Improved Spectrophotometric Assay of Nucleoside Monophosphate Kinase Activity Using the Pyruvate Kinase/Lactate Dehydrogenase Coupling System

Blondin, Catherine; Serina, Lidia; Wiesmüller, Lisa; Gilles, Anne‐Marie; Bârzu, Octavian

doi:10.1006/abio.1994.1326

Cited by 116 publications

(102 citation statements)

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“…Activity was determined as described in Blondin et al 23 Using a coupled spectrophotometric assay at 334 nm in an Eppendorf ECOM 6122 photometer. The reaction medium (0.5 ml final volume) contained 50 mM Tris-HCl pH 7.4, 50 mM KCl, 2 mM MgCl 2 , 0.2 mM NADH, 1 mM phosphoenol pyruvate and 2 units each of lactate dehydrogenase, pyruvate kinase and nucleoside diphosphate kinase.…”

Section: Spectrophotometric Binding Assaymentioning

confidence: 99%

Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

Song

Risseeuw

Froeyen

et al. 2016

Bioorganic & Medicinal Chemistry

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International audienceWe report the design and synthesis of a series of non-nucleoside MtbTMPK inhibitors (1-14) based on the gram-positive bacterial TMPK inhibitor hit compound 1. A practical synthesis was developed to access these analogues. Several compounds show promising MtbTMPK inhibitory potency and allow the establishment of a structure-activity relationship, which is helpful for further optimization

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Section: Spectrophotometric Binding Assaymentioning

confidence: 99%

Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

Song

Risseeuw

Froeyen

et al. 2016

Bioorganic & Medicinal Chemistry

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“…Enzymatic assays: Activity was determined as described previously [31] by the coupled spectrophotometric assay at 334 nm in an Eppendorf ECOM 6122 photometer. The reaction medium (0.5 mL final volume) contained Tris-HCl (50 mM, pH 7.4), KCl (50 mM), MgCl 2 (2 mM), NADH (0.2 mM), phosphoenol pyruvate (1 mM), and lactate dehydrogenase, pyruvate kinase, and nucleoside diphosphate kinase (2 units each).…”

Section: Discussionmentioning

confidence: 99%

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

et al. 2003

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] Muriel Delepierre, [d] and He ¬le ¡ne Munier-Lehmann* [a] Thymidine monophosphate kinase (TMPK) from Mycobacterium tuberculosis (TMPKmt) is an attractive target for the design of specific inhibitors. This fact is the result of its key role in the thymidine pathway and of unique structural features in the active site observed by X-ray crystallography, especially in comparison to its human counterpart (TMPKh). Different 5-modified thymidine derivatives, as well as purine and pyrimidine analogues or C-nucleosides were tested on TMPKmt and TMPKh, and the results were rationalized by docking studies. 5-Halogenated 2'-deoxyuridines are the best inhibitors of TMPKmt found and present the highest selectivity indexes in favor of TMPKmt.

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“…The in vitro assay conditions were implemented by each student group following these previously published protocols [2]. One proscribed set of assays was done as follows in a 1 mL reaction in a UV/Vis transparent plastic cuvette.…”

Section: In Vitro Assay Of Cmp Kinasementioning

confidence: 99%

“…1, reaction 1) and was chosen for several reasons. First, CMP kinase can be assayed using a coupled spectrophotometric in vitro assay [2] (Fig. 1, reactions 2 and 3) making this assay accessible to classroom laboratory teaching using commonly available equipment.…”

Section: Introductionmentioning

confidence: 99%

Doing that thing that scientists do: A discovery‐driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom

Garrett

Osmundson

Isaacson

et al. 2015

Biochem Molecular Bio Educ

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In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project. V C 2015 by The International Union of Biochemistry and Molecular Biology, 43(3): 145-153, 2015.

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Improved Spectrophotometric Assay of Nucleoside Monophosphate Kinase Activity Using the Pyruvate Kinase/Lactate Dehydrogenase Coupling System

Cited by 116 publications

References 0 publications

Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

Doing that thing that scientists do: A discovery‐driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom

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