Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transketolase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1983

The catalytic mechanism of transketolase. Thiamin pyrophosphate-derived transition states for transketolase and pyruvate dehydrogenase are not identical.

Thiamin thiazolone pyrophosphate (TTPP) has been reported to be an effective transition state analogue for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem. 251, 2863-2866). The kinetics of the interaction of TTPP with transketolase are reported here. TTPP is a competitive inhibitor, with respect to thiamin pyrophosphate, of bakers' yeast transketolase but it is neither a tight binding inhibitor nor a slow binding inhibitor. TTPP decreases the kinetically observed negative cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation of the enzyme by thiamin pyrophosphate. We conclude that thiamin thiazolone pyrophosphate is not an effective transition state analogue for the reaction catalyzed by bakers' yeast transketolase. This difference between transketolase and pyruvate dehydrogenase may be related to differences in the polarity of the active sites of the enzymes. It is conceivable that the active sites of the pyruvate decarboxylase subunit of pyruvate dehydrogenase is hydrophobic, by analogy with the known hydrophobicity of the active site of brewers' yeast pyruvate decarboxylase. This hydrophobicity would stabilize a transition state with no charge on the thiazole portion of the coenzyme, similar to the "uncharged" thiazole portion of TTPP. In contrast, the active site of bakers' yeast transketolase, which is known to contain charged amino acid side chains, should be less favorable for such an uncharged transition state. A charge-separated canonical form related to TTPP could be preferentially stabilized in the active site of transketolase.

Kinetics↗
PubMed1987

A comparison of transketolase assay and transketolase and lactate dehydrogenase activity levels in whole blood and red cell hemolysates and in leukocytes.

1. A study was made of transketolase activity in red and white blood cells and of conditions for assay for transketolase activity and for assessment of the "TPP effect" in human and rat blood. 2. The ratio of the transketolase activity in white cells to that in red cells varied between 23 and 93. 3. Red cells or white cells can both be used for assessment of transketolase activity and the "TPP effect", but the best source for evaluation of transketolase activity and the percent change on addition of thiamin diphosphate appears to be whole blood.

Animals↗
PubMed1988

Isolation of transketolase from rabbit liver and comparison of some of its kinetic properties with transketolase from other sources.

1. Rabbit liver transketolase activity was purified 56-fold using the following steps: ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-25, concentration through an Amicon ultrafiltration cell and rechromatography on DEAE-Sephadex A-25. 2. The enzyme showed an optimum PH for activity at 7.8-8.0. 3. The optimum temperature was around 40 degrees C and the activation energy calculated from the Arrhenius plot was found to be 11.4 kcal/mole. 4. The molecular weight of the enzyme, as determined by gel filtration, was found to be approximately 162,000, while the content of thiamin diphosphate was between 1.8 and 2 mumole per mole protein. 5. Addition of thiamin diphosphate and magnesium chloride did not influence the activity. 6. From the kinetic studies of the enzyme, the Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 3.8 x 10(-5) M, 9.5 x 10(-5) M and 1.1 x 10(-2) M, respectively.

Animals↗
PubMed1985

Assessment of the thiamine nutritional status. An evaluation of erythrocyte transketolase activity, the stimulated erythrocyte transketolase activity, and the thiamine pyrophosphate effect.

The most widely accepted approach to estimation of thiamine nutrition has been the measurement of the erythrocyte transketolase activity (ETKA), the ETKA stimulated in vitro with thiamine pyrophosphate (TPP) (which is suggested to be designated ETKAS) and the percentage increase of ETKA after stimulation with TPP in vitro, called the thiamine pyrophosphate effect (TPPE). In spite of 30 years of experience in the determination of these variables doubtfulness still exists of how to assess them. On the basis of a study of a group of alcoholics and a group of non-alcoholics a system of evaluation of ETKA, ETKAS and TPPE is proposed.

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↗
PubMed1988

Western blotting assay of transketolase concentration in human hemolysates.

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.

Alcohol Drinking↗
PubMed1980

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical.

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.

Glycolates↗
PubMed1989

Transketolase from human leukocytes. Isolation, properties and induction of polyclonal antibodies.

Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a D-xylulose-5-phosphate/D-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 degrees C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and D-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa. The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6-8 distinct bands within a pI range of 6.5-8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification. The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.

Antigen-Antibody Reactions↗
PubMed1993

TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.

Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.

Amino Acid Sequence↗
PubMed1971

Relationship of nervous tissue transketolase to the neuropathy in chronic uremia.

Patients with chronic uremia develop neurologic defects which are similar to the demyelinating lesions seen in thiamine deficiency. The present study describes inhibitory effects of uremic material on nervous tissue transketolase, a thiamine-dependent enzyme of the pentose phosphate pathway which has been reported to have functional importance in the metabolism of myelinated nervous structures. Transketolase activity (TKA) of normal human brain and spinal cord was measured by the conversion of ribose-5-phosphate (R5P) to sedoheptulose-7-phosphate (S7P). TKA was significantly inhibited by plasma, cerebrospinal fluid and low molecular weight dialysate fractions obtained from patients with uremic neuropathy, but not by samples from normal subjects. The specific effect on transketolase by uremic material was established by showing suppressed formation of S7P from R5P also in the presence of excess cofactor thiamine pyrophosphate and of the other substrate xylulose-5-phosphate. Uremic plasma likewise inhibited a partially purified transketolase preparation from bakers' yeast.31 of 35 chronic uremic patients with inhibition values between 10 and 84% before or during the early phase of intermittent hemodialysis had evidence of neuropathy. Data of clinical grading of the neurologic deficits and values of motor nerve conduction velocity revealed a correlation between the extent of uremic neuropathy and the degree of nervous tissue transketolase inhibition. Hemodialysis markedly reduced the inhibitory effects of the patients' plasma and the data indicate that uremic patients who received effective long-term dialysis treatment show a parallel decline of transketolase inhibition and uremic neuropathy.The findings demonstrate that in patients with chronic renal failure, low molecular weight factors accumulate and inhibit nervous tissue transketolase. This biochemical defect-uncorrectable by thiamine but reversible by dialysis-may interfere with the metabolism of myelin-supporting cells, and/or of the axonal metabolism of medullated structures, and may thus contribute to the degeneration of myelinated nerves seen with uremic neuropathy.

Adolescent↗
PubMed1995

The transketolase gene family of the resurrection plant Craterostigma plantagineum: differential expression during the rehydration phase.

Transketolases, key enzymes of the reductive and oxidative pentose phosphate pathways, are responsible for the synthesis of sugar phosphate intermediates. Here we report the first molecular analysis of transketolase genes from plants. Three distinct classes of transketolase-encoding cDNA clones were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum. One class represented by the transcript tkt3 is constitutively expressed in leaves and roots under all physiological conditions tested. By biochemical analysis and protein sequencing of purified transketolase, it was shown that tkt3 is expressed in three enzymatically active isoforms. An intriguing discovery was that accumulation of the two other transketolase transcripts, tkt7 and tkt10, is preferentially associated with the rehydration process of the desiccated plant; whereas tkt10 is only expressed in leaves, tkt7 was detected in leaves and roots. This observation suggests a possible role for these transketolases in the conversion of sugars, which are a major phenomenon in the rehydration process. Despite an abundant level of tkt7 and tkt10 transcripts in rehydrating leaves, proteins could not be isolated. This is due in part to a translational control mechanism acting on the loading of mRNAs to polysomes.

Adaptation, Biological↗
PubMed1983

Variants of transketolase from human erythrocytes.

Analytical isoelectric focusing and a stain for transketolase have been applied to partially purified samples of human erythrocyte hemolysates and have detected individual species of transketolase having pI values of 6.6, 7.3, 7.5, 7.8, 8.1, 8.2, 8.4 and 9.2. Six different patterns of these species were detected in 25 healthy subjects. The species of pI 7.5, 7.8 and 8.1 were common to all six patterns. Species isolated by electrofocusing could be rerun in the same system with identical pI value. The addition of thiamin diphosphate to the staining mixture darkened some but not all bands of transketolase activity. Thus human erythrocyte transketolase is heterogeneous and appears to share with human fibroblast transketolase heterogeneity for affinity of the cofactor. This heterogeneity might need recognition when thiamin nutritional sufficiency is assessed by the 'thiamin diphosphate effect' on erythrocyte transketolase.

Erythrocytes↗
PubMed1989

Interactions with hemoglobin: a source of error in measurements of transketolase activity in hemolysates.

Measurements of the activity of transketolase in human erythrocyte lysates by an assay coupled to NADH oxidation indicate that interactions of assay substrates with hemoglobin can give rise to overestimations of transketolase activity. Three potential sources of error are identified. Thus, in lysates containing methemoglobin, NADH oxidation can be due firstly to methemoglobin reductase activity or secondly to the monooxygenase activity of methemoglobin, for which the substrate can be ribose 5-phosphate, a substrate also of transketolase. Thirdly, the addition of high concentrations of the transketolase cofactor, TDP, to an insufficiently buffered reaction mixture can cause the aggregation and precipitation of hemoglobin: a phenomenon that may be misconstrued as an enhanced increase in absorbance at 340 nm and hence as additional transketolase activity. Although the present study concentrates on these potential artefacts in assays of transketolase activity, the findings may well be relevant to the measurement of other enzyme activities in hemolysates by procedures based ultimately on the rate of consumption or production of NAD(P)H.

Chromatography, Gel↗
PubMed1981

The interrelation transketolase and dihydroxyacetone synthase activities in the methylotrophic yeast Candida boidinii.

Crude extracts of Candida boidinii grown on glucose, xylose or ethanol gave single peaks of classical transketolase activity following chromatography, on columns of hydroxylapatite; the enzyme was heat-stable and showed no appreciable activity with formaldehyde as acceptor in place of ribose 5-phosphate. Extracts of methanol-grown cells showed two peaks of transketolase activity following chromatography on both hydroxylapatite and DEAE-cellulose. One peak was identified with that found for the cells grown on substrates other than methanol; the other peak showed dihydroxyacetone synthase activity in addition to transketolase activity. Both activities in the latter peak were very unstable and have been ascribed to one enzyme on the basis of identical rates of denaturation at all temperatures tested between 0 and 40 degrees C. It is suggested that this enzyme is a special transketolase synthesized only during methylotrophic growth of the yeast and in contrast to classical transketolase, is capable of using equally well either formaldehyde or ribose 5-phosphate as glycolaldehyde acceptor. A method based on heat treatment has been suggested for the simultaneous assay of both transketolases present in crude extracts of a methylotrophically grown yeast.

Aldehyde-Ketone Transferases↗
PubMed1995

Transketolase A of Escherichia coli K12. Purification and properties of the enzyme from recombinant strains.

Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145,000 Da (+/- 10,000 Da), judged by gel-filtration chromatography. Subunits of 73,000 Da (+/- 2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30 degrees C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 microM and 1.4 mM, respectively. Km values for the other physiological substrates of transketolase A were 90 microM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate. Hydroxypyruvate served as alternative donor (Km = 18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). The enzyme was competitively inhibited by D-arabinose 5-phosphate (K = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations. The reconstitution of the apoenzyme proceded faster in the presence of manganese ions (Kd = 7 microM at 10 microM thiamine diphosphate) than with other divalent cations.

Escherichia coli↗
PubMed1981

Erythrocyte transketolase activity in alcoholic liver disease.

Erythrocyte transketolase activity and its stimulation in vitro by the addition of thiamine pyrophosphate (TPP effect) was measured in 64 normally nourished alcoholics with well-compensated liver disease and in 20 control subjects. Biochemical evidence of thiamine deficiency as judged by low transketolase activity was found in 19 alcoholics (29.7%). In 5 of these 19 patients the TPP effect was abnormally high, indicating depleted thiamine stores. IN the other 13 patients the TPP effect was either normal or low, suggesting a deficiency or an inability to use the transketolase apoenzyme, probably as a result o long-standing thiamine deficiency or the presence of liver disease. a further eight patients (12.5%) had normal transketolase activity but a low TPP effect, perhaps reflecting failure of hte coenzyme TPP to recombine with the transketolase apoenzyme in the presence of normal thiamine stores. There was no relationship between transketolase activity and the daily alcohol consumption, the duration of alcoholism, or the histological severity of the liver disease. Thiamine should be given routinely to alcoholics even if their diet appears adequate and their liver disease is minimal or well compensated.

Aged↗
PubMed1976

Behavior of transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1) Activities in normal, neoplastic, differentiating, and regenerating liver.

The objective of this investigation was to throw light on the biological behavior and metabolic regulation of hepatic enzymes of the nonoxidative branch of the pentose phosphate pathway. The activities of transaldolase (EC 2.2.1.2) and trasketolase (EC 2.2.1.1) Were compared in biological conditions that involve modulation of gene expression such as in starvation, in differentiation, after partial hepatectomy, and in a spectrum of hepatomas of different growth rates. The enzyme activities were determined under optimal kinetic conditions by spectrophotometric methods in the 100,000 X g supernatant fluids prepared from tissue homogenates. The kinetic properties of transaldolase and transketolase were similar in normal liver and in rapidly growing hepatoma 3924A. For transaldolase, apparent Km values of 0.13 mM (normal liver) and 0.17 mM (hepatoma) were observed for erythrose 4-phosphate and of 0.30 to 0.35 mM for fructose 6-phosphate. The pH optima in liver and hepatoma were at approximately 6.9 to 7.2. For the transketolase substrates, ribose 5-phosphate and xylulose 5-phosphate, the apparent Km values were 0.3 and 0.5 mM, respectively, in both liver and hepatoma. A broad pH optimum around 7.6 was observed in both tissues. In organ distribution studies, enzyme activities were measured in liver, intestinal mucosa, thymus, kidney, spleen, brain, adipose tissue, lung, heart, and skeletal muscle. Taking the specific activity of liver as 100%, transaldolase activity was the highest in intestinal mucosa (316%) and in thymus (219%); it was the lowest in heart (53%) and in skeletal muscle (21%). Transketolase activity was highest in kidney (155%) and lowest in heart (26%) and skeletal muscle (23%). Starvation decreased transaldolase and transketolase activities in 6 days to 69 and 74%, respectively, of those of the liver of the normal, fed rat. This was in the same range as the decrease in the protein concentration (66%y. In the liver tumors, transaldolase activity was increased 1.5- to 3.4-fold over the activities observed in normal control rat liver. Transketolase activity showed no relationship to tumor proliferation rate. In the regenerating liver at 24 hr after partial hepatectomy, the activity of both pentose phosphate pathway enzymes was in the same range as that of the sham-operated controls. In differentiation at the postnatal age of 5, 12, 23, and 32 days, hepatic transaldolase activities were 33, 44, 55, and 72%, respectively, of the activities observed in the 60-day-old, adult male rat. During the same period, transketolase activ-ties were 18, 21, 26, and 55% of the activities observed in liver of adult rat. The demonstration of increased transaldolase activity in hepatomas, irrespective of the degree of tumor malignancy, differentiation, or growth rate, suggests that the reprogramming of gene expression in malignant transformation is linked with an increase in the expression of this pentose phosphate pathway enzyme...

Animals↗
PubMed1990

The relationship between the thiamin pyrophosphate effect and the saturation status of the transketolase with its coenzyme in human erythrocytes.

The thiamin pyrophosphate effect has been used as a reliable index to evaluate the nutritional status of thiamin. But there has not been any report concerning whether or not the thiamin pyrophosphate effect really reflects the saturation status of transketolase with thiamin pyrophosphate. In this report we studied the relationship between the thiamin pyrophosphate effect and the saturation status of transketolase. First, we determined the thiamin pyrophosphate concentrations, transketolase activities, thiamin pyrophosphate effects, and transketolase concentrations in human hemolysates from 16 apparently healthy subjects. The molar ratio of thiamin pyrophosphate to transketolase was in inverse proportion to the thiamin pyrophosphate effect. Second, we prepared apotransketolase preparations and reconstituted it with various concentrations of thiamin pyrophosphate. The thiamin pyrophosphate effects in these preparations were in good correspondence with the ratios of apotransketolase. These results indicate that the thiamin pyrophosphate effect really reflects the saturation status of transketolase with coenzyme.

Adult↗