ARTICLE NO.
0244
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.
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
26
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Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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Invertase Reaction Rates and Normalized Reaction Rates Obtained for NaF-Inhibited Saccharomyces cerevisiae Cells
Sucrose grown cells
Yeast strains
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.
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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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.
REFERENCES
1. Gascon, S., and Lampen, J. O. (1968) J. Biol. Chem. 243, 1567
1572.
2. Moreno, F., Ochoa, A. G., Gascon, S., and Villanueva, J. R. (1975)
Eur. J. Biochem. 50, 571579.
3. Creanor, J., May, J.W., and Mitchison, J. M. (1975) Eur. J. Biochem. 60, 487493.
4. Vitolo, M., and Borzani, W. (1983) Anal. Biochem. 130, 469470.
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05-17-96 08:20:18
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