Trypanothione synthetase enzymatic assay

Version 1

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

The SilicoTryp project aims at the creation of a “Silicon Trypanosome”, a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology. Trypanosomes are blood-stream parasites transmitted by tsetse flies; they cause African sleeping sickness in humans and livestock. Currently available drugs have severe side effects, and the parasites are rapidly developing resistance. In this project, we collect a wide range of new experimental data on the parasite in its various ...

Aim. To provide critical quantitative parameter information and to model redox balance by determining the cellular concentration of all enzymes involved in the trypanothione-dependent hydroperoxide detoxification system of trypanosomes and by performing the kinetic characterization of the involved enzymes under pseudo-physiological conditions.

The enzymes involved in the trypanothione metabolism will be studied in a uniform assay medium that mimics the intracellular milieu of the parasite.

This assay is designed to obtain the in vitro kinetic data of T. brucei recombinant trypanothione synthetase. The enzyme catalyzes the ATP-dependent ligation of spermidine (Spd) and GSH to generate glutathionylspermidine (Gsp) and also of Gsp and GSH to finally produce trypanothione (T(SH)2). The data was obtained in an spectrophotometric assay that links ADP production with NADH consumption through the piruvte kinase and lactate dehydrogenase.

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of glutathione, and spermidine.