The Absence of Cardiolipin in Hydrogenosomes of Trichomonas vaginalis and Tritrichomonas foetus

Most studies on the biochemical organization of hydrogenosomes concentrate on their enzyme content, characterizing the metabolic role of these trichomonad organelles (Miiller, 1980, Symp. Soc. Gen. Microbiol. 30: 127142). Only occasional findings are available on their chemical composition and some of these need confirmation, e.g., the report that cardiolipin is present in hydrogenosomes of Tritrichomonas foetus (Cerkasovova et al., 1976, Folia Parasitol. Praha 23: 33-37). We present here data and comment on the analysis of phospholipids in cell homogenates and hydrogenosomal preparations of T. foetus and Trichomonas vaginalis. Cells of T. foetus KV I and T. vaginalis A, strains were cultivated in Hollander's modified TYM medium with 10% new born calf serum (1976, J. Parasitol. 62: 826-828). Cultures (24 hr) were harvested and washed twice in 250 mM sucrose, containing 10 mM EDTA before being ruptured using a powered Potter homogenizer. The hydrogenosomal (large particle) fraction was prepared by differential sedimentation according to Lindmark and Miiller (1973, J. Biol. Chem. 248: 7724-7728). Homogenates and separated hydrogenosomes were suspended in 250 mM sucrose and kept frozen until biochemical analysis. For phospholipid analysis, the samples were extracted with 20 volumes of chloroformmethanol (2:1) according to Folch et al. (1957, J. Biol. Chem. 126: 497-509). After evaporation of the solvents in vacuo, lipids were dissolved in 5 ml chloroform-methanol (2:1) and stored at -20 C until used. Qualitative analysis of phospholipids was performed by TLC on plates coated with 0.5 mm silica gel PF (Merck, Darmstadt, West Germany), using chloroform-methanol-water (65:25:4) for onedimensional separation or chloroform-methanol-25% ammonia (65:35:5; first dimension) and chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5; second dimension) for two-dimensional separation (Rouser et al., 1969, Methods Enzymol. 14: 272-317). Phospholipids were visualized after spraying with 0.5% ninhydrin in methanol and heating or after spraying with molybdate reagent (Dittmer and Lester, 1964, J. Lipid Res. 5: 126127). The major phospholipid classes present in cell homogenates and in the hydrogenosomal fraction from both T. foetus and T. vaginalis are phosphatidylcholine and phosphatidylethanolamine (PE); smaller quantities of phosphatidylglycerol (PG) and N-dimethylphosphatidylethanolamine were also identified by two-dimensional TLC. Lipid extracts of T. foetus, but not of T. vaginalis contained a phospholipid which migrated on TLC ahead of PE, but behind a cardiolipin standard that was cochromatographed with the lipid extract (lane 2 in Fig. 1). With the solvent system chloroform-methanol-25% ammonia, cardiolipin remained close to the origin, whereas the Rf of the unknown phospholipid was 0.45. Cardiolipin was absent from extracts of both organisms (lanes 1 and 3 in Fig. 1). Upon mild alkaline hydrolysis, the unknown lipid gave water soluble material, which was not further characterized, and fatty acids. The fatty acid composition (mainly C6:0, C18:o0, C18:i, and C18:2) was similar to that of total T. foetus phospholipids. According to its chromatographic behaviour and lability towards alkali, the unknown lipid might be O-acylphosphatidylglycerol. It is very