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G. Rindi - One of the best experts on this subject based on the ideXlab platform.
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Thiamine pyrophosphate dependent and Thiamine metabolizing enzymes in the deafferented cerebellum and in the intact cerebral cortex of rat
Metabolic Brain Disease, 1998Co-Authors: C. Patrini, Andrea Nauti, G. RindiAbstract:The effects of chemical (CD) and surgical (SD) deafferentation of the cerebellum on Thiamine pyrophosphate (ThPP)- dependent enzymes (transketolase, TK; pyruvate-, PDH, and α-ketoglutarate, αKGDH, dehydrogenases) and Thiamine (Th) metabolizing enzymes (Thiamine Pyrophosphokinase, ThPK; Thiamine mono-, ThMPase, and pyrophosphatases, ThPPase) were evaluated in vitro in rats in steady state conditions. The enzymes were also determined in the intact cerebral cortex of the same animals. CD was carried out by i.p. injection of 3-acetylpyridine, followed by harmaline and niacinamide. SD was carried out by complete dissection of the peduncles of the left cerebellar hemisphere. Chemical and surgical cerebellar deafferentations significantly lowered the contents of both Th-metabolizing and ThPP-dependent enzymes in the cerebellum without modifying those of the cerebral cortex. ThPK and TK, which are particularly concentrated in neurons, were the most affected enzymes. The decrease in ThPP-dependent enzymes shown here is associated with the slowing down of the Th phosphorylation-dephosphorylation cycle and with no modification in the ThPP content in the cerebellum, both of which were found in a previous study. Cerebellar deafferentation seems to only affect the apoenzyme moiety of the enzymes.
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Thiamine transport by erythrocytes and ghosts in Thiamine-responsive megaloblastic anaemia
Journal of Inherited Metabolic Disease, 1992Co-Authors: G. Rindi, V. Poggi, C. Patrini, B. Vizia, D. Casirola, U. LaforenzaAbstract:A 9-year study of Thiamine metabolism and cellular transport was performed in two patients with Thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of Thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of Thiamine and Thiamine compounds were reduced by about 40% as compared to controls. Thiamine Pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of Thiamine and Thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, < 2 µmol/L, physiological concentrations of Thiamine) and the non-saturable component of Thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from Thiamine-responsive megaloblastic anaemia patients contain low levels of Thiamine compounds, probably due to their inability to take up and retain physiological concentrations of Thiamine, as a result of the lack of the saturable, specific component of transport and reduced Thiamine Pyrophosphokinase.
U. Laforenza - One of the best experts on this subject based on the ideXlab platform.
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Thiamine transport by erythrocytes and ghosts in Thiamine-responsive megaloblastic anaemia
Journal of Inherited Metabolic Disease, 1992Co-Authors: G. Rindi, V. Poggi, C. Patrini, B. Vizia, D. Casirola, U. LaforenzaAbstract:A 9-year study of Thiamine metabolism and cellular transport was performed in two patients with Thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of Thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of Thiamine and Thiamine compounds were reduced by about 40% as compared to controls. Thiamine Pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of Thiamine and Thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, < 2 µmol/L, physiological concentrations of Thiamine) and the non-saturable component of Thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from Thiamine-responsive megaloblastic anaemia patients contain low levels of Thiamine compounds, probably due to their inability to take up and retain physiological concentrations of Thiamine, as a result of the lack of the saturable, specific component of transport and reduced Thiamine Pyrophosphokinase.
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Thiamine transport by erythrocytes and ghosts in Thiamine-responsive megaloblastic anaemia
Kluwer Academic Publishers:Journals Department PO Box 322 3300 AH Dordrecht Netherlands:011 31 78 6576050 EMAIL: frontoffice@wkap.nl kluweronline@wkap, 1992Co-Authors: Rindi G, Casirola D, De Vizia B, Patrini C, U. LaforenzaAbstract:A 9-year study of Thiamine metabolism and cellular transport was performed in two patients with Thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of Thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of Thiamine and Thiamine compounds were reduced by about 40% as compared to controls. Thiamine Pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of Thiamine and Thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, less than 2 mumol/L, physiological concentrations of Thiamine) and the non-saturable component of Thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from Thiamine-responsive megaloblastic anaemia patients contain low levels of Thiamine compounds, probably due to their inability to take up and retain physiological concentrations of Thiamine, as a result of the lack of the saturable, specific component of transport and reduced Thiamine Pyrophosphokinase
Maria Rapala-kozik - One of the best experts on this subject based on the ideXlab platform.
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Enzymes that control the Thiamine diphosphate pool in plant tissues. Properties of Thiamine Pyrophosphokinase and Thiamine-(di)phosphate phosphatase purified from Zea mays seedlings
Plant physiology and biochemistry : PPB, 2008Co-Authors: Maria Rapala-kozik, Anna Golda, Marta KujdaAbstract:Abstract The pool of Thiamine diphosphate (TDP), available for TDP-dependent enzymes involved in the major carbohydrate metabolic pathways, is controlled by two enzyme systems that act in the opposite directions. The Thiamine Pyrophosphokinase (TPK) activates Thiamine into TDP and the numerous phosphatases perform the reverse two-step dephosphorylation of TDP to Thiamine monophosphate (TMP) and then to free Thiamine. Properties and a possible cooperation of those enzymes in higher plants have not been extensively studied. In this work, we characterize highly purified preparations of TPK and a TDP/TMP phosphatase isolated from 6-day Zea mays seedlings. TPK was the 29-kDa monomeric protein, with the optimal activity at pH 9.0, the K m values of 12.4 μM and 4.7 mM for Thiamine and ATP, respectively, and the V max value of 360 pmol TDP min −1 mg −1 protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24 kDa subunits, showed the optimal activity at pH 5.0 and had a rather broad substrate specificity, although TDP, but not TMP, was one of the preferable substrates. The K m values for TDP and TMP were 36 μM and 49 μM, respectively, and the V max value for TDP was significantly higher than for TMP (164 versus 60 nmoles min −1 mg −1 protein). The total activities of TPK and TDP phosphatases were similarly decreased when the seedlings were grown under the illumination, suggesting a coordinated regulation of both enzymes to stabilize the pool of the essential coenzyme.
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Modulation of Thiamine metabolism in Zea mays seedlings under conditions of abiotic stress
Journal of experimental botany, 2008Co-Authors: Maria Rapala-kozik, Ewa Kowalska, Katarzyna OstrowskaAbstract:The responses of plants to abiotic stress involve the up-regulation of numerous metabolic pathways, including several major routes that engage Thiamine diphosphate (TDP)-dependent enzymes. This suggests that the metabolism of Thiamine (vitamin B1) and its phosphate esters in plants may be modulated under various stress conditions. In the present study, Zea mays seedlings were used as a model system to analyse for any relation between the plant response to abiotic stress and the properties of Thiamine biosynthesis and activation. Conditions of drought, high salt, and oxidative stress were induced by polyethylene glycol, sodium chloride, and hydrogen peroxide, respectively. The expected increases in the abscisic acid levels and in the activities of antioxidant enzymes including catalase, ascorbate peroxidase, and glutathione reductase were found under each stress condition. The total Thiamine compound content in the maize seedling leaves increased under each stress condition applied, with the strongest effects on these levels observed under the oxidative stress treatment. This increase was also found to be associated with changes in the relative distribution of free Thiamine, Thiamine monophosphate (TMP), and TDP. Surprisingly, the activity of the Thiamine synthesizing enzyme, TMP synthase, responded poorly to abiotic stress, in contrast to the significant enhancement found for the activities of the TDP synthesizing enzyme, Thiamine Pyrophosphokinase, and a number of the TDP/TMP phosphatases. Finally, a moderate increase in the activity of transketolase, one of the major TDP-dependent enzymes, was detectable under conditions of salt and oxidative stress. These findings suggest a role of Thiamine metabolism in the plant response to environmental stress.
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Thiamine binding and metabolism in germinating seeds of selected cereals and legumes.
Plant physiology and biochemistry : PPB, 2004Co-Authors: Anna Golda, Piotr Szyniarowski, Katarzyna Ostrowska, Andrzej Kozik, Maria Rapala-kozikAbstract:The basic characteristics of Thiamine metabolism in germinating seeds of maize (Zea mays), oat (Avena sativa), faba bean (Vicia faba) and garden pea (Pisum sativum) are presented with a special emphasis of a possible Thiamine storage function of seed Thiamine-binding proteins (TBPs). Seeds were germinated for 6 d in the dark. Thiamine-binding activity in seeds decreased during germination by 50% in cereals and by 30% in legumes. The degradation of TBPs was also detected by polyacrylamide gel electrophoresis. The total Thiamine content decreased rapidly to 20-40% of the initial value in cereal seeds during first 3 d of germination while in legume seeds Thiamine content started changing from the fourth day and dropped by 50% at the sixth day. A composite pattern was found for the changes in Thiamine pyrophosphate (TPP) contribution to total Thiamine during seed germination. A peak of the coenzyme percentage was usually detected at the second day of germination. Another gain of TPP was often seen toward the sixth day of germination. The activity of Thiamine Pyrophosphokinase (EC 2.7.6.2) was high in resting legume seeds and did not significantly change during germination. In contrast, the low activity of this Thiamine-activating enzyme in cereal seeds progressively increased during germination. Thiamine phosphate synthase (EC 2.5.1.3) was also detected in seeds and was shown to contribute significantly to the balance of Thiamine compounds during seed germination.
Carsten Wrenger - One of the best experts on this subject based on the ideXlab platform.
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Chemical and genetic validation of Thiamine utilization as an antimalarial drug target
Nature communications, 2013Co-Authors: Xie Wah Audrey Chan, Ingrid B. Müller, Bärbel Bergmann, Carsten Wrenger, Katharina Stahl, Markus Winterberg, Kevin J. SalibaAbstract:Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxyThiamine, a Thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a Thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of Thiamine Pyrophosphokinase (the enzyme that converts Thiamine into its active form, Thiamine pyrophosphate) hypersensitizes parasites to oxyThiamine by up to 1,700-fold, consistent with oxyThiamine being a substrate for Thiamine Pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the Thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxyThiamine, consistent with the antimetabolite inactivating Thiamine pyrophosphate-dependent enzymes. Our studies therefore validate Thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.
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SDS-PAGE- and western blot-analysis of the recombinant enzymes of the S. aureus vitamin B1 metabolism.
2013Co-Authors: Ingrid B. Müller, Bärbel Bergmann, Leonard Amaral, Isabel Couto, Matthew R Groves, Rolf D Walter, Tadhg P. Begley, Carsten WrengerAbstract:(A) SDS-PAGE of the affinity chromatography purified recombinant enzymes and (B) corresponding western blot analysis using a monoclonal anti-Strep antibody at a dilution of 1∶5,000 according to material and methods. GTPase; TPK, Thiamine Pyrophosphokinase; TenA, thiaminase II; ThiM, THZ kinase; ThiD, HMP kinase; ThiE, TMP synthase. The arrow indicates the SaThiE protein.
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The vitamin B1 metabolism of Staphylococcus aureus is controlled at enzymatic and transcriptional levels
PLoS ONE, 2009Co-Authors: Ingrid B. Müller, Bärbel Bergmann, Leonard Amaral, Isabel Couto, Matthew R Groves, Rolf D Walter, Tadhg P. Begley, Carsten WrengerAbstract:Vitamin B1 is in its active form Thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and Thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the Thiamine Pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels.
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The human malaria parasite Plasmodium falciparum expresses an atypical N-terminally extended Pyrophosphokinase with specificity for Thiamine
Biological chemistry, 2006Co-Authors: Marie-luise Eschbach, Ingrid B. Müller, Rolf D Walter, Tim-wolf Gilberger, Carsten WrengerAbstract:Vitamin B(1) is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well as Plasmodium falciparum, are capable of synthesising vitamin B(1)de novo, whereas mammals have to take up this cofactor from their diet. Thiamine, a B(1) vitamer, has to be pyrophosphorylated by Thiamine Pyrophosphokinase (TPK) to the active form. The human malaria parasite P. falciparum expresses an N-terminally extended Pyrophosphokinase throughout the entire erythrocytic life cycle, which was analysed by Northern and Western blotting. The recombinant enzyme shows a specific activity of 27 nmol min(-1) mg(-1) protein and specificity for Thiamine with a K(m) value of 73 microM, while Thiamine monophosphate is not accepted. Mutational analysis of the N-terminal extension of the plasmodial TPK showed that it influences Thiamine binding as well as metal dependence, which suggests N-terminal participation in the conformation of the active site. Protein sequences of various plasmodial TPKs were analysed for their phylogeny, which classified the Plasmodium TPKs to a group distinct from the mammalian TPKs. To verify the location of the parasite TPK within the cell, immunofluorescence analyses were performed. Co-staining of PfTPK with a GFP marker visualised its cytosolic localisation.
C. Patrini - One of the best experts on this subject based on the ideXlab platform.
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Thiamine pyrophosphate dependent and Thiamine metabolizing enzymes in the deafferented cerebellum and in the intact cerebral cortex of rat
Metabolic Brain Disease, 1998Co-Authors: C. Patrini, Andrea Nauti, G. RindiAbstract:The effects of chemical (CD) and surgical (SD) deafferentation of the cerebellum on Thiamine pyrophosphate (ThPP)- dependent enzymes (transketolase, TK; pyruvate-, PDH, and α-ketoglutarate, αKGDH, dehydrogenases) and Thiamine (Th) metabolizing enzymes (Thiamine Pyrophosphokinase, ThPK; Thiamine mono-, ThMPase, and pyrophosphatases, ThPPase) were evaluated in vitro in rats in steady state conditions. The enzymes were also determined in the intact cerebral cortex of the same animals. CD was carried out by i.p. injection of 3-acetylpyridine, followed by harmaline and niacinamide. SD was carried out by complete dissection of the peduncles of the left cerebellar hemisphere. Chemical and surgical cerebellar deafferentations significantly lowered the contents of both Th-metabolizing and ThPP-dependent enzymes in the cerebellum without modifying those of the cerebral cortex. ThPK and TK, which are particularly concentrated in neurons, were the most affected enzymes. The decrease in ThPP-dependent enzymes shown here is associated with the slowing down of the Th phosphorylation-dephosphorylation cycle and with no modification in the ThPP content in the cerebellum, both of which were found in a previous study. Cerebellar deafferentation seems to only affect the apoenzyme moiety of the enzymes.
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Thiamine transport by erythrocytes and ghosts in Thiamine-responsive megaloblastic anaemia
Journal of Inherited Metabolic Disease, 1992Co-Authors: G. Rindi, V. Poggi, C. Patrini, B. Vizia, D. Casirola, U. LaforenzaAbstract:A 9-year study of Thiamine metabolism and cellular transport was performed in two patients with Thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of Thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of Thiamine and Thiamine compounds were reduced by about 40% as compared to controls. Thiamine Pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of Thiamine and Thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, < 2 µmol/L, physiological concentrations of Thiamine) and the non-saturable component of Thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from Thiamine-responsive megaloblastic anaemia patients contain low levels of Thiamine compounds, probably due to their inability to take up and retain physiological concentrations of Thiamine, as a result of the lack of the saturable, specific component of transport and reduced Thiamine Pyrophosphokinase.