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ANALYTICAL BIOCHEMISTRY

ARTICLE NO.

238, 2628 (1996)

0244

Assay for in Vivo Yeast Invertase Activity Using NaF


M. C. F. Silveira,* E. Carvajal,*, and E. P. S. Bon*,1
*Instituto de QuB mica/Universidade Federal do Rio de Janeiro, CT, Bloco A, Ilha do Fundao, CEP 21949-900, Rio de
Janeiro, RJ, Brasil; and Departamento de Biologia Celular e Genetica, Instituto de Biologia, Universidade Estadual do
Rio de Janeiro, Rua Sao Francisco Xavier, 524, CEP 20559-900, Rio de Janeiro, RJ, Brasil

Received February 5, 1996

The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of
sucrose into glucose and fructose. When whole yeast
cells are used in these assays, the monosaccharides
formed by the action of the periplasmic enzyme can
be taken up and metabolized, leading to errors on the
enzyme activity determination. This study reports a
method for a more accurate invertase activity measurement by blocking the glycolytic pathway. In this
method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase. This in vivo
measurement of the enzyme activity, under initial rate
conditions, was performed using cell concentrations
up to 64 mg cell/ml. The results obtained showed that
this method is particularly useful for cells with low
invertase activity. q 1996 Academic Press, Inc.

Eukaryote invertase (b-fructofuranosidase, EC 3.2.


1.26) catalyzes the hydrolysis of sucrose into glucose
and fructose. Measurement of in vivo periplasmic yeast
invertase is of great interest in metabolic studies of
Saccharomyces spp., which are widely used in food,
beverage, and ethanol fuel industries. The methods described for periplasmic invertase activity determination are based on the measurement of glucose (13) or
total reducing sugars (49). We did not detect invertase activity in Saccharomyces cerevisiae D273-10B
by performing the enzymatic reaction as described in
the literature (4). Considering that these results could
be due to the cell consumption of the glucose and fructose produced by the enzyme, one possibility to overcome this drawback was to prevent glucose metabolization. We hereby describe a method for invertase activity
determination which includes the cells preincubation
1
To whom correspondence should be addressed. Fax: (55) (21) 2904746.

with sodium fluoride, an inhibitor of the enzyme enolase (10). Data will be presented suggesting that by
blocking the glycolytic pathway, glucose uptake was
also affected (11). The glucose produced upon invertase
hydrolysis was quantified in the reaction mixture supernatant.
MATERIALS AND METHODS

Yeast strains. S. cerevisiae D273-10B and baker


yeast (Fleishmann Royal Ltd.) were used in the present
work.
Growth conditions. D273-10B cells were grown on
media containing 0.67% yeast nitrogen base without
amino acids (Difco) and 2% of either glucose or sucrose
as the carbon source in a rotatory shaker at 297C and
160 rpm. Bakers yeast was purchased from the supermarket as a pressed cell cake. Unless otherwise indicated cell concentrations were expressed as dry weight
per milliliter.
Cell suspensions. D273-10B cells from mid-log
phase at 0.7 mg cell/ml were collected by centrifugation
at 47C. Cell sediment was washed twice in cold 50 mM
sodium acetate buffer, pH 5.0, and resuspended in the
same buffer to different cell concentrations. Bakers
yeast was washed three times in the same buffer (15
ml/g wet wt) and a cell suspension of 5 mg cell/ml was
used for the enzyme activity determination.
Measurement of invertase activity in whole cells.
The inhibition step consisted of incubating 8 ml of cell
suspensions, within the concentration range of 6 to 64
mg cell/ml in 50 mM NaF, at 307C for 30 min, under
agitation. The invertase activity assay was started by
the addition of 4 ml of 300 mM sucrose solution in 50
mM sodium acetate buffer, pH 5.0. The reaction mixture was incubated at 307C, under agitation, and 1-ml
samples were withdrawn and filtered through a Millipore membrane (0.45 mm) (4) at different time intervals
up to 10 min. The kinetic zero time was performed by

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Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

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YEAST INVERTASE ASSAY USING NaF


TABLE 1

Invertase Reaction Rates and Normalized Reaction Rates Obtained for NaF-Inhibited Saccharomyces cerevisiae Cells
Sucrose grown cells

Yeast strains

Glucose grown cells

Cell concentration
(mg/ml)

Reaction rate
(mmol/min) (U)

Normalized rate
(U/mg dry wt cell)

Reaction rate
(mmol/min) (U)

Normalized rate
(U/mg dry wt cell)

6
9
25
45
64
5

0.96

3.75
6.30
8.96
9.23a
10.53

0.16

0.15
0.14
0.14
2.77a
3.19

0
0
0.45

0
0
0.01

D273-10B

Baker yeast

Note. Data are presented for different cell concentrations for sucrose- and glucose-grown cells. Values represent the averages of three
experiments.
a
Invertase rates for NaF-untreated cells.

adding sucrose after the filtration step. The glucose


concentration in the filtrate was determined with a glucose analyzer (Beckman II). The enzyme activity was
reported as mmol of glucose produced per minute per
milligram cell (U/mg cell).
Activity of a commercial invertase in the presence of
NaF. A 0.006% invertase (Sigma I-9253) solution in
50 mM sodium acetate buffer, pH 5.0, was preincubated
with 50 mM NaF at 307C for 30 min. The invertase
activity assay was started by the addition of 5 ml of a
300 mM sucrose solution in 50 mM sodium acetate
buffer, pH 5.0, to 10 ml of the treated enzyme solution.
At given time intervals up to 10 min, samples were
withdrawn and boiled for 5 min before glucose determination. For the kinetic zero time sucrose was added
after the boiling step. A control experiment was performed using an enzyme solution without NaF. Invertase activity was reported as mmol of glucose produced per minute per milligram of invertase (U/mg of
invertase).
RESULTS AND DISCUSSION

No influence of NaF on the commerical invertase activity. In order to discount any effect of NaF on invertase, the enzyme activity was measured in the presence of the salt. The enzyme activities in the presence
and absence of NaF were 32.8 and 35.4 U/mg of invertase, respectively, showing that NaF has no effect
on invertase activity.
Invertase activity in S. cerevisiae D273-10B cells.
No enzyme activity was detected in untreated cells regardless of the carbon source (glucose or sucrose) used
in the culture medium. Table 1 presents the reaction
rates and the normalized reaction rates obtained for
cell concentrations from 6 to 64 mg cell/ml, grown on
glucose and sucrose media, which were preincubated

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with NaF. Enzyme activity expressed by normalized


rates, in glucose-grown cells, was 15 times lower (0.01
U/mg cell) than in sucrose-grown cells (0.15 U/mg cell).
This result was expected, as invertase production is
known to be glucose repressed (12). The reaction rates
for sucrose-grown cells varied proportionally to the cell
concentrations within the range of 0.96 to 8.96 mmol of
glucose per minute. Therefore, first-order kinetics were
observed under the working conditions used. A normalized invertase activity within the range of 0.14 to 0.16
U/mg cell was observed for all cell concentrations.
Thus, NaF inhibition of the glycolytic pathway prevented sugar uptake (11, 13) and allowed the measurement of enzyme activity. In the data presented in Fig.
1, with inhibited cells, glucose was produced by the
invertase action at a rate of 0.15 U/mg cell. When uninhibited cells were used no glucose was detected, an
indication that under this condition glucose uptake was
at least equivalent to the rate of glucose production by
invertase action, i.e., 0.16 U/mg cell. Glucose uptake
rates within this range have been reported for S. cerevisiae (13).
Invertase activity in bakers yeast. Similar activities,
around 3.0 U/mg cell, were presented by inhibited and
uninhibited cells (Table 1). If for bakers yeast the glucose uptake rate is the same as for strain D273-10B,
i.e., 0.15 U/mg cell, and its glucose production rate is
3.0 U/mg cell, only 5.0% of the produced sugar would be
absorbed; therefore, the extracellular glucose concentration would be similar in inhibited and uninhibited cells.
This result is in accordance with the literature as the
bakers yeast invertase activity determination is usually
carried out without any previous procedure to block the
glycolytic pathway or the glucose transport system (4,
8, 9, 14).
In conclusion, a method was developed for the measurement of invertase in S. cerevisiae strains with low

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SILVEIRA, CARVAJAL, AND BON

FIG. 1. Invertase reaction rates of S. cerevisiae D273-10B cells. A cell suspension of 6 mg/ml in 50 mM sodium acetate buffer, pH 5.0,
was incubated under agitation at 307C with 300 mM sucrose solution with (l) and without (s) 50 mM NaF. Data represent the average
values of two experiments.

enzyme activity. This method, based on the inhibition


on enolase by NaF, is particularly useful when the
invertase activity cannot be detected by published
assays.
ACKNOWLEDGMENTS
This work has been supported by CNPq, CAPES, and PADCT/
FINEP.

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