J. Nutr.
THE EFFECTS INTESTINAL Setsuko
Sci. Vitaminol.,
OF MALTITOL ON DISACCHARIDASES
YOSHIZAWA, Sachiko
MORIUCHI, and
21, 31-37,
1975
RAT
Norimasa
HOSOYA1
Nutritional Laboratory, School of Health Sciences, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo (Received November 21, 1974)
Summary and
maltitol
denum.
The found
However,
low
compared
2.
For
low
compared
3.
The
equal
with
initial
was
velocity
(v)
in the
these
Furthermore,
the
maximal
velocity
maltitol,
at
of
that
in
both
and very
for
sucrose
the
presence
of
maltose
case both
and
mole
pure
fractions
maltose. maltose
The and
the
ileum
maltose and
was
Vmax
sucrose
duo
extremely
substrates
and
was
than
the
these
the
sum of the
there
is
sub initial of
the
other
competi
enzyme.
constant
(Km)
and
the
mixed
substrates,
i.e.,
of
maltose
showed
dependence
kinetic react
was maltitol
the
absence
that
same
and
and
maltitol
very
maltitol,
maltitol,
less
demonstrates
obtained
was
between
and
(vl, v2) in
the
value
sucrose
competition
maltose
Michaelis for
sucrose,
the
maltose.
of
no
for
for
maltitol
substrates
maltitol
substrates
apparent
and
and
of
This finding two
for
large
individual Thus,
the
by
maltose.
was
for
In
(Vmax)
various
fraction
evidence of
(v) rates
(v•ƒvl+v2).
between
mole
values
presence for
followed activity
sucrose
(v=v1+v2).
rates
substrate
the
observed.
individual
tion
for
activity
jejunum,
Km value
of the
v2) respectively
strates
the
disaccharidase
velocity sum
disaccharidase
in
that the
with
the
highest
the
maltitol,
to
(v1,
1. was
at
data the
single
apparent
maltose
provide active
and on
the
strong center
maltase.
Maltitol is a sugar alcohol of the disaccharide maltose and is similar to it in structure, but is not found in nature. Previous studies showed that maltitol is absorbed to a small extent by the small intestine and that the absorbed maltitol is relatively quickly excreted in the urine (1, 2). A considerable amount of maltitol is found at the surface of the intestinal mucosa (2). This is very interesting from the point of view of 1 吉 沢 節子,森
内幸 子,細 谷 憲 政
Abbreviations used: Tris, tris (hydroxymethyl) universally labeled with 14C. 31
amino methane; maltitol-U-14C, maltitol
32
S. YOSHIZAWA,
S. MORIUCHI,
and N. HOSOYA
current concepts of membrane digestion (3, 4), since the disaccharidases are also found on the intestinal surface membrane, i.e., the brush borders (3, 4). It was therefore of interest to study the effect of maltitol on the kinetics of intestinal disaccharidases in the presence of a naturally occurring disaccharide, such as maltose or sucrose.
MATERIALS Female
rats
laboratory libitum.
The The
denum,
animals
entire
and
Elvehjem in
larger
were
a
cell
strate
solution
was
of
supernatant
disaccharidase One-tenth
oxidase the using
bovine Sucrose
Maltose Hayashibara Biochemical
and
The
a
serum
of
maltitol Co.,
as were
were Ltd.,
test
the
for
the
the
the
and
ad
duo
washed
Four
performed
in
parts a Potter
homogenate to
was
remove
nuclei
experiment. of
DAHLQVIST
The
maleate
open slide.
15min
method
solution
into
slit
Then for
standard given
decapitated.
a glass was
used
(5).
concentration
buffer,
pH
contained
enzyme and
solution
Su
of
6.0.
An
a suitable
was
oxidase Protein
reagent
was
was
incubated
produced
sub
aliquot
amount
of
at
mixed 37•Ž.
determined was
determined
by
0.1ml
After with
prepared the
with
LOWRY
an
ap
Tris-glucose according
to
method
(6),
a standard.
purchased
kindly
while
were
substrates.
tube
glucose
(5).
albumin
by as
the
Tris-glucose
glucose
subdivided
pestle.
sodium
that
and
with
1,000•~g were
diluted
small
of DAHLQVIST
and
at
0.1M
being
a
were
assay.
the
amount
reagent.
procedure
the of
in
the
a teflon
given
water
before
homogenization
used
so
off
were and
intestines
assayed
in
diluted
hr
scraped
the
were
for
solution time,
was
24
removed
supernatants
0-56mM
milliliter
substrate
propriate
was
with
maltitol
activity
for
centrifuge The
was
fast
Food
subdivided
and
activity and
of
added,
debris.
maltose
the
The
150-180g,
Osaka).
quickly
mucosa
refrigerated
Disaccharidase crose,
to
homogenizer fitted
centrifuged and
made was
METHODS
weighing
Co.,
ileum. The
water
strain, Yeast
intestine
saline.
redistilled
Wistar
were
small
ice-cold
and
the
(Oriental
jejunum
with of
of
chow
AND
from
provided
glucose
by
oxidase
the the was
Kokusan
Chemical
Nikken
Chemical
obtained
from
Works, Co., the
Ltd.
Ltd.. and
Worthington
Company.
RESULTS
1. Distribution of disaccharidases in rat small intestine The activity of disaccharidases was observed in various parts of the rat small intesine (Table 1). The activities of maltase and sucrase were the highest in the jejunum, followed by those in the ileum, while the duodenum exhibited very low activity. This pattern of enzymatic activity was also observed with maltitol, al though the activity was much lower compared with that obtained with maltose
MALTITOL Table
1.
AND
Specific
INTESTINAL
activity
DISACCHARIDASES
of disaccharidases
of the small intestine
(): a The
number of disaccharidase
buffer,
pH
6.0
calculated or
determinations. activity and
from
maltitol
the
the
Mean•}S
. E. M.
b
of
1 mole
value
for
assayed
of
was
but
The
that
divided
substrate
of
by
in
0 .1M
sodium
hydrolyzed/hour/mg
produced.
glucose,
maltose
28mM
substrate
glucose
parts
of the rat.a
with
as ƒÊmoles
amount
produces
Therefore,
was
expressed
in the various
33
hydrolysis
maltose
maleate
protein. of
produces
It
1 mole 2 moles
was
of
sucrose
of
glucose.
2.
and sucrose. The jejunum portion of the small intestine was used in all subsequent enzymatic studies because its disaccharidase activity was highest. 2. Properties of disaccharidases in rat intestine The properties of disaccharidases were analyzed in the jejunum of rat small intestine. The disaccharidases activities were determined for maltose, sucrose and maltitol in the different substrate concentrations (Fig. 1). From these, the Michaelis constant (Km)and the maximal velocity (Vmax)for sucrose, maltose and maltitol were determined by the graphical method of the Lineweaver-Burk plots (Table 2). The Km value for maltitol was extremely large compared with that for maltose and sucrose. This observation demonstrates that the disaccharidase has little affinity for maltitol. Furthermore, the lowest Vmaxwas found when maltitol was used as substrate. These results indicate that maltitol is enzymatically not easily available to the rat. 3.
The
effect
of
Previous stered
in
this
consisting
the
presence If
in
the
vidual interaction
there
of
presence
of
substrates, between
the not
mucosa.
carried
of
that
was
intestinal
were
hydrolysis
to
maltitol
rat
were
the shown
bound
disaccharidases
servations in
is
study, of
the
on have
maltitol-U-14C
ever,
with
maltitol
studies
a
surface
inhibits
out
to
and
of
will
the
be
the
of
intestinal
by
considered of
how
that sucrose
sucrose
orally
mucosa
hydrolyzed
hydrolysis
determine
maltose amount
significantly It
and
sucrose
considerable
the
How
preparation
maltitol or
and
admini
(2).
combines
maltose.
maltose
Ob
hydrolyze
maltitol. no
interaction
two
substrates
v1
and
the
v2 two
between should
two be
(v=v1+v2). substrates,
On then
substrates,
equal
to the
v•ƒv1+v2
the
the sum
other
of
hand, (7).
initial those if
velocity for there
the were
(v) indi an
34
S. YOSHIZAWA,
Fig.
1. The activities of the disaccharidases activities were determined as described
Table 2.
Kinetic studies were calculated Burk.
The
effect
velocity 1.03,
of
(v=mM which
and
S. MORIUCHI,
was
maltitol,
petition
maltitol
equal
i.e.,
between
The
initial about
i.e.,
8.08
the
so-called
and
maltitol
the
velocity
and
and
sucrose
13%
0.28,
of
rate
was
observed
presence for
0mM to 56mM.
of
the
(Fig.
The
and
individual
(v=vi+v2).
2).
sucrose
substrates,
Thus,
there
initial
maltitol
was sucrose
was
no
com
maltitol. maltitol
for
the
mixture
the
sum
of
on
maltase
of
maltose
the
individual
(v•ƒvl+v2) between
the
the
respectively
respectively
at
the
Vmax and Km values of Lineweaver and
varied from
activity in
of
"competition" react
sum
and
than
0.39,
sucrase
effect
(v) less
the
The method
concentrations
produced/hr) to
0.78
Furthermore,
was
on
HOSOYA
of maltose, sucrose and maltitol. The enzymatic under MATERIALS AND METHODS.
on disaccharidases. by the graphical
Substrate
glucose
and N.
same
active
site
and of
the
and
3).
was
observed
maltitol
rates
(Fig.
maltose
activity
for These
maltitol, disaccharidase
and
was
maltose
(Fig. 7.35,
and
which maltitol,
results
demonstrate
suggest
that
"maltase."
3).
maltose
MALTITOL
Fig. 2
AND
INTESTINAL
DISACCHARIDASES
Fig.
35
3
Fig. 2. Effect of maltitol on sucrase activity. The initial velocity was determined with sucrose, maltitol, or the mixture of sucrose and maltitol. Substrate concentration was 28mM. The ordinate shows mM glucose produced and the abscissa shows incubation time in minutes. From the figure, the initial velocity for sucrose, maltitol, or mixture of sucrose and maltitol is obtained as 0.78, 0.28, or 1.03, respectively. The initial velocity was expressed as mM glucose produced/hour instead of mM substrate hydrolyzed/ hour. Fig. 3. Effect of maltitol on maltase activity. The initial velocity for maltose, maltitol, or the mixture of maltose and maltitol was determined as described in Fig. 2. That for maltose, maltitol, or the mixture of maltose and maltitol is 8.08, 0.39, or 7.35, respectively.
In order to study the competitive mechanism between maltose and maltitol, the apparent Michaelis constant Km and the apparent maximal velocity (V) were determined for single and mixed substrates at various mole fractions of maltose. Lineweaver-Burk plots of 1/v against 1/s were obtained for mixed substrate solutions having various maltose mole fractions (f) (Fig, 4-a). These plots may reasonably be regarded as being straight lines, and from them the apparent Michaelis constant (Km) and the apparent velocity (V) were calculated. The
36
S. YOSHIZAWA,
S. MORIUCHI,
and
N. HOSOYA
dependence of Km and V on the mole fraction (f) of maltose is shown in Fig. 4-b,c. Thus, the kinetic features above, obtained with the mixed substrate, maltose and maltitol, are consistent with those predicted for maltose and maltitol acting in competition for a single active center.
Fig. 4. Lineweaver-Burk plots for mixed substrates: maltose and maltitol (a) and dependence of Km (b) and V on f(C). This kinetic study was carried out by changing maltose and maltitol concentrations, but without changing the ratio of each substrate concentration. The mole fractions (f) of maltose were 0, 0.17, 0.33, 0.50, and 1.00. In this case, a mole fraction of maltose (f) was calculated as follows: f=a/a+b, where a and b represent the substrate concentrations of maltose and maltitol, respectively. (The points corres ponding to f=0 and f=1 represent the values for pure maltitol and maltose, respectively.) Km and Vmax was obtained from each fraction of maltose. V is the initial rate for the total substrate concentration(s), which is the sum of the molar concentrations of maltose and maltitol.
DISCUSSION
Previous
studies
in our
laboratory
have
shown
that
a considerable
amount
MALTITOL
AND
INTESTINAL
DISACCHARIDASES
37
of orally administered maltitol-U-14C is bound to the surface of rat intestinal mucosa, even though maltitol is not utilized by this animal (1, 2). Recently, it has been demonstrated that the disaccharides are hydrolysed on the membranes of the small intestinal mucosa (3, 4). This type of digestion is characterized by the proximity of enzymatic hydrolysis to the surface of cell membrane, which separates the intracellular and extracellular environments. The enzymes involved in these reactions are fixed onto the cell membrane. In the present paper, we report the effect of maltitol on the activity of the intestinal disaccharidases, sucrase and maltase. Our studies demonstrate that maltitol and maltose compete for the same enzyme. The kinetic features obtained with the mixed substrates, maltose and maltitol, are consistent with the kinetics predicted for the competition for a single active center on the enzyme (7). There fore, it can be reasoned that maltitol is hydrolysed by maltase. However, for maltitol, the Kmvalue is very large and the Vmaxvalue is very small, as compared to these values for maltose. Therefore, only very little hydrolysis of this synthetic sugar alcohol would occur in the small intestine. It has been reported that maltitol feeding causes growth retardation (1). Our data suggest that this reduced growth would be due to the inhibition of the digestion of maltose by maltitol. Thus, in the presence of excess maltitol, the hydrolysis of maltose, a product of natural dietary carbohydrate, would be reduced; as a result, less monosaccharides will be available for absorption and consequently the calorie intake would be decreased. We would like to thank Dr. M. Zile (Department of Biochemistry, University of Wisconsin, U. S.A.) for her kind reading of our manuscript. This research was supported by the Scientific Research Fund of the Ministry of Education of Japan (944021).
REFERENCES
1) 2) 3)
INOUE,Y., MORIUCHI,S., and HOSOYA,N., J. Jap. Soc. Food and Nutr., 23, 43 (1970). OKU, T., INOUE,Y., and HOSOYA,N., J. Jap. Soc. Food and Nutr., 24, 399 (1970). UGOLEV,A. M., Physiology and Pathology of Membrane Digestion, Plenum Press, New York (1968). 4) UGOLEV,A. M. and DELAEY,P., Biochimica et Biophysica Acta, 300,105 (1973). 5) DAHLQVIST,A., Anal. Biochem., 7, 8 (1964). 6) LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L., and RANDALL,R. J., J. Biol. Chem., 193, 265 (1951). 7) HIROMI,K., HAMAUZU,Z., and TAKAHASHI,K., J. Biochem., 59, 411 (1966).
THE EFFECTS INTESTINAL Setsuko
Sci. Vitaminol.,
OF MALTITOL ON DISACCHARIDASES
YOSHIZAWA, Sachiko
MORIUCHI, and
21, 31-37,
1975
RAT
Norimasa
HOSOYA1
Nutritional Laboratory, School of Health Sciences, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo (Received November 21, 1974)
Summary and
maltitol
denum.
The found
However,
low
compared
2.
For
low
compared
3.
The
equal
with
initial
was
velocity
(v)
in the
these
Furthermore,
the
maximal
velocity
maltitol,
at
of
that
in
both
and very
for
sucrose
the
presence
of
maltose
case both
and
mole
pure
fractions
maltose. maltose
The and
the
ileum
maltose and
was
Vmax
sucrose
duo
extremely
substrates
and
was
than
the
these
the
sum of the
there
is
sub initial of
the
other
competi
enzyme.
constant
(Km)
and
the
mixed
substrates,
i.e.,
of
maltose
showed
dependence
kinetic react
was maltitol
the
absence
that
same
and
and
maltitol
very
maltitol,
maltitol,
less
demonstrates
obtained
was
between
and
(vl, v2) in
the
value
sucrose
competition
maltose
Michaelis for
sucrose,
the
maltose.
of
no
for
for
maltitol
substrates
maltitol
substrates
apparent
and
and
of
This finding two
for
large
individual Thus,
the
by
maltose.
was
for
In
(Vmax)
various
fraction
evidence of
(v) rates
(v•ƒvl+v2).
between
mole
values
presence for
followed activity
sucrose
(v=v1+v2).
rates
substrate
the
observed.
individual
tion
for
activity
jejunum,
Km value
of the
v2) respectively
strates
the
disaccharidase
velocity sum
disaccharidase
in
that the
with
the
highest
the
maltitol,
to
(v1,
1. was
at
data the
single
apparent
maltose
provide active
and on
the
strong center
maltase.
Maltitol is a sugar alcohol of the disaccharide maltose and is similar to it in structure, but is not found in nature. Previous studies showed that maltitol is absorbed to a small extent by the small intestine and that the absorbed maltitol is relatively quickly excreted in the urine (1, 2). A considerable amount of maltitol is found at the surface of the intestinal mucosa (2). This is very interesting from the point of view of 1 吉 沢 節子,森
内幸 子,細 谷 憲 政
Abbreviations used: Tris, tris (hydroxymethyl) universally labeled with 14C. 31
amino methane; maltitol-U-14C, maltitol
32
S. YOSHIZAWA,
S. MORIUCHI,
and N. HOSOYA
current concepts of membrane digestion (3, 4), since the disaccharidases are also found on the intestinal surface membrane, i.e., the brush borders (3, 4). It was therefore of interest to study the effect of maltitol on the kinetics of intestinal disaccharidases in the presence of a naturally occurring disaccharide, such as maltose or sucrose.
MATERIALS Female
rats
laboratory libitum.
The The
denum,
animals
entire
and
Elvehjem in
larger
were
a
cell
strate
solution
was
of
supernatant
disaccharidase One-tenth
oxidase the using
bovine Sucrose
Maltose Hayashibara Biochemical
and
The
a
serum
of
maltitol Co.,
as were
were Ltd.,
test
the
for
the
the
the
and
ad
duo
washed
Four
performed
in
parts a Potter
homogenate to
was
remove
nuclei
experiment. of
DAHLQVIST
The
maleate
open slide.
15min
method
solution
into
slit
Then for
standard given
decapitated.
a glass was
used
(5).
concentration
buffer,
pH
contained
enzyme and
solution
Su
of
6.0.
An
a suitable
was
oxidase Protein
reagent
was
was
incubated
produced
sub
aliquot
amount
of
at
mixed 37•Ž.
determined was
determined
by
0.1ml
After with
prepared the
with
LOWRY
an
ap
Tris-glucose according
to
method
(6),
a standard.
purchased
kindly
while
were
substrates.
tube
glucose
(5).
albumin
by as
the
Tris-glucose
glucose
subdivided
pestle.
sodium
that
and
with
1,000•~g were
diluted
small
of DAHLQVIST
and
at
0.1M
being
a
were
assay.
the
amount
reagent.
procedure
the of
in
the
a teflon
given
water
before
homogenization
used
so
off
were and
intestines
assayed
in
diluted
hr
scraped
the
were
for
solution time,
was
24
removed
supernatants
0-56mM
milliliter
substrate
propriate
was
with
maltitol
activity
for
centrifuge The
was
fast
Food
subdivided
and
activity and
of
added,
debris.
maltose
the
The
150-180g,
Osaka).
quickly
mucosa
refrigerated
Disaccharidase crose,
to
homogenizer fitted
centrifuged and
made was
METHODS
weighing
Co.,
ileum. The
water
strain, Yeast
intestine
saline.
redistilled
Wistar
were
small
ice-cold
and
the
(Oriental
jejunum
with of
of
chow
AND
from
provided
glucose
by
oxidase
the the was
Kokusan
Chemical
Nikken
Chemical
obtained
from
Works, Co., the
Ltd.
Ltd.. and
Worthington
Company.
RESULTS
1. Distribution of disaccharidases in rat small intestine The activity of disaccharidases was observed in various parts of the rat small intesine (Table 1). The activities of maltase and sucrase were the highest in the jejunum, followed by those in the ileum, while the duodenum exhibited very low activity. This pattern of enzymatic activity was also observed with maltitol, al though the activity was much lower compared with that obtained with maltose
MALTITOL Table
1.
AND
Specific
INTESTINAL
activity
DISACCHARIDASES
of disaccharidases
of the small intestine
(): a The
number of disaccharidase
buffer,
pH
6.0
calculated or
determinations. activity and
from
maltitol
the
the
Mean•}S
. E. M.
b
of
1 mole
value
for
assayed
of
was
but
The
that
divided
substrate
of
by
in
0 .1M
sodium
hydrolyzed/hour/mg
produced.
glucose,
maltose
28mM
substrate
glucose
parts
of the rat.a
with
as ƒÊmoles
amount
produces
Therefore,
was
expressed
in the various
33
hydrolysis
maltose
maleate
protein. of
produces
It
1 mole 2 moles
was
of
sucrose
of
glucose.
2.
and sucrose. The jejunum portion of the small intestine was used in all subsequent enzymatic studies because its disaccharidase activity was highest. 2. Properties of disaccharidases in rat intestine The properties of disaccharidases were analyzed in the jejunum of rat small intestine. The disaccharidases activities were determined for maltose, sucrose and maltitol in the different substrate concentrations (Fig. 1). From these, the Michaelis constant (Km)and the maximal velocity (Vmax)for sucrose, maltose and maltitol were determined by the graphical method of the Lineweaver-Burk plots (Table 2). The Km value for maltitol was extremely large compared with that for maltose and sucrose. This observation demonstrates that the disaccharidase has little affinity for maltitol. Furthermore, the lowest Vmaxwas found when maltitol was used as substrate. These results indicate that maltitol is enzymatically not easily available to the rat. 3.
The
effect
of
Previous stered
in
this
consisting
the
presence If
in
the
vidual interaction
there
of
presence
of
substrates, between
the not
mucosa.
carried
of
that
was
intestinal
were
hydrolysis
to
maltitol
rat
were
the shown
bound
disaccharidases
servations in
is
study, of
the
on have
maltitol-U-14C
ever,
with
maltitol
studies
a
surface
inhibits
out
to
and
of
will
the
be
the
of
intestinal
by
considered of
how
that sucrose
sucrose
orally
mucosa
hydrolyzed
hydrolysis
determine
maltose amount
significantly It
and
sucrose
considerable
the
How
preparation
maltitol or
and
admini
(2).
combines
maltose.
maltose
Ob
hydrolyze
maltitol. no
interaction
two
substrates
v1
and
the
v2 two
between should
two be
(v=v1+v2). substrates,
On then
substrates,
equal
to the
v•ƒv1+v2
the
the sum
other
of
hand, (7).
initial those if
velocity for there
the were
(v) indi an
34
S. YOSHIZAWA,
Fig.
1. The activities of the disaccharidases activities were determined as described
Table 2.
Kinetic studies were calculated Burk.
The
effect
velocity 1.03,
of
(v=mM which
and
S. MORIUCHI,
was
maltitol,
petition
maltitol
equal
i.e.,
between
The
initial about
i.e.,
8.08
the
so-called
and
maltitol
the
velocity
and
and
sucrose
13%
0.28,
of
rate
was
observed
presence for
0mM to 56mM.
of
the
(Fig.
The
and
individual
(v=vi+v2).
2).
sucrose
substrates,
Thus,
there
initial
maltitol
was sucrose
was
no
com
maltitol. maltitol
for
the
mixture
the
sum
of
on
maltase
of
maltose
the
individual
(v•ƒvl+v2) between
the
the
respectively
respectively
at
the
Vmax and Km values of Lineweaver and
varied from
activity in
of
"competition" react
sum
and
than
0.39,
sucrase
effect
(v) less
the
The method
concentrations
produced/hr) to
0.78
Furthermore,
was
on
HOSOYA
of maltose, sucrose and maltitol. The enzymatic under MATERIALS AND METHODS.
on disaccharidases. by the graphical
Substrate
glucose
and N.
same
active
site
and of
the
and
3).
was
observed
maltitol
rates
(Fig.
maltose
activity
for These
maltitol, disaccharidase
and
was
maltose
(Fig. 7.35,
and
which maltitol,
results
demonstrate
suggest
that
"maltase."
3).
maltose
MALTITOL
Fig. 2
AND
INTESTINAL
DISACCHARIDASES
Fig.
35
3
Fig. 2. Effect of maltitol on sucrase activity. The initial velocity was determined with sucrose, maltitol, or the mixture of sucrose and maltitol. Substrate concentration was 28mM. The ordinate shows mM glucose produced and the abscissa shows incubation time in minutes. From the figure, the initial velocity for sucrose, maltitol, or mixture of sucrose and maltitol is obtained as 0.78, 0.28, or 1.03, respectively. The initial velocity was expressed as mM glucose produced/hour instead of mM substrate hydrolyzed/ hour. Fig. 3. Effect of maltitol on maltase activity. The initial velocity for maltose, maltitol, or the mixture of maltose and maltitol was determined as described in Fig. 2. That for maltose, maltitol, or the mixture of maltose and maltitol is 8.08, 0.39, or 7.35, respectively.
In order to study the competitive mechanism between maltose and maltitol, the apparent Michaelis constant Km and the apparent maximal velocity (V) were determined for single and mixed substrates at various mole fractions of maltose. Lineweaver-Burk plots of 1/v against 1/s were obtained for mixed substrate solutions having various maltose mole fractions (f) (Fig, 4-a). These plots may reasonably be regarded as being straight lines, and from them the apparent Michaelis constant (Km) and the apparent velocity (V) were calculated. The
36
S. YOSHIZAWA,
S. MORIUCHI,
and
N. HOSOYA
dependence of Km and V on the mole fraction (f) of maltose is shown in Fig. 4-b,c. Thus, the kinetic features above, obtained with the mixed substrate, maltose and maltitol, are consistent with those predicted for maltose and maltitol acting in competition for a single active center.
Fig. 4. Lineweaver-Burk plots for mixed substrates: maltose and maltitol (a) and dependence of Km (b) and V on f(C). This kinetic study was carried out by changing maltose and maltitol concentrations, but without changing the ratio of each substrate concentration. The mole fractions (f) of maltose were 0, 0.17, 0.33, 0.50, and 1.00. In this case, a mole fraction of maltose (f) was calculated as follows: f=a/a+b, where a and b represent the substrate concentrations of maltose and maltitol, respectively. (The points corres ponding to f=0 and f=1 represent the values for pure maltitol and maltose, respectively.) Km and Vmax was obtained from each fraction of maltose. V is the initial rate for the total substrate concentration(s), which is the sum of the molar concentrations of maltose and maltitol.
DISCUSSION
Previous
studies
in our
laboratory
have
shown
that
a considerable
amount
MALTITOL
AND
INTESTINAL
DISACCHARIDASES
37
of orally administered maltitol-U-14C is bound to the surface of rat intestinal mucosa, even though maltitol is not utilized by this animal (1, 2). Recently, it has been demonstrated that the disaccharides are hydrolysed on the membranes of the small intestinal mucosa (3, 4). This type of digestion is characterized by the proximity of enzymatic hydrolysis to the surface of cell membrane, which separates the intracellular and extracellular environments. The enzymes involved in these reactions are fixed onto the cell membrane. In the present paper, we report the effect of maltitol on the activity of the intestinal disaccharidases, sucrase and maltase. Our studies demonstrate that maltitol and maltose compete for the same enzyme. The kinetic features obtained with the mixed substrates, maltose and maltitol, are consistent with the kinetics predicted for the competition for a single active center on the enzyme (7). There fore, it can be reasoned that maltitol is hydrolysed by maltase. However, for maltitol, the Kmvalue is very large and the Vmaxvalue is very small, as compared to these values for maltose. Therefore, only very little hydrolysis of this synthetic sugar alcohol would occur in the small intestine. It has been reported that maltitol feeding causes growth retardation (1). Our data suggest that this reduced growth would be due to the inhibition of the digestion of maltose by maltitol. Thus, in the presence of excess maltitol, the hydrolysis of maltose, a product of natural dietary carbohydrate, would be reduced; as a result, less monosaccharides will be available for absorption and consequently the calorie intake would be decreased. We would like to thank Dr. M. Zile (Department of Biochemistry, University of Wisconsin, U. S.A.) for her kind reading of our manuscript. This research was supported by the Scientific Research Fund of the Ministry of Education of Japan (944021).
REFERENCES
1) 2) 3)
INOUE,Y., MORIUCHI,S., and HOSOYA,N., J. Jap. Soc. Food and Nutr., 23, 43 (1970). OKU, T., INOUE,Y., and HOSOYA,N., J. Jap. Soc. Food and Nutr., 24, 399 (1970). UGOLEV,A. M., Physiology and Pathology of Membrane Digestion, Plenum Press, New York (1968). 4) UGOLEV,A. M. and DELAEY,P., Biochimica et Biophysica Acta, 300,105 (1973). 5) DAHLQVIST,A., Anal. Biochem., 7, 8 (1964). 6) LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L., and RANDALL,R. J., J. Biol. Chem., 193, 265 (1951). 7) HIROMI,K., HAMAUZU,Z., and TAKAHASHI,K., J. Biochem., 59, 411 (1966).
