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PubMed1980

Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination.

Thiamin pyrophosphotransferase activity was present in seedling extracts from several monocot and dicot species of agronomic as well as noncultivated plants. Changes in thiamin pyrophosphotransferase activity and thiamin pyrophosphate content were followed for 6 days in soybean (Merr.) seedlings. Maximum enzyme activity occurred 48 to 96 hours from imbibition. Thiamin pyrophosphate content peaked sharply at 36 hours and was preceded by increased thiamin pyrophosphotransferase activity. Addition of pyrithiamin, an inhibitor of in vitro thiamin pyrophosphotransferase activity, to the imbibition medium at various times inhibited subsequent fresh weight gains of soybean seedlings. These results indicated that, although not among the earliest phosphorylation events after initiation of water imbibition by soybean seeds, a substantial increase in thiamin pyrophosphate content did precede the onset of rapid seedling growth and development. Since both enzyme activity and thiamin appear to be available in unimbibed soybean seeds, ATP or other nucleoside triphosphate concentration may represent an important factor in modulating thiamin phosphorylation during early seedling development.

Journal Article↗
PubMed1977

The transketolase assay of thiamine in some diseases.

Erythrocyte transketolase activity (ETKA) and the effect of adding thiamine pyrophosphate have been measured in a group of 27 healthy individuals and in 37 patients diagnosed as having diabetes mellitus, anemia, polyneuritis, or malnourishment secondary to vascular disease of the brain. The observed values for the malnourished group did not differ significantly from those for the control group. The low ETKA values in diabetes mellitus seem to be due to a reduced apoenzyme level resulting from the disease itself rather than thiamine deficiency. Polyneuritis patients had low values of ETKA. In the anemic group as a whole the values showed a difference of only marginal significance from those found in the control group, but the patients with pernicious anemia all had a highly significant elevation of the ETKA values. Although the absolute thiamine pyrophosphate effect differ, there are no significant differences in percentage of thiamine pyrophosphate effect between the groups. It appears that differences in the patient groups studied here reflect variations in apoenzyme levels rather than thiamine status.

Anemia, Macrocytic↗
PubMed1984

Escherichia coli alpha-ketoglutarate dehydrogenase complex.

The alpha-ketoglutarate dehydrogenase complex from Escherichia coli catalyzes the hydrolysis of S-succinyl-CoA to succinate and CoASH. The reaction rate is dependent upon the presence of thiamin pyrophosphate and NADH, as well as the functional integrity of the alpha-lipoyl groups associated with the enzyme. The Km value for S-succinyl-CoA is 9.3 X 10(-5) M, and the maximum velocity is 0.02 mumol X min-1 X mg of protein-1 at pH 7 and 25 degrees C. This hydrolysis can be rationalized on the basis that succinyl thiamin pyrophosphate is generated under reductive succinylation conditions. Occasional diversion of succinyl thiamin pyrophosphate to hydrolysis produces succinate.

Acyl Coenzyme A↗
PubMed1985

Pyruvate Decarboxylase from Zea mays L. : 2. Examination of Hysteretic Kinetics.

A significant lag phase was observed in the accumulation of product for the reaction catalyzed by pyruvate decarboxylase (PDC) purified from mature maize kernels. The effects of pH, pyruvate, potassium chloride, PDC concentration, and Mg(2+)-thiamine pyrophosphate upon this lag and upon the observed cooperativity were investigated. PDC preincubated with Mg(2+)-thiamine pyrophosphate for six days had Michaelis-Menten kinetics, a Hill number of 1, and no apparent lag phase. The degree of saturation of PDC with Mg(2+)-thiamine pyrophosphate appears to have a central role in controlling the lag phase and the degree of cooperativity.

Journal Article↗
PubMed1995

Improved measurement of transketolase activity in the assessment of "TPP effect".

The indirect estimation of thiamine levels in human blood by measuring thiamine pyrophosphate effect on erythrocyte transketolase activity is the method of choice in most clinical laboratories. We describe here an optimized, time-saving, and accurate method to determine the thiamine pyrophosphate effect in as many as 16 blood samples simultaneously. The method is based on a multi-point determination using a computer remote-controlled microplate reader. For multiple sample handling, three pooled reaction mixtures are freshly prepared and loaded onto a 96 well microtitre plate. A pre-written software is then initiated to remote-control the system. The data is retrieved and processed to calculate thiamine pyrophosphate effect by a self-written "macro" on a "Quattro-Pro" worksheet database. This method proves to be highly accurate (coefficient of variance: 2.7%), reproducible (coefficient of variance: 4.1%) and economical.

Analysis of Variance↗
PubMed1977

Thiamine pyrophosphokinase activity in liver, heart and brain crude extracts of control and thiamine deficient rats.

The activity of thiamine pyrophosphokinase has been measured in liver, heart muscle and brain of normal and thiamine deficient rats. The activity measurement has been performed by use of thiamine-35S as substrate and separation of the reaction products by high voltage electrophoresis. KM has been determined as 7.1.10(-6) mol/l. The activity of thiamine pyrophosphokinase is reduced in liver and heart muscle of thiamine deficient rats significantly, whereas no decrease of the enzyme activity has been found in the brain. The content of thiamine pyrophosphate has been measured in the liver of normal and thiamine deficient rats. Injection of thiamine to deficient rats normalized the content of thiamine pyrophosphate in the liver within 6 hours. Our data suggest that thiamine pyrophosphokinase is an adaptive enzyme, the activity of which depends on the thiamine content of the cells.

Animals↗
PubMed1995

Quantitative determination of dihydroxyethylthiamine pyrophosphate by the circular dichroism method.

This work is concerned with a convenient and fast method for quantitative determination of dihydroxyethylthiamine pyrophosphate in solution where it is present as the only component or as a mixture with thiamine pyrophosphate (or other optically inactive compounds). The molar coefficient is given for circular dichroism absorption of dihydroxyethylthiamine pyrophosphate. The method for determining thiamine pyrophosphate in the presence of dihydroxyethylthiamine pyrophosphate is described as well.

Anion Exchange Resins↗
PubMed1977

Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids.

The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s(20,w) of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD(+) and pyruvate; NADP(+) and other keto acids give no reaction. Mg(2+), thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria. Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg(2+), cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations.

Journal Article↗
PubMed1997

Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases.

Five mitochondrial protein kinases, all members of a new family of protein kinases, have now been identified, cloned, expressed as recombinant proteins, and partially characterized with respect to catalytic and regulatory properties. Four members of this unique family of eukaryotic protein kinases correspond to pyruvate dehydrogenase kinase isozymes which regulate the activity of the pyruvate dehydrogenase complex, an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member of this family corresponds to the branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, the most important regulatory enzyme in the pathway for the disposal of branched-chain amino acids. At least three long-term control mechanisms have evolved to conserve branched chain amino acids for protein synthesis during periods of dietary protein insufficiency. Increased expression of the branched-chain alpha-ketoacid dehydrogenase kinase is perhaps the most important because this leads to phosphorylation and nearly complete inactivation of the liver branched-chain alpha-ketoacid dehydrogenase complex. Decreased amounts of the liver branched-chain alpha-ketoacid dehydrogenase complex secondary to a decrease in liver mitochondria also decrease the liver's capacity for branched-chain keto acid oxidation. Finally, the number of E1 subunits of the branched-chain alpha-ketoacid dehydrogenase complex is reduced to less than a full complement of 12 heterotetramers per complex in the liver of protein-starved rats. Since the E1 component is rate-limiting for activity and also the component of the complex inhibited by phosphorylation, this decrease in number further limits overall enzyme activity and makes the complex more sensitive to regulation by phosphorylation in this nutritional state. The branched-chain alpha-ketoacid dehydrogenase kinase phosphorylates serine 293 of the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Site-directed mutagenesis of amino acid residues surrounding serine 293 reveals that arginine 288, histidine 292 and aspartate 296 are critical to dehydrogenase activity, that histidine 292 is critical to binding the coenzyme thiamine pyrophosphate, and that serine 293 exists at or in close proximity to the active site of the dehydrogenase. Alanine scanning mutagenesis of residues in the immediate vicinity of the phosphorylation site (serine 293) indicates that only arginine 288 is required for recognition of serine 293 as a phosphorylation site by the branched-chain alpha-ketoacid dehydrogenase kinase. Phosphorylation appears to inhibit dehydrogenase activity by introducing a negative charge directly into the active site pocket of the E1 dehydrogenase component of the branched-chain alpha-ketoacid dehydrogenase complex. A model based on the X-ray crystal structure of transketolase is being used to predict residues involved in thiamine pyrophosphate binding and to help visualize how phosphorylation within the channel leading to the reactive carbon of thiamine pyrophosphate inhibits catalytic activity. The isoenzymes of pyruvate dehydrogenase kinase differ greatly in terms of their specific activities, kinetic parameters and regulatory properties. Chemically-induced diabetes in the rat induces significant changes in the pyruvate dehydrogenase kinase isoenzyme 2 in liver. Preliminary findings suggest hormonal control of the activity state of the pyruvate dehydrogenase complex may involves tissue specific induced changes in expression of the pyruvate dehydrogenase kinase isoenzymes.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Transketolase abnormality in cultured fibroblasts from familial chronic alcoholic men and their male offspring.

We have investigated a thiamine-dependent enzyme, transketolase, in cultured fibroblasts from 41 human subjects, including patients with alcoholism-associated Wernicke-Korsakoff syndrome (n = 3), familial chronic alcoholic males (n = 7), their sons (n = 7), nonalcoholic men (n = 7), their male offspring (n = 7), and three generations of an Amish family (n = 10) without any history of alcoholism. This study was undertaken to delineate whether transketolase abnormality (i.e., high Michaelis Menton constant (Km) for thiamine pyrophosphate), previously reported in patients with Wernicke-Korsakoff syndrome is prevalent among familial chronic alcoholic men and their sons without prior history of alcohol abuse but who are at high risk for alcoholism. Our data suggest that an inborn error (i.e., high Km of transketolase for thiamine pyrophosphate) predisposing to thiamine deficiency diseases similar to those reported in Wernicke-Korsakoff syndrome may occur in the general population. However, for some as yet unexplained reason(s) this variant seems to occur more frequently among familial chronic alcoholic men and their male offspring without any history of alcoholism. The inheritance pattern of this enzyme variant as revealed from an Amish pedigree study may be autosomal recessive as previously suggested.

Adolescent↗
PubMed1977

Abnormality of a thiamine-requiring enzyme in patients with Wernicke-Korsakoff syndrome.

We studied a thiamine-requiring enzyme in cultured cells from four patients with the Wernicke-Korsakoff syndrome to determine whether these patients have a genetic predilection to thiamine deficiency. Transketolase in fibroblasts from the patients with the syndrome bound thiamine pyrophosphate less avidly than control lines. The apparent Km for thiamine pyrophosphate was 195 +/- 31 micron for transketolase in extracts of the patients' cells as compared to 16 +/- 2 micron in six control lines (means +/- S.E.M.: P less than 0.001). The ranges were 146 to 281 micron for the patients and 12 to 20 micron for the controls. The abnormality in transketolase persisted through serial passages in tissue culture in cells grown in medium containing excess thiamine and no ethanol, indicating that the aberrations were genetic rather than dietary. The abnormality of transketolase in this syndrome would presumably be clinically unimportant if the diet was adequate. These patients appear to have deleterious inborn enzymatic abnormalities of a type originally postulated by Garrod.

Alcohol Amnestic Disorder↗
PubMed1987

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation.

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.

Adenosine Diphosphate↗
PubMed1992

Thiamin contents of cerebrospinal fluid, plasma and erythrocytes in cerebellar ataxias.

Free thiamin and thiamin monophosphate have been found in the cerebrospinal fluid, plasma and in erythrocytes of patients suffering from ataxia of different origins. In erythrocytes, thiamin pyrophosphate was also measured. In a limited number of cases, uptake of 14C-thiamin by erythrocytes was found as well. Controls were hospitalized patients affected by chronic neurological diseases without any clinical sign of thiamin deficiency. The results showed a significant decrease in thiamin and thiamin monophosphate in the cerebrospinal fluid and in the plasma of ataxic subjects, in comparison to controls. In erythrocytes, only thiamin pyrophosphate levels had decreased. The uptake of 14C-thiamin by erythrocytes was similar in both ataxic and control groups. These results were comparable to those observed in thiamin-deficient individuals, like alcoholic patients, and prompted further investigation into thiamin metabolism in these diseases.

Adult↗
PubMed1995

[Some properties of multiple forms of transketolase from baker's yeast].

It has been shown that transketolase A does not differ from the enzyme earlier described in the literature by a number of properties (lack of intersubunit disulfide bonds, identical number of sulfhydryl groups, two values of Km for thiamine pyrophosphate and identity of their absolute values). Transketolase C subunits are linked together by disulfide bonds; their total number in the enzyme molecule is five (transketolase C-1) or six (transketolase C-2). The Km values of transketolase C-1 for thiamine pyrophosphate are commensurate with those of transketolase A. Transketolase C-2 has only one Km value for thiamine pyrophosphate which is close to one of the two Km values for transketolase A. The maximal rate values for transketolases C-1 and C-2 with dihydroxyacetone as substrate differ by more than one order of magnitude.

Disulfides↗