3 phosphoglycerate

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3-phosphoglycerate is a chemical compound that is an intermediate in the metabolic pathway of glycolysis. It plays a crucial role in the conversion of 1,3-bisphosphoglycerate to 2-phosphoglycerate during the energy-producing process of glycolysis. 3-phosphoglycerate is an important substrate for various enzymatic reactions in cellular metabolism.

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3-phosphoglycerate kinase (3 citations)

7 protocols using 3 phosphoglycerate

Metabolic Enzyme Assay Reagents

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Nicotine amide adenine dinucleotide (NAD) disodium salt, 3-phosphoglycerate [D-(–)-3-phosphoglyceric acid disodium salt], l-homoserine, d-homoserine, l-homocysteine, 2-oxoglutarate (α-ketoglutaric acid sodium salt), oxaloacetate, cis-aconitate, molecular weight standards (Gel Filtration Markers Kit for Protein Molecular Weights) for size exclusion chromatography analyses, and Murashige–Skoog (MS) vitamin solution were purchased from Sigma–Aldrich Co., Ltd. (St. Louis, MO, USA). The other amino acids, the salt mixture of MS medium, Good’s buffers, NADH (β-diphosphopyridine nucleotide disodium salt, reduced form), fumarate, malate (l-malate), citrate (citric acid monohydrate), and isocitrate [trisodium ( ± )-isocitrate n-hydrate] were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Okamura E, & Hirai M.Y. (2017). Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana. Scientific Reports, 7, 3533.

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Anti-inflammatory Protocols and Reagents

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LPS (Escherichia coli LPS 0111:B4; #L4391), 2DG (#D8375), TSA (#8552), 3-phosphoglycerate (#P8877), 2-phosphoglycerate (#19710), and phosphoenolpyruvate (#860077) were obtained from Sigma (St. Louis, MO, USA). Shikonin (#CAS 517-89-5) was obtained from EMD Millipore Corporation (Billerica, MA, USA). Mouse TNF-α (#410-MT-010) and IFN-γ (485-MI-100) were obtained from R&D Systems (Minneapolis, MN, USA). Mouse anti-HMGB1 2G7 neutralizing antibody was a generous gift from Kevin J. Tracey (The Feinstein Institute for Medical Research, Manhasset, NY, USA)^{32 (link)}.

Yang L., Xie M., Yang M., Yu Y., Zhu S., Hou W., Kang R., Lotze M., Billiar T.R., Wang H., Cao L, & Tang D. (2014). PKM2 Regulates the Warburg Effect and Promotes HMGB1 Release in Sepsis. Nature communications, 5, 4436.

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PHGDH Inhibition Assay Protocol

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PHGDH inhibition assay was performed in black polypropylene 96-well
plates (Sigma-Aldrich) using NAD (Sigma-Aldrich) as cofactor and
3-phosphoglycerate (Sigma-Aldrich) as substrate. Tested compounds were dissolved
in DMSO. The final concentration of DMSO in assay solutions was 10%,
which was shown to have no effect on PHGDH activity. The reaction volume per
well was 50 μL. Each assay was carried out in
triplicate. Briefly, a solution buffer (100 mM Tris HCl buffer (pH 8.8), 400 mM
NaCl, 0.2 mM DTT, 1 mM NAD, and 0.5 mM 3-PG) was added at various concentrations
of tested compounds. Then, an amount of 100 ng of PHGDH (human recombinant
protein, BPS Bioscience) is added to start the reaction. Finally, the product
formation, correlated with NADH formation, is instantly monitored for 7 min at
25 °C using a SpectraMax spectrophotometer at an excitation wavelength
of 360 nm and emission wavelength of 460 nm.

Ravez S., Corbet C., Spillier Q., Dutu A., Robin A.D., Mullarky E., Cantley L.C., Feron O, & Frédérick R. (2017). α-Ketothioamide Derivatives: A Promising Tool to Interrogate Phosphoglycerate Dehydrogenase (PHGDH). Journal of medicinal chemistry, 60(4), 1591-1597.

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Enzyme Assay Protocol with Detailed Reagents

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Ammonium chloride, ADP, DTT, EDTA, GTP, glutamate, glutamine, 2-OG, 3-phosphoglycerate, l-leucine, NAD⁺, NADP⁺, NADPH, NADH, Triton X-100, MgCl2, MOPS, HEPES, Tris, imidazole, acrylamide, GDH (no. G2626), rabbit muscle phosphorylase kinase (no. P2014), and agarose were from Sigma Chem. Co. (St. Louis, MO, USA). Anti-GDH1/2 (no. sc-160383); anti-α-tubulin (no. sc-5286); antiacetylated lysine (no. ab190479); anti-P-Ser (Q5phospho-ser; Qiagen no.37430); and anti-P-Thr (Q7phospho-Thr; Qiagen no.37420) were purchased from Santa Cruz (sc) Biotechnology (Santa Cruz, CA, USA), Abcam (ab) (Cambridge, MA, USA), or Qiagen (Venlo, the Netherlands). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, no. 105686) and HK (no. 11426362001) were from Roche (Mannheim, Germany). Recombinant phosphoglycerate kinase (PGK) was from Entamoeba histolytica (84 (link)).

Moreno-Sánchez R., Marín-Hernández Á., Gallardo-Pérez J.C., Pacheco-Velázquez S.C., Robledo-Cadena D.X., Padilla-Flores J.A., Saavedra E, & Rodríguez-Enríquez S. (2020). Physiological Role of Glutamate Dehydrogenase in Cancer Cells. Frontiers in Oncology, 10, 429.

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Investigating Glycolytic Metabolites

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D-fructose (Product No. F0127), glyceraldehyde-3-phosphate (Product No. G5251), 3-phosphoglycerate (Product No. P8877), 2-phosphoglycerate (Product No. 79480), phosphoenol pyruvate (Product No. P7002), acetate (Product No. S2889) and pyruvate (Product No. P2256) are from Sigma-Aldrich. Glucose-6-phosphate (Product No. SC-221489A) is from Santa Cruz Biotechnology. Glycerol (Product No. 3783.2) is from Carl Roth. All chemicals were tested for their purity to exclude contamination by glucose.

Bi S., Kargeti M., Colin R., Farke N., Link H, & Sourjik V. (2023). Dynamic fluctuations in a bacterial metabolic network. Nature Communications, 14, 2173.

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PGDH Activity Assay Protocol

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PGDH activity assay was carried out as described previously [13 (link)] with a modification. Briefly, PGDH activity was measured by measuring an increase in absorbance at 339 nm in the presence of NAD (Nakalai Tesque) and 3-phosphoglycerate (3PG; Sigma-Aldrich, USA). The assays were performed in triplicate and contained the reaction mixture with 100 mM Tris–HCl buffer (P. aeruginosa PGDH: pH 7.5, E. coli PGDH: pH 8.5, human PGDH: pH 9.0) containing 5 mM EDTA (Wako, Japan), 1 mM DTT (Wako), 10 mM hydrazine (Wako) and 2 mg/mL NAD. The reaction was started with 15 mM 3-phosphoglycerate and the formation of NADH was measured spectrophotometrically at 339 nm. The enzyme activity (unit per mg protein) was calculated by following the enzyme unit reported previously [14 , 15 (link)].

Okuda J., Nagata S., Yasuda M, & Suezawa C. (2019). Validating the inhibitory effects of d- and l-serine on the enzyme activity of d-3-phosphoglycerate dehydrogenases that are purified from Pseudomonas aeruginosa, Escherichia coli and human colon. Gut Pathogens, 11, 35.

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Anti-inflammatory Protocols and Reagents

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