Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge.
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 beta-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 micromol x 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.
Guimarães
Pedro M R
PM
VTT Technical Research Centre of Finland, POB 1000, FI-02044 VTT, Finland. prguimaraes@deb.uminho.pt
Multanen
Jyri-Pekka
JP
Domingues
Lucília
L
Teixeira
José A
JA
Londesborough
John
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002250:Carbon Radioisotopes / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D005947:Glucose / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase / Q000032:analysis; D007716:Kluyveromyces / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism*; D019897:Periplasm / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D027981:Symporters / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis
D002250:Carbon Radioisotopes; D027981:Symporters; D008320:Maltose; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase; D043324:beta-Fructofuranosidase; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose; D007785:Lactose