KINETIC-ANALYSIS OF HEXOSE UPTAKE IN SACCHAROMYCES-CEREVISIAE CULTIVATED IN CONTINUOUS-CULTURE

As the expression of the hexose transporters in Saccharomyces cerevisi ae is regulated by the external fructose concentration, in vivo fructo se transport was investigated at a number of different but constant fr uctose concentrations in a continuous culture. The in vitro uptake rat e and kinetic parameters were studied by uptake of C-14-labelled fruct ose. From Eadie-Hofstee plots, based on the results of the in vitro up take experiment, K-m and V-max for the different steady-state conditio ns were determined. These results demonstrate that as fructose concent rations increase, fructose consumption rates and K-m increase and V-ma x slightly decreases. However, remarkably a large discrepancy was obse rved between in vivo determined consumption rates in the continuous cu lture and the in vitro uptake rates from the C-14-labelled fructose up take experiment on cells from an identical steady-state condition. In every condition the in vivo consumption rate was approx. 2-3 times hig her than the in vitro uptake rate. A comparable discrepancy was observ ed for glucose uptake rates; however, in vivo and in vitro galactose u ptake rates were identical. These observations imply that kinetic anal yses of sugar transport mechanisms can only be performed correctly whe n accompanied by a comparison with in vivo determined consumption rate s.