Abstract
Highly purified glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact glycosomes. Three glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75 kDa membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85–100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36 kDa protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41 kDa integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3β-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids. Index Descriptors and Abbreviations: glycosome, glycosome membrane proteins, Trypanosoma cruzi, epimastigote, phospholipids, neutral lipids, sterols; LIT, liver infusion-tryptose; TMBZ, 3,3 ′,5,5 ′-tetramethylbenzamidine; GMPs, glycosome membrane proteins; HK, hexokinase, EC 2.7.1.1; PGI, phosphoglucose isomerase, EC 5.3.1.9; MDH, malate dehydrogenase, EC 1.1.1.37; GDH, α-glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; PK, pyruvate kinase, EC 2.7.1.40; PEPCK, phosphoenolpyruvate carboxykinase, EC 4.1.1.4.9; PGK, 3-phosphoglycerate kinase, EC 2.7.2.3; PPDK, phosphate pyruvate dikinase, EC 2.7.9.1; NL, neutral lipids; PL, phospholipids; TCA, trichloracetic acid
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