Ranitidine induces inhibition and structural changes in sucrase

Article abstract of DOI:10.3109/14756366.2011.601414

Ranitidine is an antagonist of histamine-2 (H2) receptor. It is employed to treat peptic ulcer and other conditions in which gastric acidity must be reduced. Sucrase is a hydrolytic enzyme that catalyzes the breakdown of sucrose to its monomer content. A liquid of yeast sucrase was developed for treatment of congenital sucrase-isomaltase deficiency (CSID) in human. In this study, the effect of ranitidine on yeast sucrase activity was investigated. Our results showed that ranitidine binds to sucrase and inhibits the enzyme in a noncompetitive manner. The Ki and IC50 values were measured to be about 2.3 and 2.2mM, respectively. Fluorescence measurement showed conformational changes after binding of ranitidine to the enzyme. The fluorescence spectra showed that ranitidine could bind to both free enzyme and enzymesubstrate complex, which was accompanied with reduction of emission intensity and red shift production.

Full text of DOI:10.3109/14756366.2011.601414

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(4): 553–557
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.601414
ReseaRch aRtIcle
Ranitidine induces inhibition and structural changes
in sucrase
Dariush Minai-Tehrani1, Mina Ghaffari1, Zahra Sobhani-Damavandifar1, Saeed Minoui2,
Sana Alavi1, Rahele Osmani1, and Shiva Ahmadi1
1Bioresearch Laboratory, Faculty of Biological Sciences, Shahid Beheshti University, G.C. Tehran, Iran and
2Environmental Sciences Research Institute, Shahid Beheshti University, G.C. Tehran, Iran
abstract
Ranitidine is an antagonist of histamine-2 (H2) receptor. It is employed to treat peptic ulcer and other conditions in
which gastric acidity must be reduced. Sucrase is a hydrolytic enzyme that catalyzes the breakdown of sucrose to its
monomer content. A liquid of yeast sucrase was developed for treatment of congenital sucrase-isomaltase deficiency
(CSID) in human. In this study, the effect of ranitidine on yeast sucrase activity was investigated. Our results showed
that ranitidine binds to sucrase and inhibits the enzyme in a noncompetitive manner. The Ki and IC values were
measured to be about 2.3 and 2.2mM, respectively. Fluorescence measurement showed conformat5io0 nal changes
after binding of ranitidine to the enzyme. The fluorescence spectra showed that ranitidine could bind to both free
enzyme and enzyme–substrate complex, which was accompanied with reduction of emission intensity and red shift
production.
Keywords: Enzyme, drug, noncompetitive, inhibitor
Introduction
mainly used for inhibition of α-glucosidase, induces
Histamine-2 (H2) receptor blockers, such as raniti-
dine and cimetidine, are a group of drugs that work by
reducing the acid secretion in stomach1. Ranitidine
and cimetidine (under the trade names Zantac and
Tagamet, respectively) are used to treat ulcers in gastro-
intestinal tract2. Most of the drugs have shown to induce
side effects in body. Ranitidine reduces the activity of
some enzymes residing in renal brush border, such
as hydrolytic enzymes3, and also duodenal mucosal
enzymes in patients with peptic ulcer4. Sucrase is a
hydrolytic enzyme that breaks down sucrose into glucose
and fructose. e human gastrointestinal system has a
kind of sucrase known as sucrase-isomaltase that resides
in the apical surface of intestinal cells. Yeast sucrase
has been shown to be used for patients with congenital
sucrase-isomaltase deficiency (CSID5,6). Some drugs can
inhibit the sucrase activity. Nojirimycin, deoxynojirimy-
cin, and acarbose have shown competitive inhibition
of intestinal sucrase7. Castanospermine, a drug that is
noncompetitive inhibition on sucrase activity8. Previous
report also indicated that codeine binds to yeast sucrase
and inhibits the enzyme via noncompetitive inhibition9.
In this study, the effect of drug ranitidine on the activity
of sucrase has been studied and kinetics of binding and
structural changes were reported. e use of sacrosidase
for treatment of CSID induces some side effects in gastro-
intestinal tract, which are similar to symptoms caused by
CSID (such as stomach ache, vomiting, diarrhea, nausea,
and constipation). As ranitidine is used to treat gastro-
intestinal disorders, this report may guide physicians to
administer ranitidine in patients under sacrosidase use,
with precaution.
Materials and methods
Yeast sucrase (3.2.1.26) was obtained from Fluka
Company. Ranitidine and cimetidine were purchased
from Sigma Company. All the chemicals used for the
Address for Correspondence: Dariush Minai-Tehrani, Ph.D., Bioresearch Laboratory, Faculty of Biological Sciences, Shahid Beheshti
University, G.C. Tehran, Iran. E-mail: d_mtehrani@sbu.ac.ir
(Received 26 May 2011; revised 25 June 2011; accepted 25 June 2011)
553

554 D. M. Tehrani et al.
enzyme assay and buffer preparation were of reagent
grade and obtained from Merck Company.
continued to reach the plateau (substrate depletion). e
Michaelis–Menten analysis was applied to determine the
reaction rate of the pure enzyme (Figure 1). Since sucrase
is more stable in pH 7, further kinetics experiments
were done at pH 7. Edie–Hofstee plot was employed
to determine the kind of inhibition in the presence
of different concentrations of ranitidine (Figure 2). A
noncompetitive inhibition in sucrase was observed after
different concentrations of ranitidine were added to the
enzyme and substrate solution.
e Km (11.5 mM) of enzyme was constant in the
presence of various concentrations of ranitidine
(Figure2),whileitsVmax decreasedbyincreasingranitidine
concentration (Figure 3). e concentration of ranitidine
producing 50% inhibition of maximal activity (IC50) was
determined to be about 2.2 mM (Figure 3). According to
Dixon plot, the inhibition constant (Ki) of ranitidine was
obtained to be 2.3 mM (Figure 4).
Enzyme activity
Enzyme activity was measured according to the method
reported in Ref. 9. Sucrase solution (0.5mg/ml) was pre-
pared by dissolving the enzyme in distilled water. e
optimum pH for enzyme activity was determined to be
4.5; however, the enzyme is more stable in pH 710. As a
matter of fact, ranitidine has been reported to be unstable
in acidic or basic solutions11; therefore, the enzyme assay
was performed in 0.1 M phosphate buffer at pH 7.
e reaction was started by adding 100 μl of sucrase
solution to the test tubes containing different concentra-
tions of sucrose (2.5–75 mM). For the assays done in the
presence of ranitidine, appropriate amounts of ranitidine
were added to the test tubes from the stock solution
(50 mM). e final volume in each test tube was always
2 ml and the concentration of ranitidine varied from 0.5
to 3 mM.
Structural changes
Sucrose hydrolyzing activity of sucrase was deter-
mined by measuring the rate of liberation of glucose and
fructose from sucrose and measured by the ferricyanide
method for determination of reducing sugars.
e enzyme was incubated with sucrose for 1, 3, 5, 10,
and 15 min in the presence and the absence of ranitidine.
e activity of enzyme was stopped at the mentioned
times by incubating the solution at 80°C. e ferricya-
nide colorimetric method was performed for the enzyme
activity12 using Perkin-Elmer visible spectrophotometer.
e results were the average of at least three separate
experiments and were expressed as mean standard
deviation.
To ensure the purity of the enzyme, sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE) was
performed which showed only a single protein bond.
Fluorescence measurements were done for determination
of conformational changes occurred after binding of sub-
strate and inhibitor to the enzyme. e excitation was per-
formed at 285nm for exciting tryptophan residues and the
emission was recorded at a region between 310 and 400nm.
Excitation of pure enzyme showed a peak at 340nm (Figure
5, graph A). Binding of substrate to the enzyme reduced the
peakintensitywithoutanyshift(Figure5,graphB).Addition
of inhibitor to pure enzyme decreased the intensity of peak,
which was accompanied by a red shift to 344nm (Figure 5,
graph C). Addition of inhibitor to enzyme–substrate com-
plex also decreased the peak intensity and induced a red
shift to 344nm (Figure 5, graph D).
Fluorescence measurements
e fluorescence spectra were taken on a Perkin-Elmer
SL45 fluorometer. e excitation wavelength was 285nm
andtheemissionspectrawererecordedinarangebetween
310 and 400nm. e enzyme solution was prepared with
a concentration of 0.04mg/ml in 0.1M phosphate buffer
(pH 7). Sucrose was used with a concentration of 30mM,
and ranitidine concentration was 6.5 μM.
Discussion
e yeast sucrase has been proposed to be applied for
treatment of patients with CSID6. Sacrosidase (a liquid
Results
Inhibition of sucrase activity by ranitidine
e effect of ranitidine and cimetidine on sucrose activity
was monitored. Although both these drugs have nearly
the similar structure and effect, no change in the enzyme
activity was observed after using different concentrations
of cimetidine. In contrast, ranitidine affected the sucrase
activity significantly. So our measurements were focused
on ranitidine and its effects on sucrase activity.
e reaction rate of enzyme on sucrose (substrate)
was determined in the absence and the presence of
ranitidine by detecting the formation of products (glucose
and fructose) at 410 nm. e incubation of enzyme with
sucrose continued to pass into the steady state and
Figure 1. Michaelis–Menten plot shows that above 30 mM sucrose
concentration, the enzyme reaches to zero-order reaction.
Journal of Enzyme Inhibition and Medicinal Chemistry

Inhibition and structural changes in sucrase 555
Figure 2. e pattern of Eadie–Hofstee plot showed noncompetitive inhibition after binding of ranitidine to the enzyme. e slope of the
lines is the same and determines a constant Km, but intercepts on the ordinate were different, which indicate that the Vmax of the enzyme
was not constant in the presence of different concentration of ranitidine (0–3mM).
are the common drugs that have been reported to
inhibit the yeast sucrase activity9,14. Some drugs such
as gentamycin increase rat intestinal sucrase activity15,
whereas nonsteroidal anti-inflammatory drugs (NSAIDs)
decrease intestinal sucrase activity16. A recent report
has been shown the crystal structure of the N-terminal
catalytic domain of sucrase–isomaltase enzyme and
has determined that some chemicals, such as kotalanol,
inhibited the enzyme by binding to this domain17.
Our results showed that ranitidine binds to sucrase
and inhibit its activity by noncompetitive inhibition.
Increasingranitidineconcentrationresultedinadecrease
in Vmax, while Km of the enzyme remained constant. e
measurement of Ki and IC50 values of the drug showed
that ranitidine binds with moderate affinity to the
enzyme. On the other hand, cimetidine, the other H2
blocker drug, had no inhibitory effect on sucrase. Some
drugs also inhibit sucrase via noncompetitive inhibi-
tion. For instance, codeine and scopolamine inhibit the
activity of yeast sucrase while castanospermine inhibits
the activity of sucrase in human enterocyte-like cell line
Figure 3. Change in maximum velocity (Vmax) of the enzyme in the
presence of different concentrations of ranitidine. Determination
of inhibitor concentration yielding 50% inhibition (IC50).
producedfromSaccharomycescerevisiae)isusedinthese
patients to lessen the outcomes of sucrose deficiency
95). Several drugs induce counter effects when admin-
istered simultaneously with sacrosidase. In this case,
some drugs such as nojirimycin, deoxynojirimycin, and
acarbose have shown competitive inhibition of intesti-
nal sucrase7.
In the present study, the effect of ranitidine on sucrase
activity was investigated. Since the stability of the enzyme
and inhibitors is achieved at pH 7, most of the enzyme
kinetics and structural studies were performed at this pH.
ere are few reports regarding the effect of therapeu-
tic drugs on sucrase activity13. Codeine and scopolamine
Caco-28,9,14
.
e fluorescence measurements showed that there
were structural changes after binding of the enzyme to the
substrate (inhibitor). Binding of the enzyme to the sub-
strate decreased the emission intensity of peak. Binding of
ranitidine to pure enzyme or enzyme–substrate complex
not only decreased the peak intensity but also revealed a
red shift. is suggests that ranitidine could bind to both
free enzyme and enzyme–substrate complex. Decreasing
the peak intensity and the red-shift production suggest
that a quenching has been occurred in free enzyme and
© 2012 Informa UK, Ltd.

556 D. M. Tehrani et al.
Figure 4. Estimation of Ki by Dixon plot in the presence of different sucrose concentrations (3.3–30mM).
Figure 5. Fluorescence spectra obtained from excitation at 285nm and emission range of 310–400nm. e pure enzyme (0.04g/ml) induces
a peak at 340nm (a). Binding of substrate (30 mM) to the enzyme reduced the peak emission intensity (b). Binding of ranitidine (6.5 μM) to
free enzyme also decreased the emission intensity and induced a red shift to 344nm (c). Binding of ranitidine to enzyme–substrate complex
also induced red shift (d). Pure ranitidine (e).
enzyme–substrate complex after binding of ranitidine.
is quenching proposed that the tryptophan residues
displace to more polar medium, which may follow con-
formational changes in the enzyme. A recent report with
fluorescence spectroscopy also indicated the structural
changes in sucrase after binding of scopolamine14.
Previous report indicated that scopolamine and
codeine bind to sucrase at the site (inhibition site)
rather than the active site14. Ranitidine may also bind
to sucrase in the same manner of scopolamine and
codeine, at the inhibition site. Considering three drugs
(ranitidine, scopolamine, and codeine), which inhibit
the sucrase with noncompetitive inhibition, a heterocy-
clic ring containing oxygen can be seen in their chemical
structure. It seems that the oxygen of heterocyclic ring
may have an important role in the binding of these drugs
to the enzyme that is associated with conformational
changes in the enzyme. However, the different groups
attached to the heterocyclic ring affect the affinity of
drugs in binding to the enzyme. Cimetidine with similar
structure and effect to ranitidine has no affinity to bind
to sucrase. Comparison between chemical structures of
cimetidine and ranitidine showed that there is no oxygen
bound heterocyclic ring in cimetidine molecule.
In conclusion, our results suggest that ranitidine binds
to both free enzyme and enzyme–substrate complex and
inhibit the enzyme by noncompetitive pattern, which is
accompanied by conformational changes in the enzyme.
Ranitidine has a heterocyclic ring containing oxygen
that may play an important role in reaction between
Journal of Enzyme Inhibition and Medicinal Chemistry

Inhibition and structural changes in sucrase 557
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Inhibitory effect of codeine on sucrase activity. Drug Metab Lett
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10. Gascon S, Neumann NP, Lampen JO. Comparative study of the
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ranitidine on the sucrase–isomaltase enzyme of human
or other mammalian gastrointestinal tract may reveal
some similarities or difference between yeast sucrase
and mammalian sucrase–isomaltase enzyme.
Declaration of interest
e authors declare no conflicts of interest.
11. Wu HL, Huang CH, Chen SH, Wu SM. Micellar electrokinetic
chromatography of scopolamine-related anticholinergics.
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