Pyruvate kinase lactic dehydrogenase

Manufactured by Merck Group

Sourced in United States

Pyruvate kinase/lactic dehydrogenase is a laboratory equipment used to measure the levels of pyruvate kinase and lactic dehydrogenase enzymes in biological samples. It plays a core role in the assessment of cellular metabolism and energy production pathways.

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Lab products found in correlation

Appliskan, by Thermo Fisher Scientific (1 mentions) Supernuclease, by Sino Biological (1 mentions) Oligodeoxynucleotides, by Sangon (1 mentions) Phosphocreatine, by Merck Group (1 mentions) Leupeptin hydrochloride, by Merck Group (1 mentions) Creatine phosphokinase, by Merck Group (1 mentions) Pyrrole, by Merck Group (1 mentions) Phosphoenolpyruvate, by Merck Group (1 mentions) Na2atp, by Merck Group (1 mentions) 3h ryanodine, by PerkinElmer (1 mentions) Mgatp, by Merck Group (1 mentions) Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)

4 protocols using pyruvate kinase lactic dehydrogenase

Quantifying ATP Hydrolysis by RNA Helicases

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The ATP hydrolysis activity of proteins was determined using an indirect NADH-coupled photometric assay as described in (41 (link)). Reactions were carried out in a buffer containing 45 mM Tris–HCl pH 7.4, 25 mM NaCl, 2 mM MgCl₂, 1.5 mM phosphoenolpyruvate, 300 μM NADH, 20 U/ml pyruvate kinase/lactic dehydrogenase (Sigma-Aldrich) and 4 mM ATP supplemented with 250 nM RNA helicase (MBP-DHX15-His₁₀ or MBP-DHX15_E261Q-His₁₀) or the above buffer as a negative control. The reactions were further supplemented with 2 μM polyU₃₂ RNA substrate and/or 1.5 μM of ZZ-GPATCH4_GP-His₇ domain where indicated. The decrease in absorbance of NADH was monitored at 340 nm every 50 sec in a Thermo Fisher Scientific Appliskan multimode microplate reader and the ATPase rate was calculated using
Kpath is the molar absorption coefficient for NADH for a given optical length, which is experimentally determined to account for background NADH decomposition.

Kanwal N., Krogh N., Memet I., Lemus-Diaz N., Thomé C.C., Welp L.M., Mizi A., Hackert P., Papantonis A., Urlaub H., Nielsen H., Bohnsack K.E, & Bohnsack M.T. (2023). GPATCH4 regulates rRNA and snRNA 2′-O-methylation in both DHX15-dependent and DHX15-independent manners. Nucleic Acids Research, 52(4), 1953-1974.

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Measuring ClpC1 ATPase Activity

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2 μg of pure ClpC1 were mixed with 100 µl of the assay buffer (50 mM TrisHCl pH 7.8; 100 mM KCl; 10% glycerol; 1 mM phosphoenolpyruvate; 1 mM NADH; 2 µl pyruvate kinase/lactic dehydrogenase (Sigma); 4 mM MgCl₂ and 1 mM ATP) and the ATPase activity of ClpC1 was followed by measuring the coupled oxidization of NADH to NAD spectrometrically at 340 nm.

Gavrish E., Sit C.S., Cao S., Kandror O., Spoering A., Peoples A., Ling L., Fetterman A., Hughes D., Bissell A., Torrey H., Akopian T., Mueller A., Epstein S., Goldberg A., Clardy J, & Lewis K. (2014). Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2. Chemistry & biology, 21(4), 509-518.

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Purification and Characterization of RecA Proteins

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The oligodeoxynucleotides were synthesized and purified by Shanghai Sangon Biological Engineering Technology & Services Co, Ltd (Shanghai, China). ATP, ATPγS (adenosine 5′-(γ-thio) triphosphate tetralithium), NADH (β-nicotinamide adenine dinucleotide, reduced disodium salt), and pyruvate kinase/lactic dehydrogenase were supplied by Sigma-Aldrich (St Louis, MO, USA). Phosphoenolpyruvate was purchased from Ruibio-bio (Ingelheim, Germany). Wild type RecA protein (E. coli) was purchased from New England Biolabs (Ipswich, MA, USA) or expressed and purified in our lab. All RecA mutants were expressed and purified in our own lab. Supernuclease was obtained from Sino Biological Inc. (Beijing, China). Ultrapure water of 18.2 MΩ cm was obtained from a Purelab Ultra Bioscience system (ELGA, UK). All other reagents and solvents were analytical grade and supplied by Beijing Chemical Reagents (Beijing, China).

Zhao B., Zhang D., Li C., Yuan Z., Yu F., Zhong S., Jiang G., Yang Y.G., Le X.C., Weinfeld M., Zhu P, & Wang H. (2017). ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination. Cell Discovery, 3, 16053-.

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Synthesis and Characterization of Organohalogens

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Organohalogens were custom synthesized as described in the Supporting Information (SI) or as described previously,^{21 (link),28 (link)} isolated from marine sponges^{38 (link)} or purchased [pyrrole (24), ethyl-4-bromopyrrole-2-carboxylate (27) and 2,3-dibromo-N-methylmaleimide (28)] from SigmaAldrich (St. Louis, MO). Names and structures for the compounds tested are summarized in Table SI1. All the HOCs were dissolved in dry DMSO (10 mM) and stored at −80 °C. Phenylmethylsulfonyl fluoride (PMSF), leupeptin hydrochloride, Mg-ATP, phosphocreatine, creatine phosphokinase, phosphoenolpyruvate, pyruvate kinase/lactic dehydrogenase, Na₂ATP, NADH were purchased from Sigma-Aldrich. Arsenazo III was purchased from Santa Cruz Biotechnology (Dallas, TE). Ryanodine was purchased from Ascent Scientific (Cambridge, MA) and dissolved in 100% ethanol and stored at 4 °C until use. [³H]Ryanodine (specific activity: 56.6 Ci/ mmol) was obtained from PerkinElmer (Santa Clara, CA).

Zheng J., McKinnie S.M., El Gamal A., Feng W., Dong Y., Agarwal V., Fenical W., Kumar A., Cao Z., Moore B.S, & Pessah I.N. (2018). Organohalogens Naturally Biosynthesized in Marine Environments and Produced as Disinfection Byproducts Alter Sarco/Endoplasmic Reticulum Ca2+ Dynamics. Environmental science & technology, 52(9), 5469-5478.

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