In this study we present the first data on the

In this study, we present the first data on the kinetic properties of the hexokinase of T. equiperdum, which we compare with similar data available for pleomorphic T. brucei (Seed and Baquero, 1965, Nwagwu and Opperdoes, 1982, Hara et al., 1997, Morris et al., 2006, Chambers et al., 2008b, Chambers et al., 2008c). We seek to establish whether the kinetic properties of hexokinases in members of Trypanozoon are tuned to ‛match’ (Bar-Even et al., 2011) the blood cox 2 inhibitor and mannose concentrations of their hosts.
Materials and methods Experiments complied with Venezuelan (“Ley para la Protección de la Fauna Doméstica Libre y en Cautiverio”, Articles 52–55), and European legislation (Directive 2010/63/EU) for the protection of research animals. Our stock of T. equiperdum (TeAp-N/D1, previously known as TEVA1, Perrone et al., 2009; referred to as T. evansi in previous studies, Sánchez et al., 2015), was originally isolated from cattle-ranch horses in the Venezuelan Llanos region (Moreno et al., 2013). Parasites were cultured in rats, trypanosomes were purified from rats, and a glycosome-rich pellet was purified from trypanosomes as in a previous study (Moreno and Nava, 2015). For resuspension of the glycosome-rich pellet, 4ml were used of pH6.5 40mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer containing 2mM MgSO4, 50mM NaCl, and a protease inhibiting cocktail (57μM PMSF, 100μM TLCK, 10μM leupeptin and 1mM benzamidine). Solubilization of the glycosome membranes was performed as in the previous study (Moreno and Nava, 2015). Centrifugation at 33,000g for 15min of the mixture so obtained produced the supernatant used in subsequent procedures. One unit (U) of enzymatic activity is defined as the quantity of enzyme required to catalyze the formation of 1μmol/min of product. Spectrophotometrical assay (340nm) of hexokinase was carried out at ~25°C in a final volume of 1ml (Misset and Opperdoes, 1984). In the case of glucose as a substrate, the catalysis was coupled to the glucose 6-phosphate dehydrogenase reaction, with NADP+ reduction being detected. In the case of mannose, the catalysis was coupled to the pyruvate kinase and lactate deshydrogenase reactions, with NADH oxidation being detected. Buffers used (Good et al., 1966) are: Buffer A, a pH6.5 solution containing 50mM Tris-HCl, 50mM NaCl, 2mM MgSO4, and 1mM dithiotreitol; MOPS buffer; Buffer B, a pH6.5 40mM MOPS buffer containing 2mM MgSO4; MES buffer; and Tris-HCl buffer.
Results
Discussion The animal trypanosome, T. b. brucei, is the de facto model for the study of human African trypanosomiasis for three reasons: it poses a negligible infection risks for researchers; it is easily maintained in the laboratory; and it is genetically and metabolically similar to the human trypanosomes, T.b.rhodesiense and T. b. gambiense. However, T. b. brucei differs from these trypanosomes in possessing an hexokinase with lower apparent K constants for hexoses (Table 2). This difference should not be overlooked because it might cause African animal trypanosomes to differ from their human counterparts in their response to chemotherapeutic agents. Apparent K constants, both our own and those published by other authors, were determined under the experimental conditions (Table S1, Supplementary material) established as optimal for the hexokinase of members of Trypanozoon (Risby and Seed, 1969); thus we assume that these apparent K constants can be compared. In addition, based on their resembling values (T. b. gambiense; Table 2), we assume that the apparent K constants determined using chemical methods can be compared with those obtained using enzymatic methods.
Acknowledgements To Jesús Molinari, for comments. This study received financial support from the Consejo de Desarrollo Científico Humanístico, Tecnológico y de las Artes de la Universidad de Los Andes, for projects C-1563-08-03-F, C-1873-14-03-B, and C-1397-06-03-B.