2 oxo 1 14c glutarate

Manufactured by PerkinElmer

2-oxo[1-14C]glutarate is a radiochemical compound used as a tracer in various biochemical and metabolic studies. It consists of a glutarate molecule with a radioactive carbon-14 atom at the 1-position. This compound can be utilized to monitor and analyze metabolic pathways and processes involving the tricarboxylic acid (TCA) cycle.

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Lab products found in correlation

Tri carb 2900tr, by PerkinElmer (1 mentions) Anti actin, by Merck Group (1 mentions) Goat anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions)

2 protocols using 2 oxo 1 14c glutarate

Enzymatic Activity Characterization of C-P4H-II

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Two assays were used to characterize the enzymatic properties of C-P4H-II and its truncated variants. First, the catalytic activity of the purified enzymes was measured by an indirect activity assay based on the hydroxylation-coupled decarboxylation of 2-oxo[1-¹⁴C]glutarate (PerkinElmer) (56 ). About 100 μM (PPG)₁₀ peptide was used as a substrate. In addition, the activities were measured with a direct assay (56 ), based on measurement of the 4-hydroxy[¹⁴C]proline formation from [¹⁴C]proline-labeled procollagen substrate, consisting of nonhydroxylated α-chain of chicken type-I procollagen. The radioactivities were determined using a Tri-Carb 2900TR (PerkinElmer) liquid scintillation counter. The results were calculated as disintegrations per minute per enzyme active site, taking into account that the number of C-P4H active sites is two per mature C-P4H-II tetramer and one per heterodimer for the truncated variants.

Murthy A.V., Sulu R., Lebedev A., Salo A.M., Korhonen K., Venkatesan R., Tu H., Bergmann U., Jänis J., Laitaoja M., Ruddock L.W., Myllyharju J., Koski M.K, & Wierenga R.K. (2022). Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer. The Journal of Biological Chemistry, 298(12), 102614.

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Purification and Enzymatic Assay of PHD2

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Flag-tagged PHD2 was expressed in insect cells and affinity-purified using anti-Flag, and the His-tagged HIF-2α oxygen-dependent degradation domain (ODDD) protein was expressed in E. coli and affinity-purified using NiNTA as described previously. 36, 33 A PHD2 enzymatic activity assay was performed to measure the radioactive CO 2 produced during the decarboxylation of 2-oxo[1-14 C]glutarate (Perkin-Elmer), which co-occurs with the substrate proline hydroxylation. 37 cycloheximide chase assay HEK cells were transfected with plasmids encoding pcD-NA3-HA-PHD2 (100-800 ng) in addition to pCMV-HA-empty vector for a total amount of 800 ng of transfected DNA. Twenty-four hours after transfection, cells were treated with cycloheximide (Sigma-Aldrich) at a final concentration of 100 μg/mL. Cells were harvested at different timepoints. An equal volume of protein lysates was analyzed by western blot assay using a mouse anti-HA antibody, anti-actin (Sigma) and a goat anti-mouse secondary antibody (Jackson Immunoresearch).

Delamare M., Le Roy A., Pacault M., Schmitt L., Garrec C., Maaziz N., Myllykoski M., Rimbert A., Karaghiannis V., Aral B., Catherwood M., Airaud F., Mansour-Hendili L., Hoogewijs D., Peroni E., Idriss S., Lesieur V., Caillaud A., Si-Tayeb K., Chariau C., Gaignerie A., Rab M., Haferlach T., Meggendorfer M., Bézieau S., Benetti A., Casadevall N., Hirsch P., Rose C., Wemeau M., Galacteros F., Cassinat B., Bellosillo B., Bento C., Van Wijk R., Petrides P.E., Randi M.L., McMullin M.F., Koivunen P., Girodon F, & Gardie B. (2023). Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis. Haematologica, 108(11).

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