EFFECTS OF AMPHOTERICIN-B ON THE GLUCOSE-METABOLISM IN SACCHAROMYCES-CEREVISIAE CELLS - STUDIES BY C-13-NMR, H-1-NMR AND BIOCHEMICAL METHODS

A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [1-C-13]glucose metabolism in glucose-grown repressed Saccharomyces cerevisiae cells were studied as a function of time using C-13, H-1-NMR and biochemical methods. The C-13 enrichment of different compounds such as ethanol, glycerol and trehalose were determined by H-1-NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1-mu-M, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The C-13 relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus, it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio (almost-equal-to 1.0) is practically constant upon addition of glucose which suggest that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1-mu-M amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.