Please cite this article in press as: Barros-Álvarez X, et al. The glycosomal-membrane associated phosphoglycerate kinase isoenzyme A plays a role in sustaining the glucose flux in Trypanosoma cruzi epimastigotes. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.04.003 ARTICLE IN PRESS G Model MOLBIO 10894 1–4 Molecular & Biochemical Parasitology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Short communication The glycosomal-membrane associated phosphoglycerate kinase isoenzyme A plays a role in sustaining the glucose flux in Trypanosoma cruzi epimastigotes Ximena Barros-Álvarez a,1 Q1 , Ana J. Cáceres a , Maria T. Ruiz a , Paul A.M. Michels a,b,2 , Juan Luis Concepción a , Wilfredo Qui ˜ nones a,∗ a Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de los Andes, La Hechicera, 5101 Mérida, Venezuela b Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels, Belgium a r t i c l e i n f o Article history: Received 14 March 2015 Received in revised form 14 April 2015 Accepted 16 April 2015 Available online xxx Keywords: Trypanosoma cruzi Phosphoglycerate kinase Glycolysis Carbohydrate metabolism regulation Osmotic shock Digitonin a b s t r a c t In Trypanosoma cruzi three isoenzymes of phosphoglycerate kinase (PGK) are found which are simulta- neously expressed: the cytosolic isoenzyme PGKB as well as two glycosomal enzymes, PGKA and PGKC. In this paper, we show that PGKA in T. cruzi epimastigotes is associated to the glycosomal membrane; it is responsible for about 23% of the glycosomal PGK activity, the fraction that remains in the pellet after osmotic shock treatment of purified organelles, in contrast to the 77% soluble activity that is mainly attributed to PGKC. Antibodies against the unique 80 amino-acid insertion of PGKA blocked almost com- pletely the glucose consumption by epimastigotes that were partially permeabilized with digitonin. These results indicate that PGKA is the predominant isoenzyme for sustaining glycolysis through the glycosomes of these parasites. © 2015 Published by Elsevier B.V. 1. Introduction, results and discussion Q2 In most trypanosomatids analyzed, phosphoglycerate kinase (PGK) is present as three isoenzymes with different subcellular locations (glycosomal PGK A and C; cytosolic PGKB) and molecular weights. The three PGK isoenzymes of Trypanosoma cruzi are simul- taneously expressed in each of its three developmental stages: amastigotes, trypomastigotes and epimastigotes [1,2]. The glycoso- mal isoenzymes A and C together were reported to be responsible for approximately 20% of the total cellular PGK activity detected in epimastigotes [1]. The presence of the three PGK isoenzymes in different compartments and their simultaneous expression, do raise the question about the role of each isoenzyme in the inter- mediary metabolism of this parasite. Based on studies of T. cruzi Abbreviations: ALAT, alanine aminotransferase; HK, hexokinase; ISDH, isocitrate dehydrogenase; PBS, phosphate-buffered saline; PGK, phosphoglycerate kinase. ∗ Corresponding author. Tel.: +58 274 2401302; fax: +58 274 2401291. E-mail addresses: wilqui@ula.ve, qwilfred@mail.com (W. Qui ˜ nones). 1 Present address: Department of Biochemistry, University of Washington, Seat- tle, WA 98195, USA. 2 Present address: Institute of Structural and Molecular Biology, School of Bio- logical Sciences, University of Edinburgh, King’s Buildings, Max Born Crescent, Edinburgh EH9 3BF, Scotland, UK. epimastigotes, we postulated previously that the principal role of PGKA is its participation in the glycolytic pathway, when a high ATP/ADP ratio within the glycosomes inhibits the PGKC activity [2]. Owing to a unique 80 amino-acid insertion in its N-terminal domain, PGKA is the largest PGK isoenzyme in kinetoplastids with a molecular weight of 56 kDa. The presence of a PGKA has been reported in Crithidia fasciculata, Trypanosoma brucei, Try- panosoma congolense and T. cruzi, but it could not be detected in Leishmania species [1,3–6]. Previous studies strongly suggested that the PGKA isoenzyme is tightly associated with the glycoso- mal membrane in epimastigotes of T. cruzi [1]. Confirmation of such association is now provided by the experiments presented in Fig. 1. When epimastigotes were treated with increasing con- centrations of the detergent digitonin, PGK activity was released from the trypanosomes in two phases: 70–80% from the cyto- sol at detergent concentrations less than approximately 0.05 mg digitonin mg protein -1 , attributed to PGKB, and 20–30% between approximately 0.04 and 0.20 mg digitonin mg protein -1 , mainly attributed to PGKC and a minor part to PGKA [1,2], as shown in Fig. 1A and B. However, the western blot of panel B, probed with a polyclonal antiserum specific for the PGKA insert (-PGKA pep- tide antiserum) shows that most of the signal corresponding to this isoenzyme is not released by this digitonin treatment, but remains in the insoluble cell fraction, even in the presence of 0.1% http://dx.doi.org/10.1016/j.molbiopara.2015.04.003 0166-6851/© 2015 Published by Elsevier B.V. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62