Indian Journal of Experimental Biology
Vol. 47, October 2009, pp. 811-815
Kinetic characteristics of brush border sucrase activation by Na+ ions in mice
intestine
Shiffalli Gupta1, Safrun Mahmood2 & Akhtar Mahmood1*
1
Department of Biochemistry, Panjab University, Chandigarh 160 014, India
Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research,
Chandigarh 160 012, India
2
Received 2 February 2009; revised 2 July 2009
The kinetics of Na+ activation of brush border sucrase (sucrose D-glucosidase E.C. 3.2.1.48) has been studied in mice
intestine. At pH 5.0, 50 mM Na+ ions stimulated sucrase activity by 84%. At pH 7.2, enzyme stimulation was reduced to
16%, whereas, at pH 8.5, 10-100 mM Na+ ions produced 18-45% inhibition of enzyme activity. Kinetic studies revealed that
at pH 5.0, the enzyme activation by Na+ ions was V-type, which changed to K-type at pH 7.2, whereas at alkaline pH (8.5),
Na+ ions inhibited the enzyme activity non-competitively. Using the non-compulsory model of Na+ ion stimulation of brush
border sucrase [Mahmood & Alvarado, Arch Bioch Biophys, 168 (1975) 585] various kinetic constants involved in
activation of sucrase by Na+ ions were determined. It is apparent that Na+ stimulation of brush border sucrase is pH
dependent, which is similar to that described for rat, rabbit and other mammalian species and conform to identical
mechanisms, at least with reference to the affinity type effects, as observed in mice intestine.
Keywords: Brush border sucrase, Mice intestine, Na+ activation kinetics, pH dependent effects
Intestinal brush border sucrase (sucrose D-glucosidase
E.C. 3.2.1.48) is an intrinsic membrane associated
protein. It is a component of the sucrase-isomaltase
enzyme complex, which is a type II integral
membrane glycoprotein of the intestinal brush border
membrane1. The enzyme is located in the vicinity of
glucose transporter, since glucose released by the
hydrolysis of sucrose has kinetic advantage for
transport over the free glucose present in the
medium2,3. The enzyme is stimulated by Na+ ions and
this activation is pH dependent and follows a kinetic
mechanism, identical for Na+ dependent organic
solute transport system in intestine4. The kinetic
mechanism of Na+ activation of brush border sucrase
varies in different animal species, which is either,
pure K type (change in Km alone), V- type (change in
Vmax alone) or a mixed type (a change both in Km and
Vmax) of the enzyme.
In guinea pig, hamster and rat, the enzyme
activation by Na+ ions follows an allosteric, affinity
type or K-kinetics, with an apparent decrease in
__________
∗
Correspondent author
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apparent Km with no change in Vmax. The sucrase
activity in Greek turtle is activated by an increase
both in Vmax and Km of the enzyme. But in rabbit and
human intestines, Na+ gives a capacity-type or Vkinetics5. However, Alvarado and Mahmood4 reported
that alkali metal ions have far more complex effects,
such that at acid pH values a biphasic effect occurs
involving both K-type activation and, at high
concentrations, K-type inhibition. Above pH 7, noncompetitive inhibition occurs6.
pH dependent conformational changes in brush
border sucrase in mice intestine have been reported7.
However, the kinetic mechanism of Na+ ion
stimulation of sucrase in murine intestine has not been
investigated. This communication reports pH
dependent effects of Na+ ions on kinetic parameters of
brush border sucrase in mice intestine.
Following the non-compulsory model of Na+ ion
stimulation of brush border sucrase, in guinea pig
intestine8, various kinetic constants involved in
enzyme-substrate (Ks) or enzyme-metal ion (Ka)
interactions, and enzyme-substrate in the presence of
Na+ (Ksa) and enzyme-Na+ in the presence of the
substrate (Kas), were determined, as described in the
following.
812
INDIAN J EXP BIOL, OCTOBER 2009
General model for the activation of brush border sucrase by alkali
metal ions.
E= Enzyme; S= Substrate; A= Activator
Materials and Methods
Chemicals and buffers―All the chemicals used
were of analytical grade. Bovine serum albumin, 4aminoantipyrine, glucose oxidase (Type V), glucose
peroxidase, D-glucose and Tris were purchased from
Sigma Chemical Company, Saint Louis, MO (U.S.A).
Various buffers of different pH (5.0, 7.2 and 8.5)
were prepared by mixing boric acid, maleic acid,
orthophosphoric acid and lithium hydroxide as
described earlier4.
Purification of brush border sucrase―Balb c mice
kept on standard pellet diet with free access to water
were used. Intestinal brush border sucrase was
purified following the method of Alvarado
and
Mahmood4. Briefly, sucrase-isomaltase activity was
solubilized by treating the microvillus membranes
with papain (2 mg/10 mg membrane protein). The
soluble enzyme preparation was subjected to
Sephadex G200 and DEAE Cellulose column
chromatography. Fractions from DEAE cellulose
column corresponding to sucrase activity were
pooled, concentrated and the final enzyme preparation
(2.5 units/mg protein) showed 155-folds enzyme
enrichment compared to tissue homogenate, with
nearly 23% enzyme yield. On SDS-Polyacrylamide
gel, the enzyme preparation yielded a single protein
band with a molecular weight around 210 kDa.
Protein estimation―Protein was determined by the
method of Lowry et al.9 using bovine serum albumin
as the standard.
Assay of sucrase activity―Sucrase activity was
assayed following a two-step procedure as described
by Dahlqvist10. After stopping the reaction with 0.5 M
Tris HCl (pH 7.2), glucose liberated by sucrose
hydrolysis, was determined by glucose-oxidase and
peroxidase system using 4-aminoantipyrene as the
chromogen.
Effect of Na+ ions―Sucrase activity was assayed in
absence and presence of different concentrations of
Na+ ions (10, 20, 40, 50, 60, 80 and 100 mM) at
different pH (5.0, 7.2 and 8.5), using the buffers
described above.
Kinetic studies―Kinetic parameters of sucrase were
determined by assaying the enzyme activity at different
sucrose concentrations (8-40 mM) in absence and
presence of 100 mM Na+ ions. The enzyme reaction
was first order under the assay conditions. The values
of kinetic parameters Km and Vmax were calculated
using Woolf plot as described earlier4.
Determination of kinetic constants―Various
kinetic constants were determined using equation
derived for non compulsory enzyme activation using
the allosteric model proposed by Mahmood and
Alvarado8. The plot of Na+/ΔK (ΔK = change in
apparent Km of the enzyme at various concentration of
Na+ ions) versus Na+ was made, which yielded a
straight line. From the intercept and slope of the line,
various kinetic constants involving enzyme, substrate
and Na+ ions were determined8.
Results
Figure 1 shows the effect of 10-100 mM Na+ ions
on sucrase activity as a function of pH. At pH 5.0,
addition of 50 mM Na+ ions activated the enzyme by
84%, but further increase to 100 mM, did not produce
any change in enzyme activity. At pH 7.2, 100 mM
Na+ ions stimulated the enzyme activity by 16% only.
However, at pH 8.5 sucrase activity was inhibited by
18-45% in presence of 10-100 mM Na+ ions
compared to the controls.
Fig. 1―pH dependent effects of sodium ions on brush border
sucrase in mice intestine. Values are mean of 2 preparations.
Enzyme assays were done in duplicate.
GUPTA et. al.: SUCRASE & Na+ IONS INTERACTIONS
Data showing the effect of Na+ on kinetic
parameters of sucrase as a function of pH is given in
Table 1. At pH 5.0, the activation of sucrase by Na+
ions was primarily a capacity effect, where the Vmax
was increased (P<0.001) from 2.65 in absence to 6.82
units/mg protein in presence of 100 mM Na+. At pH
7.2, the value of Km was reduced from 138 mM at 0
mM Na+ ions to 107.3 mM at 100 mM Na+ ions with
no change in the value of Vmax (4.65 units/mg protein).
This suggests an affinity type activation of mice brush
border sucrase by Na+ ions at pH 7.2. In contrast, at
pH 8.5, a non-competitive type of enzyme inhibition
was observed, the value of Vmax was reduced to 0.9
units/mg protein in presence of 100 mM Na+ ions
from 1.32 in the absence of Na+ ions. The value of Km
(96.7 mM) was unaffected, under these conditions.
813
As expected, the plot of (Na+/ΔK) versus (Na+)
gave a straight line where ΔK= Ks-Km and from the
values of the intercept and slope of the line (Fig. 2),
all four dissociation constants were calculated. The
value of R=Ks/Ks′ = Ka/Ka′ was also determined.
Ratios Ks/Ksa and Ka/Ksa indicated the degree of
activation of the binding of S (substrate) caused by A
(Na+) and also the converse. The kinetic constants
calculated for mice sucrase from these plots are listed
in Table 2 and compared with those reported for
sucrase in a number of other animal species. The
values of Ks and Ks′ are 138 mM and 104.7 mM
respectively, whereas, those of Ka and Ka′ are 8.79
mM and 6.67 mM respectively of mice sucrase. The
corresponding value of Ksa is 0.92 mM, in the mice.
The affinity type activation of sucrase, by Na+ ions
at pH 7.2 was further analysed according to kinetics
of non-compulsory allosteric model proposed by
Mahmood and Alvarado8. This model not only helps
to calculate the constants involving enzyme-substrate
(Ks) or enzyme-metal ion (Ka) interactions but also
the constants involving enzyme-substrate in the
presence of Na+ (Ksa) and enzyme-Na+ in the presence
of the substrate (Kas).
Table 1―Effect of sodium on kinetic parameters of brush border
sucrase at different pH in mice intestine
[Values are Mean ± SD from 3 observations]
pH
5.0
7.2
8.5
Na+=0 mM
Km
Vmax
(mM)
(μmoles/mg
protein)
130±15.8
138±8.16
96.7±1.32
2.65±0.19
4.65±0.42
1.32±0.11
Na+=100 mM
Km
Vmax
(mM)
(μmoles/mg
protein)
136.5±2.12
107.3±1.1β
90±5.1
6.82±0.11a
5.16±0.6
0.9±0.04γ
Fig. 2―Affinity type of activating effect of sodium on brush
border sucrase at pH 7.2. The data is plotted as [Na+/ΔK] = f[Na+]
following kinetics of allosteric non-compulsory model (Ref. 8).
The straight line obtained is: y=0.072 + 0.057x. ΔK= Ks – Km.
Table 2―Comparison of kinetic parameters of mice brush border sucrase with those of various other species
Parameter
Ks (mM)
Ks′ (mM)
Ka (mM)
Ka′ (mM)
Ksa (mM)
R
Ks/Ksa
Ka/Ksa
Ka/Ks
8
8
Guinea pig
(pH 6.0)
11
Hamster
(pH 5.4)
11
Rat
(pH 5.4)
13
Man
(pH 5.2)
13
Man
(pH 6.8)
4
Rabbit
(pH 5.2)
14
Rabbit
(pH 6.5)
Mice
(pH 7.2)
333
17.7
12.1
0.64
0.21
18.8
1560
56.6
0.036
1560
23.9
52.7
0.81
1.26
65.2
1240
41.8
0.034
59.4
16.1
3.4
0.93
0.055
3.69
1080
61.8
0.057
29.9
15.4
1.8
0.94
0.028
1.94
1068
64.3
0.06
70.1
8.7
7.7
0.96
0.067
8.05
1046
115
0.11
11.6
8.6
1.2
0.91
0.01
1.35
1160
120
0.1
138
104.67
8.79
6.67
0.92
1.32
150
9.5
0.063
50
4.0
1.76
0.14
0.007
12.5
7140
250
0.035
Mahmood and Alvarado (1975);
Semenza (1967)
11
Semenza (1969);
13
Semenza et al (1964); 4Alvarado and Mahmood (1979);
14
Kolinska and
814
INDIAN J EXP BIOL, OCTOBER 2009
Discussion
The present findings indicate that stimulation of
brush border sucrase by Na+ ions in mice intestine is a
pure K-type system at pH 7.2, where, the value of Km
was reduced with no change in the enzyme Vmax in
response to Na+ ions. These results are similar to
those in guinea pig which is also K type system, as
described earlier8. Analysis of various kinetic
constants involved in activation of sucrase by Na+
ions revealed that the value of Ks and Ka for mice is
close to that reported for hamster4 and rabbit11.
However, the values of Ks′ and Ka′ are quite high
compared to those described for various other animal
species. The Ksa value for mice sucrase is similar to
that of rabbit at pH 6.814. The value of R for mice was
1.32, which is also similar to that of rabbit at pH 6.5
but it is low compared to that in other animal species
examined, which suggests that Na+ is a poor activator
of brush border sucrase at pH 7.2 in mice intestine.
Semenza11 reported low value of R for K+ in hamster
intestine and which makes this ion a relatively poor
activator of sucrase activity. The value of Ka/Ksa is
quite low for the mice enzyme. These observed values
are similar to that reported for sucrase in other animal
species, which indicates that Na+ ion is a poor
activator of the enzyme in mice intestine at pH 7.2
compared to substrate activation of the enzyme in
presence of Na+ ions. The values of Ks and Ks′ were
138 and 104.7 mM respectively, whereas, those of Ka
and Ka′ were 8.79 and 6.67 mM respectively for mice
sucrase. This suggests the similarity of the enzyme to
metal ion activation in hamster and rabbit intestine.
Alvarado et al.12 reported that the kinetic constant
involved in affinity-type activation kinetics of
membrane bound rabbit sucrase is 3.1 and for the
soluble enzyme is 3.4 at pH 5.2. It was suggested, that
the two forms of the enzyme are kinetically
indistinguishable4, under these assay conditions.
The apparent values of both Ks′ and Ka′ are pH
independent whereas Ks and Ka vary hyperbolically
with a change in pH6. Comparison of various enzyme
constants calculated for mice, rabbit and other
mammalian species showed that enzyme kinetics
conform to identical mechanisms, at least with
reference to the affinity type effects, as described in
the present study. The present data also shows that
metal ion interactions with brush border sucrase in
mice intestine are pH dependent. Thus sodium
stimulation of brush border sucrase in various
mammalian species examined is modulated by pH,
suggesting the dissociation of certain key amino acid
residues to be responsible for this phenomenon.
Vasseur et al.15 suggested that dissociation of proton
in the acidic pH (pH 5.4) is involved in k-type
kinetics of sucrase activation by Na+ ions in rabbit
intestine. Although the nature of amino acid residues
responsible for such phenomenon in mice sucrase is
unknown, the enzyme kinetics suggest that metal ion
interactions with sucrase are influenced by pH, as a
consequence of ionization of certain key amino acid
residues in mice intestine. In conclusion, the data
presented herein indicate that similar to other
mammalian intestinal sucrases, the enzyme from mice
intestine exhibits pH dependent Na+ ion interactions,
which may suggest a common genetic origin of this
process.
Acknowledgement
Shiffalli Gupta was supported by senior research
fellowship from University Grants Commission, New
Delhi.
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