Pyruvate kinase lactate dehydrogenase mix

Manufactured by Merck Group

The Pyruvate kinase/lactate dehydrogenase mix is a laboratory reagent used in enzymatic assays. It catalyzes the conversion of phosphoenolpyruvate to pyruvate, which is then reduced to lactate by lactate dehydrogenase. This mix is commonly used as a coupled enzyme system for the determination of various analytes in biochemical and diagnostic applications.

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Lab products found in correlation

Amp cp, by Merck Group (1 mentions) Amppnp, by Jena Biosciences (1 mentions) Phosphoenolpyruvate pep, by Merck Group (1 mentions) Adenosine diphosphate (adp), by Merck Group (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Gen5 data analysis software, by Agilent Technologies (1 mentions) Elx808 absorbance microplate reader, by Agilent Technologies (1 mentions) Myokinase from chicken muscle, by Merck Group (1 mentions) Synergy 4, by Agilent Technologies (1 mentions) Prism 9, by GraphPad (1 mentions) Infinite m100, by Tecan (1 mentions) Phosphoenolpyruvate, by Merck Group (1 mentions)

7 protocols using pyruvate kinase lactate dehydrogenase mix

Preparation of CaMKII and EAG Proteins

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Drosophila full-length CaMKII (R3 isoform;^{58 (link)}) was a gift of Leslie Griffith (Brandeis University, Massachusetts, U.S.A.) while full-length Drosophila EAG (GenBank: M61157) was a gift of Dianne Papazian (UCLA, California, U.S.A.). Nucleotides (ATP (Sigma), AMPPNP (Jena Biosciences), ADP (Sigma) and AMPCP (Sigma)) were processed according to manufactures’ instructions. Stock solutions at 50 mM of ATP, AMPPNP and ADP were freshly prepared in either 1 M Tris pH 7.5 or 1 M HEPES pH 7.5. Stock solution of AMPCP at 50 mM in 1 M HEPES pH 7.5 was stored at −20 °C. λ-phosphatase (NEB) was processed according to manufacturer’s instructions, aliquoted and stored at −80 °C. Stock solutions of reagents used in the ATPase assay were prepared as follows: phosphoenolpyruvate (PEP; Sigma) at 50 mM in 50 mM HEPES pH 7.5; nicotinamide adenine dinucleotide, reduced (NADH; Sigma) at 6 mM in 50 mM HEPES pH 7.5; peptide syntide (GenScript) was prepared in water at 2.5 mM. All these solutions were kept aliquoted at −80 °C. The pyruvate kinase/lactate dehydrogenase mix (Sigma) was at 600–1000 units ml⁻¹ and 900–1400 units ml⁻¹ activity, respectively, and kept at −20 °C.

Castro-Rodrigues A.F., Zhao Y., Fonseca F., Gabant G., Cadene M., Robertson G.A, & Morais-Cabral J.H. (2018). The interaction between the Drosophila EAG potassium channel and the protein kinase CaMKII involves an extensive interface at the active site of the kinase.. Journal of molecular biology, 430(24), 5029-5049.

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Enzymatic NRK Activity Assay

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The coproduct of enzymatic phosphorylation of NR with ATP is ADP, which can be regenerated by PK/PEP with the formation of pyruvate, then pyruvate can be reduced to lactate with the oxidation of NADH catalyzed by LDH. The activity of Klm-NRK was assayed spectrophotometrically at 30 °C by monitoring the oxidation of NADH at 340 nm (Dölle and Ziegler, 2009 (link)). The standard assay mixture (1 mL) composed of 100 mM potassium phosphate buffer (pH 7.0), 0.5 mM NR, 0.5 mM ATP, 2 mM MgCl₂, 5 mM phosphoenolpyruvate, 0.15 mM NADH, 3.5 U PK /5 U LDH (pyruvate kinase/lactate dehydrogenase mix, Sigma) and appropriate amount of purified Klm-NRK. One unit of Klm-NRK activity was defined as the amount of enzyme catalyzing the oxidation of 1 μmol NADH per minute under above conditions.

Qian X.L., Dai Y.S., Li C.X., Pan J., Xu J.H, & Mu B. (2022). Enzymatic synthesis of high-titer nicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system. Bioresources and Bioprocessing, 9(1), 26.

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Measuring AtACS Activity via Coupled Assay

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AtACS activity was measured by coupling the acetate- and CoA-dependent formation of AMP from ATP, to the oxidation of NADH, using the reactions catalyzed by myokinase, lactate dehydrogenase and pyruvate kinase (Sofeo et al., 2019 (link)). In brief, assays were performed at 37°C in a final volume of 100 μL, in individual wells of 96-well microtiter dishes. The absorbance of NADH was measured at 340 nm using a BioTek ELx808TM Absorbance Microplate Reader, and data were collected and analyzed with Gen5TM Data Analysis software (BioTek Instruments, Winooski, VT). The reaction mix initially contained 50 mM Tris-HCl pH 7.5, 5% (v/v) ethanol, 5 mM Tris(2-carboxylethyl) phosphine hydrochloride (Thermo Fisher Scientific Inc. Waltham, MA), 6 mM MgCl₂, 5 mM ATP, 5 mM phospho(enol)pyruvate, 0.4 mM NADH, 2–20 U Pyruvate Kinase/Lactate Dehydrogenase mix (Sigma-Aldrich Co., St. Louis, MO), 1U Myokinase from chicken muscle (Sigma-Aldrich Co., St. Louis, MO) and 5 μg of recombinant atACS protein. After monitoring the initial background change A₃₄₀, ACS activity was initiated by the addition of 2.5 mM CoA, and progress of this reaction was monitored as a decrease in A₃₄₀. All data were collected from three independent assays, and all raw data collected for this study are provided in Supplementary Table S1.

Sofeo N., Winkelman D.C., Leung K, & Nikolau B.J. (2023). Modulation of plant acetyl-CoA synthetase activity by post-translational lysine acetylation. Frontiers in Molecular Biosciences, 10, 1117921.

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Enzyme-Coupled ATPase Activity Assay

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An enzyme-coupled ATPase assay based on hydrolysis of ATP coupled to oxidation of NADH was used to measure the protein ATPase activity (25 (link)). A total of 15 nM Uls1 and/or 50 μM homodimeric Top2 alone or with 100 μM DNA (purified sheared salmon-sperm DNA, Invitrogen) were mixed together in a buffer containing 50 mM Tris.HCl; pH 7.9, 100 mM KCl, 8 mM MgCl2, 5 mM beta-mercaptoethanol, 200 μg/ml BSA, 2 mM Phospho(enol)pyruvate, 280 μM NADH (Sigma, N7410), 0.5 mM ATP and 1 μl of pyruvate kinase/lactate dehydrogenase mix (Sigma, P0294). The reactions were performed in 100 μl reaction volume in a 96-well plate at 30°C. The oxidation of NADH to NAD+ was monitored by measuring of the fluorescence (Excitation—340 nm, Emission—440m) every 30 s for 30 min using a Spectramax Gemini XPS microplate reader. Titration of increasing concentration on NADH was used to obtain a standard curve for each measurement. The background signal was subtracted from each sample before plotting the results into the graph.

Swanston A., Zabrady K, & Ferreira H.C. (2019). The ATP-dependent chromatin remodelling enzyme Uls1 prevents Topoisomerase II poisoning. Nucleic Acids Research, 47(12), 6172-6183.

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Kinase Activity Characterization by NADH/ATPase Assay

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Kinase activity was characterized by using an NADH/ATPase coupled assay (52 (link)). The reactions were performed at 25 °C in a 384-well plate by using the Synergy 4 plate reader (Biotek). The reaction mix typically contained the enzyme at a concentration of 250 nM, 500 μM Peptide 7 (RRRAPSFYAK), 150 μM NADH, 300 μM phosphoenolpyruvate, 1 mM ATP, a lactate dehydrogenase/pyruvate kinase mix from Sigma (3 units/mL), 20 mM Hepes (pH 7.5), 30 mM NaCl, 10 mM MgCl₂, 1 mM CaCl₂, 1 mM dithiothreitol, and 0.01% Tween 20. The reaction was started by adding cGMP (0–200 μM) and monitored for 1 h by using the rate of NADH absorbance decrease at 340 nm, which is proportional to the rate of ATP hydrolysis.

El Bakkouri M., Kouidmi I., Wernimont A.K., Amani M., Hutchinson A., Loppnau P., Kim J.J., Flueck C., Walker J.R., Seitova A., Senisterra G., Kakihara Y., Kim C., Blackman M.J., Calmettes C., Baker D.A, & Hui R. (2019). Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation. Proceedings of the National Academy of Sciences of the United States of America, 116(28), 14164-14173.

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Measuring Mot1 ATP Hydrolysis Activity

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For determination of the ATP hydrolysis rate of Mot1, an assay coupling ATP hydrolysis to NADH oxidation was performed as previously reported⁴⁴. Either Mot1 (150 nM) alone or added to preformed TBP (180 nM):DNA (240 nM) complexes were incubated for 10 minutes at 4°C. Reconstituted complexes were mixed with 0.5 mM phosphoenolpyruvate, 1 mM ATP or ATPγS, 0.1 mM NADH, 25 U/ml lactate dehydrogenase pyruvate kinase mix (Sigma) and ATPase buffer (20 mM HEPES pH 8.0, 60 mM KCl, 1 mM DTT, 5 mM MgCl₂, 0.1 mg/ml BSA) in a final volume of 50 μl. Changes in fluorometrical absorbance of NADH at 340 nm were monitored in non-binding black 96-well plates (Greiner Bio-One) with a Tecan Infinite M100 (Tecan) using 343 nm for excitation and emission of 448 nm at 30 °C. ATP turnover was calculated using maximal initial linear rates, corrected for a buffer blank. The data was statistically analyzed and visualized by Graphpad PRISM 9.5.0.

Woike S., Eustermann S., Jung J., Wenzl S.J., Hagemann G., Bartho J., Lammens K., Butryn A., Herzog F, & Hopfner K.P. (2023). Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1. Nature structural & molecular biology, 30(5), 640-649.

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ATPase Activity Quantification Protocol

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ATPase reactions were carried out side-by-side from the same master mix as the fluorescence-based unwinding assays. In separate reactions, NADH (Sigma), phosphoenolpyruvate (Sigma or Alfa Aesar) and lactate dehydrogenase/pyruvate kinase mix (Sigma) were added to unwinding reactions to a final concentration of 2 mM, 2 mM and 1/250 (v/v), respectively. NADH turnover was monitored by measuring absorbance at 340 nm. Obtained absorbance data were converted to the concentration of NADH using condition and machine specific ϵ (NADH) of 0.62 mM⁻¹. ATPase rates were obtained from a linear fit to the experimental data using Prism (GraphPad 7, 8 or 9).

Schmidt T., Dabrowska A., Waldron J.A., Hodge K., Koulouras G., Gabrielsen M., Munro J., Tack D.C., Harris G., McGhee E., Scott D., Carlin L.M., Huang D., Le Quesne J., Zanivan S., Wilczynska A, & Bushell M. (2023). eIF4A1-dependent mRNAs employ purine-rich 5’UTR sequences to activate localised eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation. Nucleic Acids Research, 51(4), 1859-1879.

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