologically relevant, since the enzyme functions as a peripheral membrane enzyme. Previous studies showed that proteolytic activation of pyruvate oxidase results in substantial changes in the absorption spectrum of the oxidized form of the bound flavin. Up to this time, similar studies of the lipid-activated form of the enzyme have not been feasible, since it is necessary to reduce the flavoprotein in order to induce binding to the lipids. In this paper, glutaraldehyde cross-linking of the lipid-activated enzyme is used to trap the enzyme in this form. Spectroscopic studies show alterations of the flavin spectrum similar to those observed upon proteolytic activation. This alteration in the flavin binding site is consistent with kinetic studies which suggest that activation results from an acceleration in the rates of electron transfer both into and out of the bound flavin, which appears to be more "accessible" in the activated forms of the enzyme.

Cross-Linking Reagents↗
PubMed1996

Conserved residues are functionally distinct within transketolases of different species.

Most of the amino acid residues which interact with thiamine pyrophosphate are highly conserved among enzymes which use this cofactor. The possible roles of several such residues in cofactor binding, catalysis, and/or substrate binding were examined for human transketolase. Mutations in H110 resulted in dramatic reductions to 2% or less of the normal activity. No alterations were found in the K(m)app's for the cofactor or for the donor and acceptor substrates. Alterations in Q428 resulted in a less severe loss of activity and also no changes in the K(m)app's. On the basis of the results, H110, an invariant residue, is proposed to function as a base which abstracts a proton from the protonated 4'-iminopyrimidine ring. The deprotonated 4'-imino moiety is required for generation of the C2-thiazolium carbanion which attacks the donor substrate. Interestingly, the function in the human enzyme of this invariant histidine is distinct from its role in yeast transketolase in which it aids in binding donor substrate and in subsequent catalytic events. Q428 is suggested to play a supportive role by stabilizing and orientating a water molecule which mediates the interaction between the 4'-amino group and H110. In other TPP-utilizing enzymes, the equivalent residue of Q428 is a histidine and is thought to deprotonate the 4'-amino group.

Binding Sites↗
PubMed1987

Beneficial effects of cocarboxylase in the treatment of experimental myocardial infarction in dogs.

Cocarboxylase, or thiamine pyrophosphate, is an essential coenzyme in the catabolism of pyruvate. The authors evaluated the effects of a stable cocarboxylase solution in the treatment of an experimentally created acute myocardial infarction in 14 healthy mongrel dogs. The left anterior descending artery was ligated for 60 minutes and data were collected at the following points: A) prior to ligation, B) 15 minutes after ligation, C) 30 minutes after ligation, and D) 60 minutes after ligation. In one group (Group II), cocarboxylase (150 mgm/kg) was given systematically via a central line 15 minutes and 45 minutes after ligation, while in Group I an equal amount of D5W was given. Hemodynamic data include heart rate, systolic and mean arterial pressure, pulmonary wedge pressure, right arterial pressure, and cardiac output. Myocardial O2 consumption was determined by the method of Rooke and Feigl. Electrocardiographic data were also monitored throughout the experiment. In both groups, preligation (point A) hemodynamic data were similar. In Group II, there were beneficial hemodynamic changes versus Group I (expressed as percentage recovery of hemodynamic performance from preligation) at points C and D, with significant (P less than 0.05) decreases in heart rate, increased stroke volume, decreased systemic vascular resistance, and decreased myocardial O2 consumption. EKG criteria also showed improvement in Group II versus Group I. In conclusion, this experiment suggests that cocarboxylase may be beneficial to ischemic canine myocardium by virtue of its favorable systemic hemodynamic effects.

Animals↗
PubMed1983

Albumin requirement of Treponema denticola and Treponema vincentii.

Treponema denticola and Treponema vincentii were found to require albumin, oleic acid, and thiamine pyrophosphate (TPP) for growth. Previous studies indicated that commercial human alpha globulin, which is 50% albumin, was the only serum fraction that supported growth of these two oral treponemes. The alpha-globulin proteins were separated from the contaminating albumin with Affi-Gel Blue affinity chromatography. Both the albumin fraction and one of the alpha-globulin fractions were required for growth of T. denticola. Oleic acid was supplied by the alpha-globulin fraction and the albumin functioned as a chelator to maintain a low level of free fatty acid in the medium. Purified serum albumin (bovine or human) could substitute for the alpha-globulin fraction that contained albumin. Optimal growth of T. denticola and T. vincentii was in a medium supplemented with 0.4% (w/v) delipified albumin, 0.08 mg/mL of sodium oleate, and 25 micrograms/mL of TPP. Serum albumin tightly bound TPP (0.5 microgram of TPP per milligram of albumin). Optimal growth of T. denticola was only in an albumin-oleate supplemented medium with sufficient TPP to saturate the albumin binding sites and provide excess free TPP. Albumin bound long-chain fatty acids and thus detoxified the medium. Neither starch- nor charcoal-treated Tween 80 (polysorbitan monooleate) replaced albumin for optimal growth. Short-chain fatty acids supported only limited growth of T. denticola when added to a medium with TPP or to a medium that contained 0.4% delipified albumin and TPP.

Animals↗
PubMed1980

The binding of a fluorescent activator 2-(N-decyl)aminonaphthalene-6-sulfonic acid to pyruvate oxidase.

E. coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme which has been purified to homogeneity. In vivo the oxidase resides on the inner surface of the cytoplasmic membrane and is coupled to the bacterial electron transport chain. In vitro, the purified oxidase requires lipids for full enzymatic activity. Previous studies have characterized the conformational and energetic coupling between the lipid-binding site(s) and the catalytic active site. The affinity of the enzyme for phospholipids and detergents is significantly enhanced when the flavoprotein is in the reduced form, i.e., in the presence of pyruvate and the required cofactor, thiamin pyrophosphate. The lipid-binding studies were hindered due to the complicating factor of the self-association of the substrate-reduced flavoprotein. In this paper, fluorescence techniques are employed to measure the binding of a detergent-like activator to the oxidase. The experiments are performed at much lower protein concentrations than previously employed, so that protein aggregation is not a problem. The chromophore on the activator, 2-(N-decyl)aminonaphthalene-6-sulfonic acid is effective at quenching the pyruvate oxidase intrinsic tryptophan fluorescence. Quenching titrations are used to obtain the binding isotherm. AT DNS concentrations less than 10(-5) M, the results show a larger amount of DNS binding to the reduced flavoprotein than to the oxidized form of the enzyme. This is the concentration range where DNS is an effective activator of the enzyme. This represents a class of binding sites specifically found on pyruvate oxidase and not apparent in other proteins such as lysozyme or aldolase. At the DNS concentration which is optimum for activation approx. 20 molecules of DNS are bound per enzyme tetramer in the absence of the substrate. The pyruvate-reduced form of the enzyme binds about 40--50 molecules of DNS per tetramer. Qualitatively, the results are similar to what was previously found for both sodium dodecyl sulfate and cetyl trimethylammonium bromide. However, in both these cases, the amount of bound detergent was nearly an order of magnitude less than the values obtained using DNS.

Enzyme Activation↗
PubMed1975

The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components.

Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.

Azotobacter↗
PubMed1978

The action of inorganic phosphate on thiamin transport by rat everted jejunal sacs.

Rat everted jejunal sacs were incubated at 37 degree C in Krebs-Henseleit buffer, pH 7.4, containing thiazole-2- 14C-thiamine (0.2 micrometer), in the absence or in the presence of inorganic phosphate at a final concentration of 6mM. Control experiments under the same conditions, but without labeled thiamin were also carried out. In the intestinal sac wall, the content of labeled and endogenous (unlabeled) thiamin in the free and phosphorylated form was measured, while in the serosal fluid, total transported thiamin (labeled and endogenous) was evaluated. Accumulation in the enterocyte of inorganic phosphate, an inhibitor of thiamin pyrophosphate enzymatic hydrolysis, lowered the dephosphorylation of endogenous thiamin phosphates, both in the presence and in the absence of labeled thiamin. In the presence of labeled thiamin, the following results were observed: a) decreased labeled thiamin uptake and accumulation, both in the phosphorylated and free form; b) reduction of labeled thiamin transport to the serosal side; c) increase of endogenous thiamin exit from the intestinal cell. These findings seem to point out an important role of thiamin phosphorylation-dephosphorylation coupling in thiamin intestinal transport in vitro.

Animals↗
PubMed2000

Thiamine intestinal transport and related issues: recent aspects.

In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes place primarily in the proximal part of the small intestine by means of a dual mechanism, which is saturable at low (physiological) concentrations and diffusive at higher. Thiamine undergoes intracellular phosphorylation mainly to thiamine pyrophosphate, while at the serosal side only free thiamine is present. Thiamine uptake is enhanced by thiamine deficiency, and reduced by thyroid hormone and diabetes. The entry of thiamine into the enterocyte, as evaluated in brush border membrane vesicles of rat small intestine in the absence of H+ gradient, is Na+- and biotransformation-independent, completely inhibited by thiamine analogs and reduced by ethanol administration and aging. The transport involves a saturable mechanism at low concentrations of vitamin and simple diffusion at higher. Outwardly oriented H+ gradients enhance thiamine transport, whose saturable component is a Na+-independent electroneutral uphill process utilizing energy supplied by the H+ gradient, and involving a thiamine/ H+ 1:1 stoichiometric exchange. The exit of thiamine from the enterocyte, as evaluated in basolateral membrane vesicles, is Na+-dependent, directly coupled to ATP hydrolysis by Na+-K+-ATPase, and inhibited by thiamine analogs. Transport of thiamine by renal brush border membrane vesicles is similar to the intestinal as far as both H+ gradient influence and specificity are concerned. In the erythrocyte thiamine transport is a Na+-independent, electroneutral process yet with two components: saturable, prevailing at low thiamine concentrations, and diffusive at higher. The saturable (specific) component is missing in patients of the rare disease known as thiamine-responsive megaloblastic anaemia (TRMA), producing a general disturbance of thiamine transport up to thiamine deficiency. The TRMA gene is located in chromosome 1q23.3. Recently, the thiamine transporter has been cloned: it is a protein of 497 amino acid residues with high homology with the reduced-folate transporter.

Absorption↗
PubMed1976

Effect of Thiamine on Ethanol and Pyruvate Production in Helminthosporium maydis.

Growth of the fungus Helminthosporium maydis race T in a basal glucose-l-asparagine liquid medium, pH 5, is inhibited by thiamine-HCl. Analysis of the media for organic acids reveals that the extracellular pyruvate concentration decreases as the thiamine-HCl concentration of the medium increases. Extracellular ethanol, in contrast to pyruvate, increases in concentration as the thiamine-HCl concentration of the medium increases under both aerobic and anaerobic conditions.The changes in ethanol and pyruvate levels in the presence of thiamine-HCl occur via a thiamine-mediated increase in the activity of pyruvate decarboxylase but not alcohol dehydrogenase. This increase in pyruvate decarboxylase activity appears to be due to an increase in the quantity of enzyme present rather than an activation of pre-existing enzyme. Whereas thiamine-pyrophosphate stimulates pyruvate decarboxylase activity in vitro, thiamine-HCl has no effect. Neither thiamine derivative affects alcohol dehydrogenase activity. The increase in pyruvate decarboxylase activity which accompanies an increase in the thiamine-HCl concentration of the medium is correlated with a decrease in the level of intracellular pyruvate.

Journal Article↗
PubMed1975

Subacute necrotizing encephalomyelopathy. Clinical, ultrastructural, biochemical and therapeutic studies in an infant.

Subacute necrotizing encephalomyelopathy (SNE) has been observed in an infant with regressing psychomotor development. The concentrations of alanine, pyruvate and lactate were increased in the serum and blood as well as in the cerebrospinal fluid. Pyruvate carboxylase activity was reduced in the liver tissue. An inhibitor of thiamine-pyrophosphate-ATP-phosphotransferase was present in the urine. Thiamine treatment was followed by a decrease of serum alanine and blood pyruvate and lactate, but there was no clinical improvement during a period of 17 months. Ultrastructural investigations revealed high glycogen levels in liver tissue and skeletal muscle. These findings contrast with decreased gluconeogenesis, which is suggested by the diminished pyruvate carboxylase activity. Therefore it is concluded that reduced hepatic pyruvate carboxylase activity is not the primary cause of SNE.

Alanine↗
PubMed1993

Thiamin-responsive maple syrup urine disease in a patient antigenically missing dihydrolipoamide acyltransferase.

Maple syrup urine disease results from inherited defects in human nuclear genes for branched chain alpha-ketoacid dehydrogenase, a mitochondrial multienzyme complex. Thiamin pyrophosphate is necessary for complex activity and a thiamin-responsive form of maple syrup urine disease is known. Here we demonstrate the use of [1-13C]leucine oxidation to [13C]O2 quantified in breath samples as a means of assessing whole body leucine oxidation. Analysis of cultured cells from this patient shows the antigenic lack of the E2 subunit, yet she gained branched chain alpha-ketoacid dehydrogenase activity in response to diet supplementation with pharmacologic doses of thiamin. These cultured cells were used to seek a molecular basis for the observed thiamin response. Despite normal thiamin transport in these cells, medium supplementation of up to 1000 thiamin/liter failed to increase complex activity or cause the antigenic appearance of the missing protein. This lack of response in cultured cells suggests that the observed whole body response to thiamin must be a tissue-specific effect in liver, muscle, or kidney. In addition, allele-specific detection of paternal and maternal mutations was used to genotype family members in this pedigree.

Acyltransferases↗
PubMed1988

Determination of thiamin status during protein calorie malnutrition in rats.

To clarify the influence of protein calorie malnutrition (PCM) on thiamin metabolism in rats, the effects of three diets containing different amounts of protein and sucrose were evaluated by three tests in 20 young male Wistar rats equally divided into four groups. Three of the groups, the controls (group 1), those fed high protein (group 2), and one of the two groups fed a low protein diet (group 3), were fed ad libitum. A second group (group 4) on the same low protein diet was fed 40% less than that consumed by group 3, which consumed less than the controls or high protein rats because of poor appetite. The sucrose contents of the experimental diets were inversely related to the protein contents. Thiamin concentrations were determined in blood, brain, heart, liver, kidney and spleen, and in 24-hr urine and stools. Tissue transketolase activity (TTKA) was measured in brain and liver and in erythrocytes (ETKA), the latter in association with the thiamin pyrophosphate (TPP) effect. Malnutrition caused decreased 24-hr urinary thiamin excretion, decreased ETKA, with corresponding increased TPP effect and decreased TKA of brain. As tissue thiamin concentrations were not decreased, the results suggest that malnutrition causes functional thiamin deficiency.

Animals↗
PubMed2003

Reversal of refractory congestive heart failure after thiamine supplementation: report of a case and review of literature.

Patients with refractory heart failure comprise a very important subgroup of patients with congestive heart failure. Before assuming that this condition simply reflects advanced, perhaps terminal, myocardial dysfunction, potentially reversible factors should be sought carefully. We describe a 58-year-old Hispanic man with a diagnosis of idiopathic dilated cardiomyopathy who presented with symptoms of severe congestive heart failure, glossitis, and peripheral neuropathy. His hemodynamic profile was characterized by refractory low-output cardiac failure and decreased vascular resistance. Thiamine deficiency was documented by a high thiamine pyrophosphate effect. His clinical condition was quickly reversed with thiamine administration. This response to thiamine administration supports the diagnosis and indicates that thiamine deficiency may play an important etiologic role in the deterioration of cardiac function in some patients with congestive heart failure.

Cardiac Output, Low↗
PubMed1978

Red blood cell transketolase activity and the effect of thiamine supplementation in patients with chronic liver disease.

Biochemical evidence of thiamine deficiency was found in 58% of patients with chronic liver disease, the incidence being higher in alcoholic than in non-alcoholic patients. Daily supplementation with high doses of thiamine hydrochloride (200 mg/day) for one week restored levels of thiamine pyrophosphate (TPP), the active co-enzyme form of thiamine, to normal in all cases. Such supplementation also stimulated synthesis of the TPP dependent enzyme transketolase. Because of the essential role of TPP as a co-factor in intermediary metabolism, it is concluded that high doses of thiamine should be included in the routine nutritional management of patients with severe chronic liver disease.

Ascorbic Acid↗
PubMed1998

Thiamin-binding protein from sunflower seeds.

A thiamin-binding protein was isolated from sunflower seeds. Its molecular mass was estimated to be 230 kDa by gel filtration. The protein was suggested to be composed of six subunits, which consisted of polypeptides linked by disulfide bond(s). The protein contained a large amount of glutamine or glutamic acid (19.9 mol%) and asparagine or aspartic acid (11.1 mol%). The levels of tryptophan and valine in the protein were low. These properties of the thiamin-binding protein were similar to those of helianthinin. Optimum pH for the thiamin-binding activity of the protein was 8.0 to 9.0. The thiamin-binding activity was not inhibited by thiamin monophosphate, thiamin pyrophosphate, oxythiamin, or pyrithiamin. These properties of the thiamin-binding protein from sunflower seeds were similar to those from buckwheat seeds, but not to those from rice seeds and sesame seeds.

Asparagine↗
PubMed1978

Membrane bound pyrophosphatases in Entamoeba histolytica.

Entamoeba histolytica homogenates are capable of hydrolyzing a range of inorganic and organic pyrophosphates. Two separate activities are present: an inorganic pyrophosphatase hydrolyzing inorganic pyrophosphate and linear tripolyphosphate, and a nucleoside diphosphatase hydrolyzing thiamine pyrophosphate and nucleoside diphosphates (ADP, GDP and UDP). The inorganic pyrophosphatase has an acid pH optimum, a relatively high KM (congurent to 1 micrometer) and is markedly heat stable and lacks a metal requirement. The nucleoside diphosphatase also has an acid pH optimum but displays a much higher affinity for substrate (KM congurent to 50 micrometer), is unstable to heating and is activated by Ca ions. Both pyrophosphatases distinct from the acid phosphatase activity which is also present. All three hydrolases are sedimentable and latent suggesting their association with membrane bounded organelles. No soluble inorganic pyrophosphatase activity could be demonstrated.

Animals↗