[Parameters of the succinate transport into Saccharomices cerevisiae cells after prolonged cold preincubation]

A nonconventional approach to the measurement of succinate transport through plasmalemma is proposed. It is based on the conditions in which the succinate oxidation rate is limited by transport through plasmalemma. Impermeable specific inhibitor of plasmalemma dicarboxylate transporter was employed as a tool to optimize conditions for the transport activity assay. For this purpose yeast culture was grown in synthetic medium under selected conditions. After aerobic preincubation of S. cerevisiae cells at 0 degree C (instead of incubation at 15 degrees C), the rate of endogenous respiration decreased substantially and was stabilized during measurements at a level that was five times lower than oxidation rates in the presence of exogenous substrate. This approach allowed for the reproducible determination of K(M) for the dicarboxylate transporter (7.3 +/- 2.1 mM) within a half-hour period. The advantages and drawbacks of this fast, but indirect, assay of slow substrates transport into the cell are compared with conventional methods.