scholarly journals 5,10-Methylenetetrahydrofolate Reductase (MTHFR) Assay in the Forward Direction: Residual Activity in MTHFR Deficiency

2002 ◽  
Vol 48 (6) ◽  
pp. 835-843 ◽  
Author(s):  
Terttu Suormala ◽  
Gertraud Gamse ◽  
Brian Fowler
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Residual Activity ◽  
Forward Direction ◽  
Reverse Direction ◽  
Control Activity ◽  
Reliable Determination ◽  
Protein Assay ◽  
Control Value ◽  
Mthfr Deficiency

Abstract Background: Assay of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in homocysteine metabolism, is important for the study of severe and mild deficiency states. Because the conventional assay measures in the reverse direction, lacks sensitivity, and uses nonphysiologic substrates, the exact measurement and characterization of residual activity in easily accessible tissues have been difficult. Methods: To measure MTHFR in the physiologic direction, we determined the NADPH-dependent conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate by use of HPLC with fluorescence detection. Results: MTHFR activity in control fibroblast in the presence of FAD was maximal between pH 6.3 and 6.9, increased linearly up to 40 min and 80 μg protein/assay, and showed Kms of 30 μmol/L for NADPH and 26 μmol/L for 5,10-methylenetetrahydrofolate. Intraassay variation (CV) was 10%, interassay variation was 7.2%, and variation among 10 subcultures of the same cell line was 18%. Mean (SD) control activity was 431 (150) μU/mg protein (range, 242–910; n = 75), which is 2.5-fold higher than that with the reverse assay. After heat treatment (46 °C for 5 min), the activity showed a trimodal distribution corresponding to the 677TT (thermolabile; 15%), 677CT (35%), and 677CC (51%) genotypes. We found clearly measurable activity ranging from 2.6% to 25.6% of the mean control value in 15 patients with MTHFR deficiency, including 11 cell lines with zero activity in the reverse assay. Ten patients had complete enzyme deficiency. Conclusion: This assay allows reliable determination of residual activity in mutant fibroblasts and characterization of kinetic parameters for natural substrates.

scholarly journals Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency

2015 ◽  
Vol 39 (1) ◽  
pp. 115-124 ◽  
Author(s):  
Martina Huemer ◽  
Regina Mulder-Bleile ◽  
Patricie Burda ◽  
D. Sean Froese ◽  
Terttu Suormala ◽  
...  
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Residual Activity ◽  
Clinical Pattern ◽  
Mthfr Deficiency

scholarly journals Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase

2021 ◽  
Vol 22 (8) ◽  
pp. 4231
Author(s):  
Giulia Murtas ◽  
Giorgia Letizia Marcone ◽  
Alessio Peracchi ◽  
Erika Zangelmi ◽  
Loredano Pollegioni
Keyword(s):  
Cancer Progression ◽  
Forward Direction ◽  
Biochemical Properties ◽  
Reverse Direction ◽  
Phosphate Binding ◽  
Biophysical Characterization ◽  
E Coli ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Metabolism

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

Immobilization and Characterization of Tannase and its Haze-removing

2009 ◽  
Vol 15 (6) ◽  
pp. 545-552 ◽  
Author(s):  
Erzheng Su ◽  
Tao Xia ◽  
Liping Gao ◽  
Qianying Dai ◽  
Zhengzhu Zhang
Keyword(s):  
Kinetic Parameter ◽  
High Activity ◽  
Green Tea ◽  
Storage Stability ◽  
Residual Activity ◽  
Black Tea ◽  
Optimum Temperature ◽  
Oolong Tea ◽  
Optimum Ph

Tannase was effectively immobilized on alginate by the method of crosslinking-entrapment-crosslinking with a high activity recovery of 76.6%. The properties of immobilized tannase were investigated. Its optimum temperature was determined to be 35 ° C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH of 5.0 did not change. The thermal and pH stabilities of immobilized tannase increased to some degree. The kinetic parameter, Km, for immobilized tannase was estimated to be 11.6 × 10-4 mol/L. Fe2+ and Mn2+ could activate the activity of immobilized tannase. The immobilized tannase was also applied to treat the tea beverage to investigate its haze-removing effect. The content of non-estern catechins in green tea, black tea and oolong tea increased by 52.17%, 12.94% and 8.83%, respectively. The content of estern catechins in green tea, oolong tea and black tea decreased by 20.0%, 16.68% and 5.04%, respectively. The anti-sediment effect of green tea infusion treated with immobilized tannase was significantly increased. The storage stability and reusability of the immobilized tannase were improved greatly, with 72.5% activity retention after stored for 42 days and 86.9% residual activity after repeatedly used for 30 times.

scholarly journals Brain-wide mapping of water flow perception in zebrafish

2020 ◽  
Author(s):  
Gilles Vanwalleghem ◽  
Kevin Schuster ◽  
Michael A. Taylor ◽  
Itia A. Favre-Bulle ◽  
Ethan K. Scott
Keyword(s):  
Water Flow ◽  
Lateral Line ◽  
Reverse Flow ◽  
Forward Direction ◽  
Brain Regions ◽  
Reverse Direction ◽  
Departure Point ◽  
Forward Flow ◽  
Larval Zebrafish ◽  
The Brain

AbstractInformation about water flow, detected by lateral line organs, is critical to the behavior and survival of fish and amphibians. While certain specific aspects of water flow processing have been revealed through electrophysiology, we lack a comprehensive description of the neurons that respond to water flow and the network that they form. Here, we use brain-wide calcium imaging in combination with microfluidic stimulation to map out, at cellular resolution, all neurons involved in perceiving and processing water flow information in larval zebrafish. We find a diverse array of neurons responding to forward flow, reverse flow, or both. Early in this pathway, in the lateral line ganglia, these are almost exclusively neurons responding to the simple presence of forward or reverse flow, but later processing includes neurons responding specifically to flow onset, representing the accumulated volume of flow during a stimulus, or encoding the speed of the flow. The neurons reporting on these more nuanced details are located across numerous brain regions, including some not previously implicated in water flow processing. A graph theory-based analysis of the brain-wide water flow network shows that a majority of this processing is dedicated to forward flow detection, and this is reinforced by our finding that details like flow velocity and the total volume of accumulated flow are only encoded for the simulated forward direction. The results represent the first brain-wide description of processing for this important modality, and provide a departure point for more detailed studies of the flow of information through this network.Significance statementIn aquatic animals, the lateral line is important for detecting water flow stimuli, but the brain networks that interpret this information remain mysterious. Here, we have imaged the activity of individual neurons across the entire brains of larval zebrafish, revealing all response types and their brain locations as water flow processing occurs. We find some neurons that respond to the simple presence of water flow, and others that are attuned to the flow’s direction, speed, duration, or the accumulated volume of water that has passed during the stimulus. With this information, we modeled the underlying network, describing a system that is nuanced in its processing of water flow simulating forward motion but rudimentary in processing flow in the reverse direction.

Characterization of six novel mutations in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with homocystinuria

2000 ◽  
Vol 15 (3) ◽  
pp. 280-287 ◽  
Author(s):  
Sahar Sibani ◽  
Benedicte Christensen ◽  
Erin O'Ferrall ◽  
Irfan Saadi ◽  
Fran�ois Hiou-Tim ◽  
...  
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Mthfr Gene ◽  
Novel Mutations

Cloning and characterization of a thermostable glutathione reductase from a psychrophilic Arctic bacterium Sphingomonas sp.

2019 ◽  
Author(s):  
Hai VuThi ◽  
Sei-Heon Jang ◽  
ChangWoo Lee
Keyword(s):  
Thermal Stability ◽  
Glutathione Reductase ◽  
Redox Homeostasis ◽  
Residual Activity ◽  
Reducing Power ◽  
Composition Analysis ◽  
Psychrophilic Bacterium ◽  
Tertiary Structures

Abstract Glutathione reductase is an important oxidoreductase that helps maintain redox homeostasis by catalyzing the conversion of glutathione disulfide to glutathione using NADPH as a cofactor. In this study, we cloned and characterized a glutathione reductase (referred hereafter to as SpGR) from Sphingomonas sp. PAMC 26621, an Arctic bacterium. SpGR comprises 449 amino acids, and functions as a dimer. Surprisingly, SpGR exhibits characteristics of thermophilic enzymes, showing optimum activity at 60°C and thermal stability up to 70 °C with approximately 50% residual activity at 70 °C for 2 h. The amino acid composition analysis of SpGR showed a 1.9-fold higher Arg content (6%) and a 2.7-fold lower Lys/Arg ratio (0.75) compared to the Arg content (3.15%) and the Lys/Arg ratio (2.01) of known psychrophilic glutathione reductases. SpGR also exhibits its activity at 4°C, and circular dichroism and fluorescence spectroscopy results indicate that SpGR maintains its secondary and tertiary structures within the temperature range 4–70°C. Taken together, the results of this study indicate that despite its origin from a psychrophilic bacterium, SpGR has high thermal stability. Our study provides an insight into the role of glutathione reductase in maintaining the reducing power of an Arctic bacterium in a broad range of temperatures.

Tyrosine Biosynthesis in Sorghum bicolor: Characteristics of Prephenate Aminotransferase

1986 ◽  
Vol 41 (1-2) ◽  
pp. 79-86 ◽  
Author(s):  
Daniel L. Siehl ◽  
James A. Connelly ◽  
Eric E. Conn
Keyword(s):  
Reaction Rate ◽  
Deae Cellulose ◽  
Forward Direction ◽  
Reverse Direction ◽  
Aminotransferase Activity ◽  
Forward Reaction ◽  
High Affinity ◽  
Sorghum Plant ◽  
Arogenate Dehydrogenase ◽  
Tyrosine Biosynthesis

Abstract A stable activity which transfers the amino group from glutamate to prephenate was extracted from 4-day old etiolated shoots of sorghum. The activity was retained on DEAE cellulose and eluted as a single peak. Prephenate aminotransferase co-eluted with a very abundant α-ketoglutarate: aspartate aminotransferase, but heating at 70 °C resulted in loss of α-ketoglutarate: aspar­tate activity with nearly full retention of prephenate: glutamate aminotransferase activity. The heated enzyme displayed high affinity and specificity for prephenate. Among 7 donors tested, only glutamate, and aspartate at less than 20% the rate with glutamate, supported prephenate aminotransferase activity. In the reverse direction, a reaction rate comparable to that in the forward direction was unchanged as the concentration of α-ketoglutarate was reduced from 1.0 to 0.09 mᴍ. The apparent Km for arogenate was 0.8 mᴍ. The forward reaction was unaffected by the inclusion of tyrosine, phenylalanine or tryptophan. Together with the discovery of arogenate dehydrogenase in sorghum [3], these data indicate that, in the sorghum plant, tyrosine derives from prephenate by transamination and aromatization. rather than the reverse sequence.

scholarly journals Folate Insufficiency Due to MTHFR Deficiency Is Bypassed by 5-Methyltetrahydrofolate

2020 ◽  
Vol 9 (9) ◽  
pp. 2836
Author(s):  
Maša Vidmar Golja ◽  
Alenka Šmid ◽  
Nataša Karas Kuželički ◽  
Jurij Trontelj ◽  
Ksenija Geršak ◽  
...  
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Fold Increase ◽  
Mthfr Gene ◽  
Folate Supplementation ◽  
Disease States ◽  
Metabolic Defects ◽  
Key Enzyme ◽  
Cell Processes ◽  
Activity Groups ◽  
Mthfr Deficiency

Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs (n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells’ metabolic activity and intracellular 5-Me-THF levels, we conclude supplementation of FA is sufficient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.

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