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Results for “Transketolase”

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At least 19 recordsLinked to original sources

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↗

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↗

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↗

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↗

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↗

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↗

PubMed1988

Studies of transketolase abnormality in Alzheimer's disease.

The partially purified transketolase from each of eight well-nourished patients with Alzheimer's disease contained significantly less heat-stable component with a significantly longer half-life of heat inactivation than that from eight controls. Immunochemical studies utilizing antibodies to the purified human liver transketolase did not distinguish between red blood cell transketolases of patients with Alzheimer's disease and those of controls. However, three brains from patients with Alzheimer's disease that were deficient in transketolase activity lacked a 69-kilodalton form on immunoblots. Subtle structural abnormalities of transketolase appear to occur in a high proportion of patients with Alzheimer's disease.

Alzheimer Disease↗

PubMed1992

DNA sequence of the yeast transketolase gene.

Transketolase (EC 2.2.1.1) is the enzyme that, together with aldolase, forms a reversible link between the glycolytic and pentose phosphate pathways. We have cloned and sequenced the transketolase gene from yeast (Saccharomyces cerevisiae). This is the first transketolase gene of the pentose phosphate shunt to be sequenced from any source. The molecular mass of the proposed translated protein is 73,976 daltons, in good agreement with the observed molecular mass of about 75,000 daltons. The 5'-nontranslated region of the gene is similar to other yeast genes. There is no evidence of 5'-splice junctions or branch points in the sequence. The 3'-nontranslated region contains the polyadenylation signal (AATAAA), 80 base pairs downstream from the termination codon. A high degree of homology is found between yeast transketolase and dihydroxyacetone synthase (formaldehyde transketolase) from the yeast Hansenula polymorpha. The overall sequence identity between these two proteins is 37%, with four regions of much greater similarity. The regions from amino acid residues 98-131, 157-182, 410-433, and 474-489 have sequence identities of 74%, 66%, 83%, and 82%, respectively. One of these regions (157-182) includes a possible thiamin pyrophosphate (TPP) binding domain, and another (410-433) may contain the catalytic domain.

Aldehyde-Ketone Transferases↗

PubMed1992

High control coefficient of transketolase in the nonoxidative pentose phosphate pathway of human erythrocytes: NMR, antibody, and computer simulation studies.

The degree of control exerted by transketolase over metabolite flux in the nonoxidative pentose phosphate pathway in human erythrocytes was investigated using transketolase antiserum to modulate the activity of that enzyme. 31P NMR enabled the simultaneous measurement of the levels of pentose phosphate pathway metabolites following incubation of hemolysates with ribose 5-phosphate. The variations in metabolic flux which occurred as the transketolase activity of hemolysate samples was altered indicated that a high degree of control was exerted by transketolase. Investigations using transaldolase-depleted hemolysates showed that transaldolase exhibits a lesser degree of control over pathway flux. Experimental data were compared with simulations generated by a computer model encompassing the reactions of the classical nonoxidative pentose phosphate pathway. The sensitivity coefficients (also called "control strengths" or "flux-control coefficients") calculated from the computer simulations were 0.74 and 0.03 for transketolase and transaldolase, respectively.

Blotting, Western↗

PubMed1991

Effect of chronic alcohol administration on transketolase in the brain and the liver of rats.

To estimate the nutritional and the pathological states in thiamin-deficiency-related diseases, especially Wernicke-Korsakoff syndrome, we studied the relationship among transketolase activity, transketolase concentration, and thiamin phosphate esters in rats chronically fed alcohol. In the brain of alcohol-fed rats, the enzyme activity and concentration decreased although there was no positive correlation between the two. On the contrary, transketolase activity in the liver correlated positively with concentration, and both transketolase activity and concentration were decreased in the thiamin-deficient groups. These findings suggest that transketolase in the brain may be different from that in the liver and that the alteration of the enzyme activity in the brain may be based on the conformational change of the protein molecule caused by chronic alcohol administration.

Animals↗

PubMedDate not supplied

[Transketolase activity in rabbit myocardium and erythrocytes in health and in allergic heart diseases].

Transketolase activity was determined in myocardium and erythrocytes of rabbits by following formation of D-sedoheptuloso-7-phosphate (S-7-P), when the convertion of the substance was blocked in a transaldolase reaction by addition of 3-phosphoglycerol aldehyde dehydrogenase. In the transaldolase reaction consumption of the S-7-P did not exceed 5% of the amount of this compound, produced in the transketolase reaction. In allergic impairment alterations in the transketolase activity occurred simultaneously with a change in the level of plastic metabolism in myocardium. The transketolase activity was decreased when destructive processes were stimulated. But the transketolase activity was distinctly increased under the conditions, characterized by activation of protein biosynthesis.

Animals↗

PubMed1977

Effect of ethanol administration on thiamine metabolism and transketolase activity in rats.

To clarify the effect of ethanol on thiamine metabolism and activity of transketolase, a thiamine dependent enzyme, in vivo and in vitro experiments were carried out. For the in vivo experiment, rats were separated into four groups: Group 1, thiamine sufficient diet with no ethanol administered; group 2, thiamine sufficient diet and ethanol administered by stomach tube; group 3, thiamine deficient diet with no ethanol administered; group 4, thiamine deficient diet and ethanol administered. After three weeks, the rats were killed, then thiamine levels and the activity of transketolase were assayed. Thiamine concentration in brains and livers of ethanol fed rats were lower than that in control rats. Urinary thiamine was higher in ethanol administered rats than in controls. The increase in the liver transketolase activity by the in vitro addition of cocarboxylase (TPP effect) was higher in group 3 than in group 4 although original transketolase activity was lower in group 4. For the in vitro experiment, various amounts of ethanol or acetaldehyde were added to the supernatant of brain and liver of intact rats, and transketolase activity was determined. Inhibition by acetaldehyde to transketolas was about 10 times higher than that by ethanol.

Acetaldehyde↗

PubMedDate not supplied

[The transketolase activity in erythrocytes as a criterion for thiamine balance in the body].

In deficiency of thiamin the transketolase activity was decreased in patients and in experimental animals. The data obtained showed the high sensitivity of transketolase from erythrocytes to hypovitaminosis B1. At the same time, in diseases, especially accompained by hypoxia (and also by avitaminosis B1), the transketolase activity was shown to be increased. The effect was observed in relation to an increase in amount of young erythrocytes, which contained more transketolase. In these cases it was reasonable to estimate not only the transketolase activity but also other characteristics of thiamin metabolism (content of the vitamin in food, urine and blood).

Animals↗

PubMed1986

Effect of ring substituents on the transketolase-catalyzed conversion of nitroso aromatics to hydroxamic acids.

Transketolase catalyzed the conversion of eight different aromatic C-nitroso compounds into the corresponding N-glycolyl derived hydroxamic acids. Three of the nitroso compounds were also found to be converted in part to the arylhydroxylamines by a reductive process. A correlation was found for the rates of production of these metabolites with the electronegativities of substituent groups that were present on the aromatic ring. The rates of reaction of these substituted nitroso substrates with transketolase and D-fructose-6-phosphate were found to decrease in the order 4-NO2 much greater than 4-CF3 greater than 3-CF3, unsubstituted greater than 4-Cl greater than 4-CH3, 4-phenyl greater than 4-OC2H5. N,N-Dimethyl-p-nitrosoaniline was not metabolized by transketolase under the conditions employed for the other substrates. Those substrates possessing the strong electron-withdrawing groups 4-NO2, 4-CF3 and 3-CF3 were the only substrates that were found to undergo enzymatic reduction to the hydroxylamines as a competing process. A mechanism was proposed that involves a redox reaction between the nitroso substrate and the enzymatic intermediate "active glycolaldehyde" at the active-site of transketolase.

Animals↗

PubMed1986

Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells.

Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.

Amino Acids↗