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

Abstract

ABSTRACT 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 [ 14 C]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.