Limiting factors involved in CO₂ production by starved and X‐irradiated starved yeast cells

Abstract

Studies of reduced COZ production by starved yeast cells were carried out to localize the site limiting this process and responsible for a greater production by irradiated cells. Cell-free extracts prepared before and after starvation of cells, and from irradiated and unirradiated cells, showed similar hexokinase activity and produced similar amounts of COz. These results demonstrated that rate limiting glycolytic enzymes did not decay during starvation, were not induced during a lag period i n COz production which could be overcome by glucose incubation, and were not responsible for differences in COz production between irradiated and unirradiated cells. Possible limiting factors involved in these differences include glucose transport as a consequence of differential decay during starvation, restricted cofactor synthesis and a n enzymic binding or compartmentalization.We have reported that irradiated yeast cells after a period of starvation (incubation overnight in water) were better able to produce COe, use oxygen and sugar and take up P3' than unirradiated cells similarly starved (Spoerl et al., '60). The possibilities by which radiation effects combined with starvation might produce these results were discussed and examined in connection with pathways of sugar dissimilation (Spoerl and Quiner, '61) and with leakage or permeability of the mernbranes of the cells (Spoerl et al., '64). The relation between the inhibition of cell division, a concomitant effect of the irradiation and a primary interest in this series of studies, and the above observations is still to be clarified.The present experiments were carried out to examine further the fermentative capacity of starved cells and cell extracts to clarify whether sugar transport, a glycolytic deficiency, or an interaction of these processes is limiting COz production. The results indicate clearly that adequate glycolytic enzyme capacity exists in the starved cells. Consequently, some aspect of the processes which make sugar available to or useable by fermentation centers presumably accounts for the differences between irradiated and unirradiated cells. MATERIALS AND METHODSOur culture of Saccharomyces cerevisiae was grown and handled as previously de- scribed (Spoerl et al., '60). Cultures were irradiated for two hours during exponential growth at a dose of 2.0 kr/hr as measured with a benzoate dosimeter. Following irradiation, cells were washed twice with distilled wa.ter by centrifugation, resuspended in distilled water at one-half their original volume and incubated on a reciprocal shaker overnight (21 hours) at 28.5"C as the means of starvation. Stasved cells were washed twice with water before resuspension in buffer for further use. Unirradiated cells were handled in parallel except for the irradiation. Measurements made with starved cells have shown a greater variability than those made with unstarved cells. Consequently, irradiated were always paired with unirradiated cells and preparations to obtain the greater statistical sensitivity of the re...

Inhibition by Methylphenidate of Transport Across the Yeast Cell Membrane

“…Organism and hiandlintg. Yeast cells (Saccharomyces cerevisiae) were grown as described previously (10), except that temperature was adjusted to 28.5 C. For most measurements, the cells were harvested during exponential growth at a count of 2 X 107/ml and washed twice by centrifugation with distilled water before experimental use (11).…”

Selective enhancement of glycolytic activity by incubation of yeast cells in sugars and polyols

“…The data in the last column of Table V show that the capacities of cell extracts to produce C 0 2 from glucose were similar, except where a marked decrease in the capacity to decarboxylate pyruvate was found. Previous measurements showed that rates of C 0 2 output by extracts of starved and unstarved cells also are similar (15). Incubation in solutions of readily used sugars at high concentrations (0.1 M glucose or 0.2 M maltose) reduced the capacity to decarboxylate pyruvate, whereas other compounds did not affect this capacity or increased it.…”

“…Elimination of the lag by ethyl but not by methyl alcohol, however, is consistent with an effect unrelated to a metabolic use (additions of 0.1 M ethyl alcohol, but not 0.1 M methyl alcohol, also removed the lag from cells incubated in 0.2 M lactose and in 0.2 M sorbose). Thus, removal of the lag period may be interpreted as an alteration in intracellular membrane restrictions so that required intermediates, or glucose which enters the cell immediately (15), are able to move to appropriate cellular sites. The components necessary for C 0 2 production evidently were present within starved cells; they simply needed to be brought together.…”