Monosaccharide
inhibition
DAVID
.and
H.
ALPERS
JOHN
of human
E.
GERBER
intestinal
lactase*
St. Louis, No., and Boston, Muss.
Lactase has been partially
purified from human intestinal
mucosa. Lactase was
inhibited by glucose, galactose,
and fructose with the use of a radioisotopic
assay; sucrase was inhibited
by glucose and fructose; and maltase was inhibited
by glucose alone. However, the Ki for glucose was the same (about 30 mM) for
all 3 enzymes. No obvious structural requirements
could be determined
for
inhibition
of lactase, but naturally
occurring sugars were the best inhibitors.
Inhibition
by sugars of all disaccharidases
was pH dependent,
with a pH
optimum of 6. It was suggested that inhibition
of lactase by sugars might be
physiologically
important
and provide one mechanism for the production
of
lactose intolerance.
T
he intestinal mucosa contains 3 different P-galactosidases, only two of
which hydrolyze lactose itse1f.l The other heterogalactosidase is present in the
soluble fraction of the homogenate and has no activity against lactose.2 Of the
remaining two lactases one is found in the supernatant fraction of the homogenate and is located in lysosomes .3 The other lactase is a brush border enzyme4
and is probably responsible for the hydrolysis of dietary lactose. This enzyme is
the one usually referred to as intestinal lactase. Lactose intolerance is a common
finding in hospitalized patients5 and many ethnic groups and is usually correlated with a high incidence of lactase deficiency. However, there have been few
studies designed to investigate inhibition of lactase activity as a factor involved
in lactose intolerance. The present study provides evidence that human intestinal
membrane-bound
lactase is inhibited by the three major naturally
occuring
monosaccharides at concentrations which could be physiologically
significant.
From
the Departments
of Medicine
and the Gastrointestinal
Units,
Barnes
Hospital,
Washington
University
School of Medicine
and Massachusetts
General
Hospital,
Harvard
Medical
School.
This paper was supported
by Research
Grant AM 14038 and Training
Grants AM-04501
and
AM-05280
from the National
Institutes
of Health,
United
States Public
Health
Service.
Received
for publication
Feb. 3, 1971.
Accepted
for publication
May 19, 1971.
Reprint
requests :
*A preliminary
report
of this work appeared
in abstract
form in Clin Res 17: 296, 1968.
265
266
Alpers
Table I. Partial
I
II
III
IVt
V
VIt
J. Lnb. Clin. Med.
Aupust, I971
ct al.
purification
Homogenate
Papain treatment
105,000 x g Supernatant
Acetone (21 to 56%)
PEI cellulose
Preparative
polyacrylamide
of human lactase*
electrophoresis
2,990
942
928
900
743
378
38,752
2,950
1,720
416
92
IS
100
31.5
31
30
24.8
12.7
0.077
0.3?2
0.54
a.2
8.1
21.0
*Purifkation
was carried out as described in the Methods section. Activity
is reported as
units of absorbance at 530 mp per milligram of protein.
tFractions
IV and VI were dialyzed against 0.05M potassium phosphate buffer (PI-I G.0)
Prior to use as the source of enzyme for experiments.
Methods
Intestine was obtained from 2 patients at autopsy within 4 hours of the time of death.
The jejunum was removed from the ligament of Treitz half way to the ileocecal valve. Patient
H. C. was a 6%year-old Caucasian woman with malignant
melanoma who died of cerebral
metastases after a terminal illness. Patient R. W. was a %O-year-old man who died of malignant
hypertension
and an intracerebral
hemorrhage. The intestine was washed in cold normal saline
and the mueosa scraped off with glass slides. The scrapings were homogenized with a PotterElvejhem
homogenizer
in O.lM potassium phosphate buffer (pH 7.4) and centrifuged
at
105,000 x g for 1 hour at 4” C. The supernatant
fluid, containing
about 10 per cent of the
lactase activity
(presumably
of lysosomal origin),
was discarded, and the pellet resuspended
twice with buffer and re-centrifuged.
The final pellet was diluted with phosphate buffer to a
protein concentration
of 2 to 3 mg. per milliliter.
Papain (0.1 mg. per milliliter)
and cysteine
HCl (0.1 mg. per milliliter)
were added and the solution was incubated at 37” C. for 15
minutes.
The reaction
was stopped by adding p-chloromercuribenzoate
(lo-4M)
and chilling
rapidly.
The resulting
mixture
was centrifuged
at 105,000 x g for 60 minutes and the
supernatant
fraction
dialyzed against O.OlM potassium phosphate buffer (pH 6.0). Further
purification
was carried out as previously
described with the exception
that acetone precipitates
from 21 to 56 per cent were used. The eluate from (PEI)
cellulose columns containing lactase and some sucrase activity
was subjected to preparative
acrylamide
electrophoresis with the use of a Canalco apparatus
and an imidazole
buffer system.6 Protein
(18 mg.) was applied at one time to a 7.5 per cent separating
gel and a constant current
was applied beginning
at 3 ma. and increasing
to 12.5 ma. by the end of the run. Eluate
was collected in 5 ml. fractions.
All the lactase activity
was present in 3 fractions
(27 to
29) but 27 and 28 contained moderate sucrase activity.
Fraction
29 contained only a small
amount of sucrase activity
but still showed 4 contaminating
protein bands on polyaerylamide disc electrophoresis.
Thus, although
the final lactase preparation
was considerably
purified over the homogenate,
it was not a pure protein. Table I shows the purification
of
human lactase as used in these studies. This enzyme had a pH optimum of 6, and is the
lactase which is thought to be responsible
for intralumenal
hydrolysis
of lactose.
Maltase and suorase were purified by procedures which were identical
to those used
for lactase. Maltase eluted earliest of the 3 enzymes and had only one minor contaminating
protein. This enzyme is probably
identical
with the glucoamylase
found in rat intestine.7
Suorase eluted next and contained one major contaminating
band which was identical
with
the maltase.
Protein
was determined
by the method of Lowry and associates.6 For pH curves of
enzyme activity,
the following
buffers were used: sodium or lithium
acetate (pfI 4.5 to
5.8) potassium or lithium phosphate (pH 6 to 7.8).
Volume 78
Number 2
Monosaccharide
inhibition
2 67
Lactose-l-l4C
(1.55 pc per micromole)
(Nuclear
Chicago, Des Plaines, Ill.), sucrose-14C
(u.1.) (5.1 pc per micromole),
and maltose I-14C (0.2 to 1 cc per micromole)
(New England
Nuclear
Corp., Boston, Mass.) were used to assay for disaccharidases
in the presence of
monosaccharides.
&-Lactose was used for all assays of lactase activity.
For studies of K,
or K,, substrate
concentration
varied from 5 to 50 mM. Buffer concentration
was 50 mM
in a final assay volume of 0.08 ml. Each assay contained 0.05 lc of the original
radioactive
substrate.
After
incubation
for 30 minutes, the reaction
was stopped by boiling for one
minute or adding acid ethanol
(0.02 ml., l.ON HCl in 70 per cent ethanol).
A quantity
of 0.05 ml. was transferred
in 0.01 ml. aliquots
to 3 MM (Whatman)
chromatography
paper (46 by 57 cm.). Electrophoresis
was carried
out in 0.05M sodium borate buffer
(pH 9.2)9 at 22 V. per centimeter
for 90 minutes with the use of a Savant flat plate
(Model FP-22A).
The electrophoresis
was run at 4” C. with the use of a refrigerated
circulator. Sugar standards
(50 pg) were run simultaneously
at each edge of the paper. The
paper was completely
dried after electrophoresis.
Mono- and disaccharide
areas were identified first by cutting the paper in strips and assaying for radioactivity
in a radiochromatographic scanner (Packard
Model 7201). When it became apparent that in all the samples
the monosaccharide
regions had identical
Rf values on a given electrophoretic
run, the two
outer strips containing
sugar standards were cut off, stained with aniline phthalate,O and
the appropriate
regions were cut out from the unstained radioactive
strips. The paper was
then added to a scintillation
vial with toluene (15 ml.) containing
0.4 per cent p-bis-2,5
diphenyloxazole
and 0.005 per cent p-bis-2’.(5’-phenyloxazolyl)
benzene and counted in a
liquid spectrometer
(Packard
Model 3320) with an efficiency
of 71 per cent. The monosaccharide
region was used to determine
those glucose equivalents
which were liberated.
Assays were performed
in duplicate or triplicate
with a maximal variation
of ? 10 per cent.
In order to obtain values for K,, and K, in all studies, the data were used to obtain
a line by the method of least squares. Analysis
of enzyme kinetics was performed
by using
the method of Alberty.10
Results
Validity of the radioisotopic assay. As the studies to be reported necessitated
the use of an assay which would not be affected by added sugars, it was felt
important to demonstrate the validity of this assay. Radiochromatography
of
the electrophoretic separation of mono- and disaccharides demonstrated a wide
separation and virtually
no radioactivity
between the two sugars, Thus, it was
easy to cut out the appropriate areas for counting. The hydrolysis of lactose
(50 mM) was linear for 60 minutes, at which time 20 per cent of the original
substrate had been hydrolyzed (Fig. 1, a). Similar results were obtained with
maltase and sucrase. The hydrolysis of lactose (50 mM) was dependent upon the
amount of enzyme added, at least up to 20 per cent hydrolysis of the original
substrate (Fig. 1, b). Optimal substrate concentration
has been previously
reported to be important?
because of the phenomenon of substrate inhibition.
No such substrate inhibition was noted for lactose hydrolysis at concentrations
up to 200 mM (Fig. 1, c) . For maltase and sucrase, there was also no evidence
of substrate inhibition with the use of concentrations up to 200 mM. Because at
high sugar concentrations
(over 100 mM) there may be some smearing and
distortion of the electrophoretic patterns, substrate concentrations of 5 to 50 mM
were chosen for subsequent enzyme assays.
Intestinal disaccharidases are also transglucosylases.12 Thus, it was possible
that once radioactive monosaccharides were liberated by hydrolysis, they could
be reincorporated
into higher saccharides and be removed from the monosac-
268
Alpers
et al.
a.
I
I
I
I
20
40
Time (Minutes)
I
100
Lactose
200
I
60
1
b
Glucose
(p moles/m in)
x103
[mM]
Fig. 2. Radioisotopic
assay of lactase activity.
The assay was performed as described in the
methods section, with the use of the eluats from preparative
polyacrylamide
electrophoresis
(Fraction
VI). (a) Shows the kinetics of the reaction;
(a) the response to increasing protein
concentration;
and (c) the rate of reaction with increasing substrate concentration.
Incubation in b and G was for 30 minutes. The concentration
of lactose in a and b ww 50 mM.
Table II. Lack of transglucosylation
Lactose added
fmJf)
10
25
50
60
80
during disaccharidase assay”
Radioactivity
in monosaccharide
(6.p.m. x 10-J)
10.2
10.3
10.6
10.5
10.4
10.3
*Fraction IV, during guriflcation,
was used as the source of enzyme. To tbe assay mixture
containing lactase and nonradioactive
lactose was added glucose-l-W
(0.01 &cc). Incubation was at 37” C. for 30 minutes. EIectrophoresis
and assay of monosaccharide
peal&
were performed as described in the Methods section.
eharide region. By this mechanism, hydrolysis of disaccharides could be underestimated. To test this possibility,
a known quantity
of glucose-l-t4C was
incubated with nonradioactive
lactose and intestinal lactase. Table II demonstrates that even at high lactose concentrations no glucose was converted to
higher glycosides, but that all added glucose could be accounted for as the
Volume 8
Number 2
Monosaccharide
inhibition
269
8.3mM
30 mM
Fig. 8. Glucose inhibition of lactase activity. The source of enzyme was the eluate from preparative polyacrylamide electrophoresis (Fraction VI). Incubation was for 30 minutes at 37”
C., with the use of 50 mM glucose. Radioactive enzyme assay was performed as described in
the Methods section. The substrate concentration was 10 mM. The open circles refer to control
experiments, the closed circles to those with glucose added.
monosaccharide. Thus, no transglucosylation
was occurring under the conditions
used in this assay, and the radioactivity
in the monosaccharide peak could be
interpreted as the result of substrate hydrolysis.
Inhibition
of lactase by major dietary sugars. By using the radioactive
assay as outlined above, it was possible to test the inhibitory
effects of various
sugars. Fig. 2 shows the inhibition
of partially purified lactase by glucose at
pH 6.0. Inhibition
was competitive and the K, for glucose was 30 mM, as compared with a K, for lactose of 8 to 10 mM. Similar results were obtained for
sucrase and maltase, where the Ki for glucose was 28 and 25 mM, respectively.
Somewhat different results were obtained by using fructose and galactose
(Fig. 3). Both sugars were competitive inhibitors of lactase activity. Fructose
was as potent an inhibitor of lactase activity as was glucose, with a Ki of 33 mM.
However, galactose was not so inhibitory
(Ki = 74 mM) . Fructose was also an
inhibitor of sucrase activity, giving values for K1 of 34 mM. Galactose did not
inhibit sucrase activity at all. Neither fructose nor galactose inhibited maltase
activity.
Inhibition
of lactase by other sugars. Lactase activity was not only inhibited
by the three major dietary sugars but also by potassium gluconate (Fig. 4).
Lactase activity was markedly inhibited by potassium gluconate at concentrations
which did not affect either sucrase or maltase activity. A variety of other sugars
were tested for their ability to inhibit lactase activity. Using 10 mM lactose and
80 mM inhibitor, only n-arabinose was inhibitory
among sugars which included
hexoses substituted in the C,., and C, positions, pentoses, tetroses, and disaccharides. This inhibition
was also competitive, but the Ki for arabinose was 82
mM. Sugars which did not inhibit lactase activity were D-xylose, D-erythrose,
D-erythritol, n-mannose, n-mannitol, cu-methylglucoside, n-sorbitol, n-glucosamine,
n-galactosamine, 3-0-methylglucose, 2-deoxyglucose, n-ribose, D-threose, L-glucose,
sucrose, and maltose. By using a similar concentration of glucose, lactase activity
was inhibited by over 60 per cent. Under similar conditions, n-arabinose inhibited
sucrase and maltose only 18 and 14 per cent, respectively.
The effect of pH on inhibition
of lactase activity. All the experiments
270 Alpers ct al.
c
Km= 0.0133
30
Ki 20.074
I/S (lactose)
Fig. 3. Fructose and galactose inhibition of lactase activity. The experiments were performed
as described in Fig. 2. The open circles refer to control experiments, the closed circles to those
with inhibitor added (50 mM) .
presented on inhibiton of lactase were performed at pH 6, which corresponds
to the pH optimum of the enzyme and to the intraluminal
pH of the intestine.
However, the degree of inhibition
was markedly dependent upon pH. Fig. 5
demonstrates the effect of raising the pH upon the inhibition of lactase by glucose
when compared with the results obtained at pH 6 (Fig. 2). Both the K1 and K,,,
rose with increasing pH. Similar results were obtained by lowering the pH and
with the use of sucrase or maltase as the enzyme. The effect of pH upon lactase
activity was then examined by the kinetic analysis of Alberty.l”
The ionization
constants for the enzyme itself were 4.95 and 6.88, the constants for the enzyme
substrate complex were 4.81 and 6.95, and the constants for the enzyme-inhibitor
complex were 4.96 and 7.15. It is evident that the addition of neither the substrate nor the inhibitor
(which are not themselves ionized), changed the ionization constants of the enzyme complexes.
Discussion
The radioactive assay described here seems to measure disaccharidase activity
accurately and, as the reaction is not a coupled one (as with glucose oxidase
assays), would seem theoretically better for studies of inhibition, However, “in-
Volume 78
Number 2
Monosaccharide inhibition
27 1
Fig. 4. Potassium gluconate inhibition
of lactase activity.
Experiments
were performed
as
described in Fig. 2. The open circles refer to control experiments, the closed circles to those
with inhibitor
added (50 mM).
K,= lZ.SmM
Kl’ 80mM
pH 7.4
I(,,,,=24 mM
>xKI=177mM
150
50
‘/s
Fig. 5. Glucose inhibition
of lactase activity as a function of pH. Experiments
were performed
as described in Fig. 2. The open circles refer to control experiments,
the closed circles to
those with glucose added.
hibition” of disaccharidases by glucose has been claimed to be partly the result
of transglycosylation. l3 Table II clearly demonstrates that this is not the case.
Moreover, when transglycosylation has been demonstrated by using intestinal
disaccharidases, the conditions employed have been very different from those in
the assay described. Carnie and Porteous14reported that a sucrose concentration
of O.lM was required with rather large amounts of enzyme and incubation for
10 to 24 hours to form 1 to 4 per cent oligosaccharides. Similarly, Dahlqvist and
272
Alpers et al.
Borgstromf2 used 30 per cent sucrose at 37O C. for 20 hours or longer to demotestrate transglycosylation.
The inhibition reported here is probably not the result
of transglycosylation.
In addition, fructose, which should not be a substrate for
transglycosylation,
still inhibits lactase activity.
By using a radioactive assay, no substrate inhibition can be demonstrated for
any disaccharidases. This result is in agreement with the results of previous
worker,+* who noted no substrate inhibition
of sucrase at concentrations up to
0.5M. Most likely the “inhibition”
noted is related to inhibition
of the glucose
oxidase reaction by disaceharides.15
We have demonstrated that lactase activity is inhibited by a variety of monosaccharides. Earlier, Wallenfels and FischerI demonstrated monosaccharide inhibition of calf intestinal lactase with the use of 0-nitro-phenyl
&galactoside as
a substrate. They also found that glucose was more inhibitory than was galactosc.
Larner and Gillespie I7 demonstrated glucose inhibition of intestinal maltase and
sucrase but did not determine a value for Ki. Although in OUT studies the Ki
for any individual sugar, such as glucose, is the same for all disaccharidases, onl?
lactase is inhibited by all three major dietary sugars-glucose,
galactose, and
fructose. Thus, lactase inhibition is not seen with products of hydrolysis only, as
fructose is not a product of lactose hydrolysis. However, sucrase and maltase arc
most inhibited by hydrolytic products of their natural substrate.
Since neither the substrate nor the inhibitors of lactase are themselves ionized, it is not surprising that the analysis of the effect of pH on inhibition
(Fig.
5) reveals that binding of neither substrate nor inhibitor changes the ionization
eonstants for intestinal lactase. Interestingly,
the pK, and pKb of 4.8 and 6.9 are
consistent with previous data in the literature. Wallenfels and FischeP” found
values of 4.0 and 6.9 for calf lactase, and Larner and Gillespiel? found a value
of 6.9 for the neutral ionization constant of suerase. The acid ionization constant
is probably not significantly different from that obtained for the calf. These data
are consistent with the previous interpretation Is, lg that an imidazole group may
be involved at the active center of human lactase.
No obvious structural requirements for inhibition
of lactase are clear from
this study. Glucose, fructose, and arabinose have identical structures in the last
four carbon positions. This is suggestive of the data of Kelemen and WhelanI
who found that the carbon positions C3 t0 6 were important in the inhibition
of
plant glucosidases. I8 Xylose, which is identical to glucose in the positions C, to ,:
does not inhibit lactase activity. However, mannose, methyl glucoside, 2-deoxyglucose, and sorbitol, which all differ from glucose only in the C, or C, position,
do not inhibit lactase activity, but galactose, a C, isomer, is inhibitory.
Keleman and WhelanI also noted that many polyols inhibited enzyme activity (glycerol, erythritol, n-threitol, ribitol, and xylitol)
although the inhibition was more related to structural similarity to the substrate glycon than to the
number of hydroxyl groups. This same inhibitory
structure does not apply to
lactase, because sorbitol (a polyol differing from glucose only at the C, position)
and mannose (a C, isomer of glycose) do not inhibit at all, Moreover, the glyeon
of the substrate binds poorly to plant glucosidases,18 yet is bound best by in-
Volume 78
Number 2
Monosaccharide inhibition
27 3
testinal disaccharidases. Recently, Swaminathan and Radhakrishnan10 have reported on the properties of monkey intestinal lactase. They found that pentaerythritol, a structural deaminated analogue of Tris, does not inhibit. These results
confirm the present data (see above) that polyols alone do not inhibit intestinal
lactase.
The inhibition by gluconate is of interest, since Kraml and otherszOhave
reported that intestinal lactase from the rat is inhibited by galactono (14)
la&one, but not by sodium galactonate. However, the lysosomal enzyme was inhibited by both sugars. There was no evidence, by substrate specificity, pH
optimum, or presence of other acid hydrolases, that the lactase used in these
studies was contaminated by lysosomal enzymes. Kraml and othersZo concluded
that the action of the neutral lactase was more specific toward substrates and inhibitors. The present data are in agreement with those conclusions.
Therefore, the requirements for inhibition are very specific and naturally
occurring sugars are the most inhibitory. Inhibition is maximal at pH 6.0, the
pH normally found in the upper intestine and perhaps even at the brush border,
since most brush border hydrolases have pH optima in the range of 5 to 6.21It is
possible that some of the discrepancies between abnormal lactose tolerance tests
and normal or borderline-normal intestinal lactase activity might be explained
by the mechanism of sugars which inhibit lactose hydrolysis in vivo.
Monosaccharide inhibition of lactase may be of some physiologic importance.
This suggestion is supported by the fact that after a standard meal the concentration of glucose in the jejunal lumen is about 20 to 40 n-i&I,22a concentration in
the same range as the Ki for glucose. In the ileum, where disaccharidase activity
is lower, luminal glucose concentrations were also lower (1 to 15 mM) . Gray and
IngelfingeP have demonstrated that 40 mM galactose can inhibit the hydrolysis
of 40 mM sucrose in man. Moreover, recent experiments in our laboratoryz4 have
provided evidence that sugars can inhibit lactose hydrolysis in a perfused intestinal loop in the rat. The K, for inhibition by glucose was 11 mM. This Ki
agrees quite well with the Ki found for the enzyme lactase in vitro (30 mM),
considering the fact that perfusion experiments cannot be performed with the
same precision as enzyme assays in vitro. Gray and Ingelfinger23 postulated that
galactose inhibition of sucrose hydrolysis could be due to inhibition of the enzyme
by either galactose or glucose (or galactose) which accumulates as the result of
galactose (or glucose) competing for active transport. We have found in the
perfused rat intestine24 that fructose also inhibits lactose hydrolysis. The mechanism for this inhibition can be reasonably explained by a direct inhibition of
the enzyme, as fructose does not compete for active transport with glucose or
galactose. It is possible that competition for transport by products of hydrolysis
leads to accumulation of these products at a site adjacent to the brush border
with further inhibition of enzyme activity. Whether this inhibition actually
occurs
during the course of digesting a meal, or can only be produced by ingesting large amounts of monosaccharides, is still unclear.
The authors are especially grateful to Dr. Kurt J. Isselbacher for his many discussions
and suggestions and to Miss Marilyn N. Cote for expert technical assistance.
272
Alpers
et al.
J. Lab. Clin. Meil.
.“tigust, I’,7 I
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