A simple and sensitive assay for ascorbate using a plate reader

Vislisel, Jesse M.; Schäfer, Freya; Buettner, Garry R.

doi:10.1016/j.ab.2007.03.002

Cited by 69 publications

(73 citation statements)

References 19 publications

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“…The final reaction volume was 100 l and the reaction temperature was 35°C unless otherwise stated. The concentration of the reaction substrate, DK-MTP-1-P, was calculated on the basis of the reaction characteristics between 2,3-butanedione and o-phenylenediamine (37,38). The concentration of the reaction product, 2-hydroxy-3-keto-5-methylthiopentenyl 1-phosphate was monitored spectrophotometrically at 280 nm (20,24,33,34).…”

Section: Expression and Purification Of Recombinant Rlps And Mutated mentioning

confidence: 99%

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

Saito

Ashida

Sakiyama

et al. 2009

Journal of Biological Chemistry

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The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO 2 fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg 2؉ for catalysis and, like RuBisCO, was stimulated by CO 2 . Four residues that are essential for the enolization reaction of RuBisCO, Lys 175 , Lys 201 , Asp 203 , and Glu 204 , were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys 123 , the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K i value of 103 M. RuBP and D-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) 4 catalyzes the carboxylation and oxygenation reactions of ribulose 1,5-bisphosphate (RuBP) in photosynthesis (1-4). This enzyme is the sole CO 2 -fixing enzyme in plants; however, it has certain inefficiencies. It has a very low turnover rate, a low affinity for the substrate, CO 2 , and low specificity between the carboxylation and oxygenation reactions (5-7). Thus, the intrinsic enzymatic properties of RuBisCO are inadequate for efficient incorporation of CO 2 into organic matter in photosynthesis (7). However, plants have overcome these disadvantages by investing a huge amount of leaf nitrogen in RuBisCO synthesis (8).In nature, there are wide variations in the properties and primary sequences of RuBisCO among different photosynthetic organisms (9 -12). The primary sequences vary as much as 73% without loss of activity. The relative specificity ranges from ϳ0.5 in a small subunitless RuBisCO to 238 in a red algal, hexadecameric RuBisCO (13,14). The affinity for CO 2 varies some 100-fold (15). Comparisons ...

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Section: Expression and Purification Of Recombinant Rlps And Mutated mentioning

confidence: 99%

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

Saito

Ashida

Sakiyama

et al. 2009

Journal of Biological Chemistry

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“…We have performed direct comparisons of the results obtained with the colorimetric ascorbate assay described above and the fluorometric ascorbate determination of Vislisel and colleagues 36 . We found that both assays gave identical results for the intracellular ascorbate determination assay (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…The molecular identities of the plasma membrane conduits involved in VSOAC formation remain to be identified 33,34 . Although many assays have been developed for the determination of ascorbate in biological samples, which include spectrophotometric, fluorometric and chromatographic assays 35,36 , there is much variability in specificity, sensitivity, interference by chemical contaminants, effective linear range and stability of the endpoint analyte. Additionally, other significant factors that influence the choice of assay are rapidity, ease of use and access to relatively specialized equipment such as a high-performance liquid chromatography (HPLC) apparatus.…”

Section: Introductionmentioning

confidence: 99%

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Lane

Lawen

2014

JoVE

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Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes -a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra-and extracellular ascorbate in cell culture. Video LinkThe video component of this article can be found at

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“…Blood samples were collected from the heart and urine samples from the bladder using syringes. Ascorbic acid content was measured by the fluorescent method detailed by Vislisela et al (18). The data are presented either as µmol/l for body fluids (blood plasma and urine) or as nmol/g wet tissue for tissue (heart and liver homogenates).…”

Section: Methodsmentioning

confidence: 99%

Nutrient supplementation modulates angiotensin II-mediated atherosclerosis in ApoE KO mice

Ivanov

Cha²,

Иванова³

et al. 2010

Mol Med Rep

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Abstract.We studied the effect of nutrient/herbal extract mixture (nM) supplementation prior to angiotensin ii (angii) administration on apoe Ko mice. angii-mediated injuries, which included increased atherosclerotic lesion size (by 31%) and severity (by 200%), as well as an increased number of necrotic cores (by 44%), were attenuated by nM supplementation, resulting in the reduction of lesion size by 20% (p=0.09), lesion severity by 31% (p=0.13) and the average number of necrotic cores per arterial tree by 39% (p=0.35), compared to mice fed the regular diet. The frequency of Type B thoracic aortic dissections and abdominal aneurysms in angii-treated mice was reduced from 55.6% to 14.3 and 42.9%, respectively, with nM supplementation. Macrophage density in the aortic lesions of nM-supplemented angii-treated mice was 20% lower (p=0.033) than in mice on the regular diet, whereas collagen density in nM mice was increased by 208% (p=0.026) compared to that in mice fed the regular diet, making for a more stable plaque type. nM-supplemented mice showed a significant reduction in total cholesterol (32% decrease, p=0.0001) and ldl (66% decrease, p<0.0001), commonly measured risk factors for cardiovascular disease. in conclusion, nM consumption prior to angii administration in ApoE KO mice minimized aortic dissections, inflammatory monocyte recruitment to plaques, plaque mass and abdominal aortic dilation.

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A simple and sensitive assay for ascorbate using a plate reader

Cited by 69 publications

References 19 publications

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Nutrient supplementation modulates angiotensin II-mediated atherosclerosis in ApoE KO mice

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