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PubMed1987

Hydrolysis and synthesis of thiamin triphosphate in bacteria.

Thiamin triphosphate (ThTP) in early stationary phase cells of Escherichia coli grown in nutrient broth with 0.1% yeast extract was found to constitute approximately 5-7% of cellular thiamin diphosphate (ThDP) or around 5 nmol/g cell. Nearly the same level of ThTP was obtained in a Bacillus strain. When E. coli was loaded with an excess of ThTP or ThDP, cellular ThTP was found to be controlled in the course of the long term to maintain its ratio to the amount of cellular ThDP. The ThTP vs. ThDP ratio in E. coli cells after short-term ThDP uptake was found to be a function of the cellular growth phase. The ratio in early exponential phase E. coli cells was found to be approximately 4% and it became lower (less than 3%) when cell growth proceeded to the late exponential stage. Two phosphatases specific for ThTP (ThTPase) among thiamin phosphates were detected in E. coli. One required Mg2+ and was found mainly in the soluble fraction, while the other was Mg2+-independent and originated from the membrane. The two ThTPases were similar to their rat tissue counterparts.

Bacillus↗
PubMed2005

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1999

Prolonging cell-free protein synthesis with a novel ATP regeneration system.

A new approach for the regeneration of adenosine triphosphate (ATP) during cell-free protein synthesis was developed to prolong the synthesis and also to avoid the accumulation of inorganic phosphate. This approach was demonstrated in a batch system derived from Escherichia coli. Contrary to the conventional methods in which exogenous energy sources contain high-energy phosphate bonds, the new system was designed to generate continuously the required high-energy phosphate bonds within the reaction mixture, thereby recycling the phosphate released during protein synthesis. If allowed to accumulate, phosphate inhibits protein synthesis, most likely by reducing the concentration of free magnesium ion. Pediococcus sp. pyruvate oxidase, when introduced in the reaction mixture along with thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), catalyzed the generation of acetyl phosphate from pyruvate and inorganic phosphate. Acetyl kinase, already present with sufficient activity in Escherichia coli S30 extract, then catalyzed the regeneration of ATP. Oxygen is required for the generation of acetyl phosphate and the H(2)O(2) produced as a byproduct is sufficiently degraded by endogenous catalase activity. Through the continuous supply of chemical energy, and also through the prevention of inorganic phosphate accumulation, the duration of protein synthesis is extended up to 2 h. Protein accumulation levels also increase. The synthesis of human lymphotoxin receives greater benefit than than that of chloramphenicol acetyl transferase, because the former is more sensitive to phosphate inhibition. Finally, through repeated addition of pyruvate and amino acids during the reaction period, protein synthesis continued for 6 h in the new system, resulting in a final yield of 0.7 mg/mL.

Adenosine Triphosphate↗
PubMed1999

[Vitamin levels in the serum and erythrocytes during erythropoietin therapy in hemodialyzed patients].

BACKGROUND: The treatment of anaemia by erythropoietin is the method of choice in chronic renal failure patients without dialysis treatment, in hemodialyzed patients and during CAPD. Erythropoietin treatment influenced supplementary doses of vitamins especially those participating in haem formation. OBJECTIVES: The aim of the study was to investigate the influence of erythropoietin treatment during long-term 15 months study on serum vitamin A levels and its protein carriers, prealbumin and retinol-binding protein, on erythrocyte vitamin B1, B2 and B6 and folic acid and on serum or plasma vitamins B12, C and E levels in hemodialyzed patients. METHODS: The authors investigated the influence of erythropoietin on the biochemical parameters mentioned above in 30 hemodialyzed patients during the period of 15 months. Patients were divided into two groups. Group A was formed by patients without erythropoietin treatment and group B was treated by subcutaneous administration of erythropoietin. Total weekly dose of erythropoietin was 50-100 U/kg of body weight. All patients received perorally pyridoxine 5 mg/day. B group of patients was supplemented by pyridoxine 20 mg/day and by folic acid 10 mg/day from the 12th to 15th month of the study. Erythrocyte vitamins B1, B2 and B6 values were determined by indirect enzymatic methods and were expressed as the effects of their coenzymes, thiamine pyrophosphate, flavine adenine dinucleotide and pyridoxal-5-phosphate in per cents. RESULTS: Long-term erythropoietin treatment led in the 12th month to a significant decrease of serum vitamin A and its protein carriers which were significantly increased in both groups of patients during the whole study. Erythrocyte vitamin B6 and folic acid significantly decreased due to erythropoietin treatment. Supplementary doses of pyridoxine and folic acid given perorally to the patients in B group during the last three months of our study influenced the deficiency of erythrocyte vitamin B6 and folic acid. CONCLUSIONS: Supplementations of pyridoxine in the dose of 20 mg/day and of folic acid 5 mg/week in hemodialyzed patients during erythropoietin treatment are necessary. (Tab. 5, Ref. 28.)

Anemia↗
PubMed2000

Thiamine status in liver and brain of rats genetically selected for different sensitivity to hypnotic effect of alcohol.

BACKGROUND: The mechanisms of the different sensitivity or resistance of animals and humans to alcohol are still not completely understood. For further biochemical characterization of animals genetically selected for high-alcohol sensitivity (HAS) and low-alcohol sensitivity (LAS) with the hypnotic effect of alcohol, the thiamine status and thiamine metabolizing enzymes in these animals have been studied. METHODS: We investigated thiamine diphosphate and thiamine triphosphate levels as well as the activity of thiamine-dependent enzyme, transketolase, and thiamine-metabolizing enzymes, thiamine kinase, and thiamine triphosphatase in the liver and brain of HAS, LAS, and CAS (control) rats by standard biochemical techniques. RESULTS: It was found that the activity of transketolase, and the level of the coenzyme form of thiamine, thiamine diphosphate (TDP), were significantly lower in HAS versus LAS rats. The activation of transketolase by the exogenous TDP (TDP-effect) was significantly higher in the liver and brain regions of HAS rats compared with LAS rats. The level of TDP in the liver and cerebellum of HAS rats was significantly lower compared with LAS rats. These results indicate a severe deficiency of TDP in HAS rats. HAS rats have a significantly lower activity of thiamine triphosphatase, the additional source of TDP. Accordingly, HAS rats have much higher thiamine triphosphate levels in the liver and brain, compared with LAS rats. There were no significant differences between groups with respect to the thiamine diphosphatase and thiamine kinase activity. Most of the above parameters had the intermediate values in CAS rats, compared with LAS and HAS rats. These data indicate the possible role of the thiamine phosphate esters and related enzymes in the mechanisms that bring about the differential sensitivity to the hypnotic effect of alcohol. CONCLUSIONS: HAS rats have the genetically mediated thiamine diphosphate deficiency and increased thiamine triphosphate levels, probably due to reduced activity of thiamine triphosphatase in the liver and brain, compared with LAS rats. It can be related with the higher initial sensitivity of HAS rats to hypnotic effect of ethanol.

Alcohol Drinking↗
PubMedDate not supplied

[Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis].

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.

Acetolactate Synthase↗
PubMedDate not supplied

[The role of transaminases in realizing the protective effect of L-aspartate in hypoxia].

Activation of aspartate aminotransferase and alanine aminotransferase of mitochondria introduced to the incubation medium of pyridoxal-5'-phosphate (40 microM) is approximately 2 times higher than that of the corresponding cytoplasmic forms. At hypoxia aspartate aminotransferase activity in mitochondria and postmitochondrial supernatant tends to an increase while that of alanine aminotransferase decreases (above 2 times). The protection from hypoxic damage when using L-aspartate (100 mg/kg subcutaneously 3-5 min before hypoxia) intensifies an adaptive increase of aspartate aminotransferase activity and removes a decrease of alanine aminotransferase activity. Under these conditions stimulating effect of pyridoxal-5'-phosphate on transaminases activity in vitro weakens. A simultaneous administration of vitamin-coenzyme complex (thiamine pyrophosphate, lipoate, sodium 4-phospho-pantothenate, flavin-mononucleotide, nicotinate) intensifies these metabolic shifts and protective action of L-aspartate.

Alanine Transaminase↗
PubMed2005

[Riboswitches].

Riboswitches are RNA structures able to bind small molecules and regulate gene expression at both, transcriptional and translational level. They are present in a wide variety of bacterial species. In Bacillus subtilis more than 2% of the genome is regulated by riboswitches. Metabolite binding is highly specific and can be provided in cooperative manner. Several riboswitches has been identified and characterized to be specific for flavin mononucleotide, thiamine, thiamine pyrophosphate, adenosylcobalamin, S-adenosylmethionine, lysine, glycine, adenine, guanine and glucosamine-6-phosphate. Some of them have been found also in fungi (Neurospora crassa) and plants (Oryza sativa, Arabidopsis thaliana). In human only one riboswitch with binding capacity for 2-aminopurine, has been found. Occurrence of riboswitches in all of the phylogenetic groups suggests that they are one of the oldest regulatory systems. It provokes also the question about their involvement in regulation of gene expression in human.

Animals↗
PubMed2006

[alpha-Oxidation of 3-methyl-branched fatty acids: unraveling of a pathway].

Peroxisomes have an important role in lipid metabolism e.g. beta-oxidation of long and very long chain fatty acids, 2-methyl-branched fatty acids, dicarboxylic fatty acids, prostanoids and bile acid intermediates, and synthesis of ether lipids. Also the process of alpha-oxidation of 3-methyl-branched fatty acids, with phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) as the best known example, occurs in peroxisomes. alpha-Oxidation is a process in which fatty acids are shortened by one carbon atom. The alpha-oxidation sequence of 3-methyl-branched fatty acids starts with an activation to the corresponding CoA-ester. Subsequently this acyl-CoA-ester undergoes a 2-hydroxylation by the peroxisomal phytanoyl-CoA hydroxylase (PAHX). In a third step the peroxisomal 2-hydroxyphytanoyl-CoA lyase (2-HPCL) splits the carbon carbon bond of the 2-hydroxy-intermediate into a 2-methyl(n-1)aldehyde and formyl-CoA, which is subsequently converted to formate and CO2. Finally the aldehyde is dehydrogenated by an aldehyde dehydrogenase to the corresponding acid, which, after its conversion to the acyl-CoA ester, can be a substrate for beta-oxidation. 2-HPCL is the first thiamine pyrophosphate dependent peroxisomal enzyme in mammals. Apart from 2-hydroxy-3-methylacyl-CoAs also 2-hydroxyacyl-CoAs are substrates for this enzyme. This indicates that the 2-hydroxy function but not the 3-methyl function of acyl-CoA esters is needed for 2-HPCL-activity. Long and very long chain 2-hydroxy fatty acids are constituents of brain cerebrosides and sulfatides, which mainly occur in myelin.

Acyl Coenzyme A↗
PubMedDate not supplied

[Metabolism of thiamine and its di- and triphosphorus esters in the blood of patients with neurological symptoms of osteochondrosis].

Concentrations of phosphate esters in blood of patients with vertebral osteochondrosis and activities of enzymes involved in thiamine metabolism have been investigated. The pathological processes were characterized by a decrease in content of thiamine triphosphate while concentration of coenzyme, thiamine diphosphate, was relatively constant. The enzymes involved in the initial and final steps of biosynthesis and degradation of thiamine phosphorylated derivatives play the main role in maintaining vitamin B1 metabolic homeostasis.

Adult↗
PubMed1976

Hypophosphatasia: a cytochemical study of phosphatase activities.

Skeletal abnormalities with defective formation of mature calcified bone are the most prominent clinical features of hypophosphatasia. Low concentrations of serum and tissue alkaline phosphatase and elevated plasma and urinary levels of phosphorylethanolamine (PEA) are also present. Although PEA is hydrolyzed by serum alkaline phosphatase, the relationship between PEA and the deficiency is unclear. PEA has not previously been tested as a cytochemical substrate for the in situ demonstration of human alkaline phosphatase activity. We have studied alkaline phosphatase activity in hypophosphatasia in tissue sections, utilizing PEA and adenosinetriphosphate (ATP) as well as the usual beta-glycerophosphate and naphthol phosphate substrates. Neutral and acid phosphatase activities were also examined. Our results demonstrate that PEA is a substrate for the localization of alkaline phosphatase in normal human tissue, but is not hydrolyzed in hypophosphatasia in the liver, brain or costochondral junction under alkaline conditions. In the kidney in hypophosphatasia only the straight segments of proximal tubules that rim the medullary rays are reactive with PEA. Similar results in hypophosphatasia were obtained at an alkaline pH with ATP, beta-glycerophosphate, and naphthol phosphate. However, the defect in hypophosphatasia is not a generalized deficiency of membrane-associated phosphatases because membranes that were deficient in alkaline phosphatase activity demonstrated normal reactivity with ATP at neutral pH. In addition, thiamine pyrophosphate was also split by Golgi membranes within the cytoplasm. Acid hydrolysis of beta-glycerophosphate by lysosomes was normal.

Alkaline Phosphatase↗
PubMedDate not supplied

[Functionally different states of the "hydrophobic pocket" of the myosin ATPase center].

The influence of an increased temperature (39 degrees C) on a denaturation of 50 kDa-fragment of myosin subfragment 1 was studied in the presence of different nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP). The degree of the denaturation was appreciated evaluated from its trypsinolysis depth. According to their protective influence NTP and NDP were shown to arrange in lines ATP greater than or equal to CTP greater than UTP greater than GTP and ADP greater than GDP greater than CDP greater than UDP, correspondingly. The results received and the literature data allow to suggest that there are at least two states of ATPase site hydrophobic pocket, one of which in responsible for sharp ATPase reaction slowing-down on the stage of macroergic bonding splitting.

Adenosine Diphosphate↗
PubMedDate not supplied

[Free amino acids of the liver and the characteristics of the amino acid metabolism in the liver and brain after cyanamide administration to rats].

A single administration to rats of cyanamide (60 mg/kg, for 1 hour) was found to decrease the contents of cysteate, serine, glutamate, glycine, alanine, valine, methionine, isoleucine, tyrosine, ethanolamine, ornithine and histidine that may be considered as a manifestation on the drug hepatotoxicity. The activities of transaminases, glutamate dehydrogenase, pyruvate dehydrogenase remained unchanged. Cyanamide effects were considerably abolished by the supplementary ethanol administration (0.5 g/kg). Cyanamide failed to affect vitamin-dependent enzymes reflecting thiamine pyrophosphate, pyridoxal phosphate and flavine adenine dinucleotide status of the rat organism.

Amino Acids↗
PubMed1990

Cross-linking of pyruvate decarboxylase. Characterization of the native and substrate-activated enzyme states.

In order to demonstrate the role of the protein component of pyruvate decarboxylase in the mechanism of substrate activation, we have isolated and characterized two states of the enzyme, the non-activated and the substrate-activated state, by covalent linking with bifunctional reagents. Because of the fact that modification of the reactive amino groups by 2,4,6-trinitobenzenesulfonic acid or methyl propionimidate influences neither the catalytic nor the regulatory properties of pyruvate decarboxylase, we used bisimidates of different chain length in the modification experiments. Both the non-activated and the substrate-activated enzyme states could be characterized separately. The lag phase of product formation as a typical property of the native enzyme disappeared completely when the enzyme had been cross-linked in the presence of the substrate. The permanently activated enzyme state shows 85% of the activity of native pyruvate decarboxylase and is exclusively stabilized by intra-subunit links. Elimination and subsequent reincorporation of the cofactors thiamine pyrophosphate and magnesium ions resulted in a complete regaining of the properties of the permanently activated enzyme form. An inactive enzyme form was obtained after cross-linking of non-activated pyruvate decarboxylase at low ionic strength (less than 0.01). Using a disulfide-containing linker we could prove that the inactivity of the obtained enzyme preparation was only the result of the incorporated cross-links and not that of denaturation.

Cross-Linking Reagents↗
PubMed1990

Characteristics of a novel transport system for folate compounds in wild-type and methotrexate-resistant L1210 cells.

The growth requirements and transport characteristics of folate compounds in a methotrexate-resistant L1210/R81 cell line were compared with parental cells. Concentrations for half-maximal growth of the resistant cells with folate (350 nM) and 5-formyltetrahydrofolate (20 nM) were found to be higher by 2.7-fold and 20-fold, respectively, relative to the parent, suggesting that changes had occurred in the transport of these folate compounds. Transport measurements revealed that the resistant cells have lost the capacity to transport methotrexate and other folate compounds via the reduced-folate transport system but that a second previously undescribed transport system is present. Uptake of folate via this second route is energy dependent, exhibits saturation kinetics, can be inhibited by substrate analogues, and is activated by a reduction in pH. The pH effect is substantial since uptake at 5.0 microM folate can be increased 10-fold by decreasing the pH from 7.4 to 6.2. The observed Kt for half-maximal influx of folate at pH 6.8 was 5.2 microM, and the Vmax was 0.55 pmol/min/mg of protein. Ki values for methotrexate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were 10.2, 3.2, and 2.7 microM, respectively. Transport activity was not affected by depleting internal stores of folate by growth in folate-free medium, and this same transport system was also present in comparable amounts in wild-type cells. In the latter case, interfering uptake via the reduced-folate transport system was blocked by bromosulfophthalein and thiamine pyrophosphate. Methotrexate and 5-formyltetrahydrofolate were also transported via this alternative, pH-dependent route, whereas little or no uptake of 5-methyltetrahydrofolate could be detected. The results indicate that antifolate resistance in L1210/R81 cells was induced by inactivating the reduced-folate transport system and by utilizating a secondary route which has a low capacity for transporting methotrexate but whose activity is sufficient to permit cell growth in medium supplemented with folate or 5-formyltetrahydrofolate.

Animals↗
PubMed1986

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis.

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

Cytosolic adenylate kinase catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate.

An attempt was made to purify a porcine skeletal muscle enzyme catalyzing the formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP), requiring ATP, Mg2+ and a cofactor (creatine). As the purification proceeded, the reaction requirements for ATP and creatine were lost and then a requirement for ADP was manifested. The activity responsible for TTP synthesis from TDP, ADP, and Mg2+ was found to be copurified with adenylate kinase [EC 2.7.4.3] activity, and was finally purified to a single band on SDS-PAGE. Antiserum obtained against the purified enzyme preparation inhibited both adenylate kinase activity and the TTP-synthesizing activity to exactly the same extent. These results indicate that adenylate kinase catalyzes TTP formation from TDP in vitro.

Adenylate Kinase↗