Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Cheleski, Juliana; Freitas, Renato Ferreira de; Wiggers, Helton José; Rocha, Jansle Vieira; Araújo, Ana Paula Ulian de; Montanari, Carlos Alberto

doi:10.1016/j.pep.2010.11.013

Cited by 15 publications

(12 citation statements)

References 25 publications

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“…Cell lysis was carried out by bead-beating using 0.1 mm zirconia beads in a Precellys-24 homogenizer (Biolabo, Switzerland). Glyceraldehyde-3-phosphate dehydrogenase activity was measured in whole cell protein extracts by following NADH formation measuring absorbance at 340 nm, as per the procedure described previously51. Assay was performed at 37 °C in 200 μl of assay buffer comprising 0.015 M sodium pyrophosphate buffer pH 8.5, 0.03 M sodium arsenate, 2.5 mM NAD + , 2.5 mM glyceraldehyde-3-phosphate.…”

Section: Methodsmentioning

confidence: 99%

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

et al. 2014

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Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3–4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.

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Section: Methodsmentioning

confidence: 99%

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

et al. 2014

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“…Protein/peptide interactions with polymers and nanoparticles Nucleic acid interactions other than with proteins Enzyme activity and kinetics Other unclassified studies (including, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…367-380 (x) Nucleic acid interactions other than with proteins 381-421 (xi) Enzyme activity and kinetics. [422][423][424][425][426][427][428][429] (xii) Other unclassified studies (including, e.g. small molecule interactions and micelle formation).…”

Section: Introductionmentioning

confidence: 99%

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Ghai

Falconer

Collins

2011

J of Molecular Recognition

163

103

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Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.

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“…Enzyme preparation: recombinant TcGAPDH enzyme was expressed and purified as described elsewhere [40].…”

Section: Itc Assaysmentioning

confidence: 99%

“…The kinetic parameters of G3P (K M , k cat and V max ) were determined according to a protocol previ ously established in our laboratory [40]. Briefly, the syringe was filled with 5 mM d,lG3P in 100 mM HEPES buffer at pH 8.0.…”

Section: Itc Runsmentioning

confidence: 99%

Integration of Methods in Cheminformatics and Biocalorimetry for the Design of Trypanosomatid Enzyme Inhibitors

et al. 2013

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Nequimed125, the most potent inhibitor to act upon TcGAPDH so far, which sits on the G3P site without any contact with the co-factor (NAD(+)) site, underpins the result obtained by ITC that it is a G3P-competitive inhibitor. Molecular dynamics simulation provides biding poses of TcGAPDH inhibitors that correlate with mechanisms of inhibition observed by ITC. Overall, a new class of dihydroindole compounds that act upon TcGAPDH through a competitive mechanism of inhibition as proven by ITC measurements also kills T. cruzi.

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Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Cited by 15 publications

References 25 publications

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Integration of Methods in Cheminformatics and Biocalorimetry for the Design of Trypanosomatid Enzyme Inhibitors

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