An improved automated assay of serum glutamic-oxalacetic transaminase

Lombarts, A.J.P.F.; Peters, Helmut

doi:10.1016/0009-8981(71)90191-4

Cited by 4 publications

(5 citation statements)

References 17 publications

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“…Conversely, Fast Red KL and Fast Ponceau L were most stable over a pH range of 4.2-4.6 [26,28,45,46] . Additionally, Fast Violet B has a broad absorption peak between 490 and 540 nm compared to the narrower absorption peaks of Fast Red KL and Fast Ponceau L (455-467 nm respectively), enabling greater flexibility in the assay parameters [26,28] . The red-shifted absorbance wavelength of FVB also reduces the likelihood of interference with small molecule effectors.…”

Section: Discussionmentioning

confidence: 94%

“…While diazonium salts have previously been used to detect the production of OAA, they have not specifically been applied to assay PC activity. Diazonium salts such as Fast Red KL, Fast Ponceau L and Fast Violet B have been used to detect OAA production from aspartate transaminase [26,28,45,46] . Fast Violet B, in particular, has also been utilized to assess phosphoenolpyruvate carboxylase activity and has previously been optimized in a microtiter plate assay [27,29] .…”

Section: Discussionmentioning

confidence: 99%

“…The choice of Fast Violet B over other diazonium salts in this assay is based on several factors. Fast Violet B is stable over a pH range of 7.0-7.5 and tolerates the addition of both EDTA and detergent [27,28,45,47] . Conversely, Fast Red KL and Fast Ponceau L were most stable over a pH range of 4.2-4.6 [26,28,45,46] .…”

Section: Discussionmentioning

confidence: 99%

“…Fast Violet B is stable over a pH range of 7.0-7.5 and tolerates the addition of both EDTA and detergent [27,28,45,47] . Conversely, Fast Red KL and Fast Ponceau L were most stable over a pH range of 4.2-4.6 [26,28,45,46] . Additionally, Fast Violet B has a broad absorption peak between 490 and 540 nm compared to the narrower absorption peaks of Fast Red KL and Fast Ponceau L (455-467 nm respectively), enabling greater flexibility in the assay parameters [26,28] .…”

Section: Discussionmentioning

confidence: 99%

“…1). Diazonium salts have previously been used to detect OAA formation in other enzymatic systems such as phosphoenolpyruvate carboxylase and aspartate transaminase [[26], [27], [28], [29], [30]] , but FVB has never been applied to determine PC activity. The assay readout is stable for an extended time period and accurately reports on enzyme activity from a single data point.…”

Section: Introductionmentioning

confidence: 99%

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A high-throughput screening assay for pyruvate carboxylase

Wyatt

Arnold

Maurice

2018

Analytical Biochemistry

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Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A high-throughput screening assay for pyruvate carboxylase

Wyatt

Arnold

Maurice

2018

Analytical Biochemistry

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Analysis of complexes of metabolites with europium tetracycline using capillary electrophoresis coupled with laser-induced luminescence detection

Craig

Hiebert

2017

Biometals

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Glycolysis and Krebs cycle intermediates were incubated with Eu-tetracycline and separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. 3-phopshoglycerate, phosphoenolpyruvate, adenosine diphosphate, phosphate, citrate and oxaloacetate were detected at a concentration of 100 μM or lower. When all these detected metabolites were contained within the same sample it was found that they interfered with one another. Of all the metabolites, oxaloacetate showed the highest detectability. The system was found to yield a linear response for oxaloacetate from 50 nM to 10 μM. The injected volume of sample was 400 pL. This corresponds to 2 × 10 mol of injected oxaloacetate from the 50 nM sample. As an application, the system was used to assay the enzyme aspartate aminotransferase, for whom oxaloacetate is a product. After a 1 h incubation period, 1.2 × 10 M (3.3 μU/mL) enzyme was sufficient to form a detectable product signal. Extension of this incubation to 18 h permitted the detection of the activity of 1.2 × 10 M (330 nU/mL) enzyme. This is the equivalent of 4.8 ymoles (2.9 molecules) of enzyme in the 400 pL injection volume. The enzyme's catalytic rate was determined to be 240 s under the conditions used. In a second application, homogenates of Drosophila melanogaster were analyzed for metabolites, providing several peaks, including one which had the same retention time as citrate.

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1 Clinical Enzymology

Goldberg¹

1976

Progress in Medicinal Chemistry

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An improved automated assay of serum glutamic-oxalacetic transaminase

Cited by 4 publications

References 17 publications

A high-throughput screening assay for pyruvate carboxylase

A high-throughput screening assay for pyruvate carboxylase

Analysis of complexes of metabolites with europium tetracycline using capillary electrophoresis coupled with laser-induced luminescence detection

1 Clinical Enzymology

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