Molecular & Biochemical Parasitology 118 (2001) 111 – 121 The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi  Juan Luis Concepcio ´n a , Christiane A. Adje ´ b,1 , Wilfredo Quin ˜ ones a , Nathalie Chevalier b , Michel Dubourdieu a , Paul A.M. Michels b, * a Unidad de Bioquimica de Parasitos, Facultad de Ciencias, Uniersidad de Los Andes, Me ´rida, Venezuela b Research Unit for Tropical Diseases, Christian de Due Institute of Cellular Pathology and Laboratory of Biochemistry, Uniersite ´ catholique de Louain, ICP-TROP 74.39, Aenue Hippocrate 74, B-1200 Brussels, Belgium Received 14 May 2001; received in revised form 28 August 2001; accepted 3 September 2001 Abstract In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle’s membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma cruzi ; Phosphoglycerate kinase; Isoenzymes; Glycosomes; Membrane attachment www.parasitology-online.com. 1. Introduction In Trypanosomatidae, glycolysis is compartmental- ized. The glycolytic enzymes responsible for the conver- sion of glucose into phosphoglycerate are sequestered in peroxisome-like organelles called glycosomes [1–4]. Among these trypanosomatid enzymes, phosphoglycer- ate kinase (PGK; EC 2.7.2.3) is peculiar because it is represented by different isoenzymes. In Trypanosoma brucei, three PGKs have been detected [5–8], each encoded by a different gene [6,8–11]. The three genes (A, B and C ) are tandemly arranged in the genome [9]. PGKC codes for an isoenzyme with a subunit M r of 47 kDa and is present only in the glycosomes, whereas PGKB encodes a cytosolic enzyme of 45 kDa [9]. The PGKC protein is abundant in the mammalian blood- stream stage of the parasite’s life cycle, but is not detected in the procyclic insect stage, while the reverse is the case for PGKB [9,10]. The PGKA gene is ex- pressed throughout the life cycle, but only at very low levels [10]. Its product was located in the glycosomes [6,12]. The amino-acid sequences of PGKB and C are 95% identical, but exhibit two notable differences; the glycosomal enzyme has a much higher isoelectric point than the cytosolic enzyme (pI 9.3 vs. 6.3) and it con- tains a 20 amino-acid long C-terminal extension [5,9,13]. The last 3–5 residues of this extension are Abbreiations: ALAT, alanine aminotransferase; HXK, hexokinase; MDH, malate dehydrogenase; PEPCK, phosphoenolpyruvate car- boxykinase; PGI, glucose-6-phosphate isomerase; PGK, 3-phospho- glycerate kinase.  Note: Nucleotide sequence data reported in this paper are avail- able in the EMBL, GenBank™ and DDJB data bases under the accession numbers AJ311044 (PGKA) and AJ311045 (PGKB/C). * Corresponding author. Tel.: +32-2764-7473; fax: +32-2762- 6853. E-mail address: michels@trop.ucl.ac.be (P.A.M. Michels). 1 Present address: Project Retro CI, Abidjan, Cote d’Ivoire. 0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S0166-6851(01)00381-4