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Stimulation of Zero-trans Rates of Lactose and Maltose Uptake into Yeasts by Preincubation with Hexose To Increase the Adenylate Energy Charge

VTT Technical Research Centre of Finland, POB 1000, FI-02044 VTT, Finland,¹ Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal²

Received 21 January 2008/ Accepted 23 March 2008

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular β-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [¹⁴C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 µmol·g dry yeast^–1). Stimulation by glucose affected the transport V_max values, with smaller increases in K_m values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H⁺ symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H⁺ symports. A short exposure to glucose allows a closer approach to the sugar/H⁺ symport capacity of actively metabolizing cells.

Published ahead of print on 31 March 2008.