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Appl. Environ. Microbiol. doi:10.1128/AEM.00188-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

STIMULATION OF ZERO-TRANS RATES OF LACTOSE AND MALTOSE UPTAKE INTO YEASTS BY PRE-INCUBATION WITH HEXOSE TO INCREASE THE ADENYLATE ENERGY CHARGE

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

^* To whom correspondence should be addressed. Email: prguimaraes{at}deb.uminho.pt.

	Abstract

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabelled sugars for 5 – 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth media, washed, suspended in nutrient-free buffer and stored on ice before assay. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae harvested from lactose fermentations were 3- to 8-fold smaller than the specific rates of lactose consumption during fermentation. No significant extracellular -galactosidase was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before starting the ¹⁴C-lactose uptake reactions. Short (1 – 7 min) pre-incubation of the yeasts with 10-30 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, with smaller increases in K_m. Similar observations were made for maltose transport by a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H⁺ symports is significantly smaller in the starved yeast suspensions used for zero-trans assays than in actively metabolising cells. Zero-trans assays with such starved yeast preparations can seriously underestimate the capacity of sugar/H⁺ symports. Short exposure to glucose allows a closer approach to the sugar/H⁺ symport capacity of actively metabolising cells.