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31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae. Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase. 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region. 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates. Other metabolites observed were ATP, ADP, NAD(P)+, phosphoenolpyruvate and low molecular weight polyphosphates (PPn, n less than 20). The presence of high-molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH. 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx. pH 7.2 +/- 0.1, using the orthophosphate resonance as an indicator. The cells responded to the addition of glucose (final concentration approx. 35 mM) with a decrease in pH, both internal (delta pH = -0.9 (55 min)-1) and external (delta pH = -1.3 (15 min)-1). Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.

Proton nuclear magnetic resonance (NMR) spectroscopy was used to follow glucose metabolism in Crithidia luciliae. Parasites were grown aerobically and anaerobically in culture, with glucose as the major carbon source and 1H NMR spectra were acquired for the cell free medium. The 1H NMR resonances of metabolites utilised and produced during cell growth were identified by difference spectroscopy, and quantitated from standard curves using 3-trimethylsilyl propionate-2,2,3,3-d4 sodium salt as an internal standard. The major metabolites produced by C. luciliae grown aerobically on 8 mM glucose were succinate, pyruvate, acetate and ethanol, in final concentrations in the media when the cells entered stationary phase of 8.5 +/- 0.5, 5.0 +/- 0.3, 2.1 +/- 0.2 and 2.5 +/- 0.6 mM, respectively. The production of succinate and pyruvate, but not acetate and ethanol, followed closely the growth curve of the parasites. Succinate was also measured enzymically and glucose using an autoanalyser. In both cases the results correlated well with the NMR data. The amounts of end products formed were greater than could be accounted for by the utilisation of glucose or any other metabolite observable in the 1H NMR spectra. There was approximately one extra atom of carbon for each molecule of succinate formed, supporting the view that succinate is produced via phosphoenolpyruvate carboxykinase and carbon dioxide fixation. Anaerobically the same major metabolites were produced, but with a decreased ratio of succinate to acetate and ethanol. The formation of glycerol from glucose was not observed under these conditions.

Crithidia fasciculata cells grown in defined medium are sensitive to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR). When cells are challenged with 2-5 microM MTX, cell division ceases after 3-4 divisions and the cells become rounded and immotile for approximately 60 h, with a 40% decrease in cell viability occurring during this period. The cells then recover normal morphology and cell division resumes. Cells which undergo this treatment can be transferred directly into high levels of the drug (1-2 mM). The resistance phenotype is stable in the absence of the drug. Resistance correlates with impaired uptake of [3H]MTX, which in wild-type cells is taken up by a carrier-mediated process. There is no indication of gene amplification at the DNA level or at the level of DHFR activity, as occurs in the case of MTX-resistant Leishmania major. Several lines of MTX-resistant L. major which show gene amplification also exhibit impaired uptake of [3H]MTX.