Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge

Pedro M. R. Guimarães (Corresponding Author), Jyri-Pekka Multanen, Lucilia Domingues, Jose A. Teixeira, John Londesborough

Research output: Contribution to journalArticleScientificpeer-review

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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 [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 Vmax values, with smaller increases in Km 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.
Original languageEnglish
Pages (from-to)3076-3084
JournalApplied and Environmental Microbiology
Volume74
Issue number10
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

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Hexoses
Maltose
hexoses
Lactose
maltose
lactose
yeast
Yeasts
yeasts
uptake mechanisms
sugar
sugars
Ion Transport
energy
glucose
Glucose
Fermentation
cells
Saccharomyces cerevisiae
fermentation

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Guimarães, Pedro M. R. ; Multanen, Jyri-Pekka ; Domingues, Lucilia ; Teixeira, Jose A. ; Londesborough, John. / Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge. In: Applied and Environmental Microbiology. 2008 ; Vol. 74, No. 10. pp. 3076-3084.
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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. / Guimarães, Pedro M. R. (Corresponding Author); Multanen, Jyri-Pekka; Domingues, Lucilia; Teixeira, Jose A.; Londesborough, John.

In: Applied and Environmental Microbiology, Vol. 74, No. 10, 2008, p. 3076-3084.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge

AU - Guimarães, Pedro M. R.

AU - Multanen, Jyri-Pekka

AU - Domingues, Lucilia

AU - Teixeira, Jose A.

AU - Londesborough, John

PY - 2008

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N2 - 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 Vmax values, with smaller increases in Km 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.

AB - 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 Vmax values, with smaller increases in Km 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.

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DO - 10.1128/AEM.00188-08

M3 - Article

VL - 74

SP - 3076

EP - 3084

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

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