Kinetic analysis of hexose uptake in Saccharomyces cerevisiae cultivated in continuous culture.

As the expression of the hexose transporters in Saccharomyces cerevisiae is regulated by the external fructose concentration, in vivo fructose transport was investigated at a number of different but constant fructose concentrations in a continuous culture. The in vitro uptake rate and kinetic parameters were studied by uptake of 14C-labelled fructose. From Eadie-Hofstee plots, based on the results of the in vitro uptake experiment, K(m) and Vmax for the different steady-state conditions were determined. These results demonstrate that as fructose concentrations increase, fructose consumption rates and K(m) increase and Vmax slightly decreases. However, remarkably a large discrepancy was observed between in vivo determined consumption rates in the continuous culture and the in vitro uptake rates from the 14C-labelled fructose uptake experiment on cells from an identical steady-state condition. In every condition the in vivo consumption rate was approx. 2-3 times higher than the in vitro uptake rate. A comparable discrepancy was observed for glucose uptake rates; however, in vivo and in vitro galactose uptake rates were identical. These observations imply that kinetic analyses of sugar transport mechanisms can only be performed correctly when accompanied by a comparison with in vivo determined consumption rates.