Muzaiyan Ahmed Khan IRJP 2012, 3 (1)
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
www.irjponline.com
ISSN 2230 – 8407
Research Article
SODIUM INSENSITIVE BRUSH BORDER MEMBRANE SUCRASE IN SHEEP INTESTINE
Muzaiyan Ahmed Khan*
Associate Professor, Biotechnology and Biochemistry, Rajeev Gandhi College, Bhopal, M.P., India
Article Received on: 03/11/11 Revised on: 14/12/11 Approved for publication: 19/01/12
*Email: [email protected]
ABSTRACT
Dietary sugars and intermediate products of starch digestion are hydrolyzed by small intestinal epithelial cell enzymes associated with the brush border
membranes. The BBM consists of enzyme sucrase and isomaltose, responsible for hydrolyzing the sucrose and maltose, constitutes approximately 10% of the
intrinsic protein of brush border membrane. Dietary sugars regulate the activity of sucrase during ageing. Dietary control is particularly important in evaluating
the maintenance of sucrose enzyme. It is observed that in ruminants the sucrase is either absent or present with feeble activity. The sucrase activity observed in
the brush border membrane, which is of microbial origin and very much insensitive to the high concentrations of sodium. Sucrase and maltase activities are
reported to be induced by dietary carbohydrates in the jejunum of rodents while, this phenomenon was lacking in sheep. During ageing the activity of sucrase
is diminished in humans and rodents, but in contrast to this, such phenomenon is yet to be observed. The influence of age and high carbohydrate
concentrations in the intestine on sucrase induction in sheep is yet to be established.
Key words: Sucrase, Brush Border Membrane, Ruminants, Small Intestine.
INTRODUCTION
Dietary sugars and intermediate products of starch digestion
are hydrolyzed by small intestinal epithelial cell enzymes
associated with the brush border membranes. BBM [Brush
border membrane] lining the enterocytes constitutes one of
the most important cellular membranes owing to its role in
digestive and absorptive functions, which are carried out by
hydrolytic enzymes and transport enzymes respectively. The
enzyme sucrase-isomaltase, which is responsible for
hydrolyzing sucrose and maltose, constitutes approximately
10% of the intrinsic proteins of brush border membrane1. In
humans and rats the disaccharidase activities are diminishing
during the process of ageing.2 However, data from animals
maintained under defined dietary conditions are unavailable.
Dietary carbohydrate appears to be a primary regulator of the
activity of the sucrase3. Jejunal sucrase and maltase activities
are induced by dietary carbohydrate in young rats4. Dietary
control is particularly important in evaluating the
maintenance of sucrase. So far, scanty information is available
on the brush border Sucrase enzyme as the data reveals other
disaccharidases except sucrase.
Small Intestine: The small intestine extends from the pyloric
orifice, where it is continuous with the stomach, to the ileocecal
junction, where it continues with into large intestine. The
circular concentric folds as known as 'villi', small finger like
projections of mucus membrane, 0.5 to 1.5mm length are seen in
intestine. Between these villi are depressions called 'Crypts of
Intestinal Glands 'or 'Lieberkuhn'5. Each enterocyte has two
structurally and functionally distinct membrane domains, the
MVM (microvillus membrane) and the BLM (basolateral
membrane). The specialized apical striated border consists of
closely packed array of microvilli. The outer lining of
microvillus separating the luminal environment from epithelium
is acknowledged as microvillus membrane [MVM] or Brush
Border Membrane [BBM]6. The brush border membrane is a
trilaminar structure contains lipid-protein complexes and
carbohydrates. The epithelial surface is covered with a
carbohydrate-rich layer ‘glycocalyx’ or ‘fuzzy coat’7.
Carbohydrates constitute nearly 10% of the MVM mass8.
Studies in MVM confined to processes glycosylation,
sialylation, and fucosylation, due to functional roles in the
intestine9-11, and have been implicated in pathological
conditions of the intestine12. In earlier studies extensive research
efforts has been directed toward elucidating the developmentassociated changes in the intestine13. Evidences pointed out to an
important role of glycosylation in the receptor-ligand
interactions on the cell surface14. The existence of lipid bilayer
was confirmed by X-ray diffraction15. The membrane covering
the microvilli is covered with a polysaccharide coat
‘Glycocalyx’ consists of glycolipids, and glycoproteins. It is
highly negative charged, due to sialic acid and sulphate residues
on the carbohydrate moieties. The MVM is a mosaic of
structural and functional proteins intercalated in the lipid bilayer
with transport carriers, γ-Glutamyl transpeptidase, Sucrase, and
Alkaline Phosphatase16.
Chemical composition of BBM
Brush border preparation contains 60% proteins, 30% lipids and
10% carbohydrates expressed on the basis of dry mass. The
molar ratio of Cholesterol to Phospholipid to Glycolipid17 is1:1:2. The chief glycolipid is 2-Monohexosyl Ceramide,
contains Glucose, Galactose, Fucose, and Hexosamines18.
Sugars in MVM are Hexosamines and Sialic acid. Extrinsic
enzyme activity encompasses Leucine Amino peptidase,
Lactase, and γ-Glutamyl transpeptidase. In rats the BBM
glycosylation processes are reliant on endogenous factors i.e.,
distribution across crypt-villus unit19 distribution along the
intestinal tract20, hormones21, and age22.
Aims And Objectives
Earlier in rodents, our colleagues had reported BBM sucrase
enzyme activity. The aim was to elucidate in sheep Microvillus
membrane: Chemical composition, Brush Border Microvillus
Sucrase enzyme, and other Brush Border Membrane enzymes.
MATERIALS AND METHODS
All chemicals used were of analytical grade. Sheep [Total
N=25 sheep, of 1-1.5 yr age; out of which twenty sheep for
normal study and five sheep for starvation studies] of average
weight, kept in animal house under standard conditions for
studies. Five fully grown albino rats of six months, in a
separate steel cage were maintained on standard animal diet
with ad-libitum free access to water and food. Starting from
the ligament of Treitz, entire small intestine was taken,
flushed with ice-cold saline, dried and weighed. Intestinal
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Muzaiyan Ahmed Khan IRJP 2012, 3 (1)
cells were cut into three equal parts lengthwise and measured
by Savaiano23 method, utilizing three collection periods of
10, 15, and 25 min. Cell pellets were suspended in
physiologically buffered saline for light microscopy and for
grit of cell numbers and sucrase activity. Cells collected
during the first two collection period had high colored and
characteristic morphology of differentiated villus epithelial
cells. Cells collected during the twenty five min. collection
period had morphologic and enzymatic characters of crypt
cells in agreement with previous reports. Sucrase activity was
determined by method of Dahlquist24 Enzyme activity was
expressed as-'A unit of enzyme hydrolyzes 1μmol of
disaccharide per minute'. Reactions with substrate were in
triplicate and run. All procedures were carried out between 040C, in 50mM Sodium Maleate buffer (pH-6.8). Proteins by
Lowry’s25 method, Blood glucose by Winkers and Jacob26
method, Intestinal Brush Border Microvillus preparation by
Kessler27et al method, Alkaline Phosphatase by
Bregmeyer28method, Lactase by Dahlquist29method, Leucine
Amino peptidase by Goldberg and Rutenberg30method, γGlutamyl transpeptidase by Naftalin31 et al method, Sialic
acid by Skoza and Mohe32 method, Fucose by Dische and
Shettle33method, Total hexose by Roe34 method,
Hexosamines by Gatt and Berman35 method, Total
Cholesterol by Zlatkis36method, Free Cholesterol by
Courchains37method, Triglycerides by Van Handel38method.
Statistical Analysis
Data was analyzed by two way analysis of variance and
student-t-test.
RESULTS AND DISCUSSION
The body weight and small intestine weight were not
significantly altered at the initiation and end of experimental
period. The protein contents of the cell isolate was high in
normal fed sheep than that of malnourished group (62.7 vs
51.5mg). The enzyme activity of BBM in sheep and rat is
shown in Table. In this experiment, first of all recognizing
the lack of sucrase activity in sheep, although, observed in
rats. The sucrase activity did not change significantly in the
sheep in any of the three segments of small intestine. In
intestine it was lost near zero or extremely feeble. On
addition of sodium, the enzyme activity was unaltered.
Sucrase in very low concentrations is insensitive to sodium.
This leads to hypothesis that sucrase is absent rather than
very feeble. Sucrase is insensitive to sodium in goats31.
Sucrase in sheep may be of microbial origin. It can be said as
a ‘free sucrase’ rather than embedded. Density gradient
ultracentrifugation revealed that the analogous cytosol
enzymes migrated behind those of brush border3. A reduction
of total number of villus epithelial cells in old rats and rabbits
has been reported, and evaluated cell count/villi and Villi
count/small intestine2. Scanty information on sheep is
available. Rats fed both high and low-carbohydrate diets were
able to sustain the enzyme activity of disaccharidases. The
assessment of carbohydrate digestion capacity of sheep small
intestinal villus epithelial cells under the defined dietary
conditions was performed. It is now confirmed that in sheep
the brush border sucrose enzyme activity is total absent. A
small activity can be recognized in intestinal cell cytosol,
intracellular sucrase differs from their brush border
membrane counterparts physically and immunologically the
enzyme which is present is very much insensitive to sodium.
ACKNOWLEDGMENT
I would like to thank my Dean Professor Dr E Hibshey
Department of Biochemistry and Chemistry, College of
Science, AlJabal AlGharby University Ghariyan, Libya for
providing all the necessary facilities to accomplish this
research work. Thanks to Dr Ravinder K Gill Assistant
Professor Physiology, California Medical College, UIC, USA
and Prof. Dr. A. Mahmood Emeritus Scientist, Biochemistry,
Panjab University Chandigarh for the valuable suggestions,
and making completion of my manuscript.
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Table: SHOWING STATUS OF BBM IN SHEEP
BRUSH BORDER MEMEBRANE
Normal Diet
Enzyme Activity[Unit/mg proteins]
Body weight [kg]
29.5 ± 5.3
Intestinal weight [kg]
0.62 ± 0.042
SHEEP
Diet Deficient
25.6 ± 5.3
0.59± 0.065
3
Intestinal length [ft]
19.5 ±4.3
16.8 ±3.3
4
Blood glucose [mg/dl]
69.8 ±11.5
58.7 ± 15.5
5
Serum protein [mg/ml]
1
Lactase
0.84±0.088
0.581±0.07
2
3
4
Alkaline phosphatase
Leucine amino peptidase
Sucrase
1.1945±0.185
0.754±0.016
0.01±0.0001
1.081±0.08
0.584±0.075
0.01±0.001
5
γ- Glutamyl transpeptidase
0.48±0.034
0.42±0.06
104.8 ± 16.5
Status Of Memrbane Enzymes
115 ± 13.4
Membrane Composition
1
Sialic acid [nmol/mg protein]
197± 10.5
182± 4.2
2
Fucose [nmol/mg protein]
85.4± 7.8
81.2± 8.2
3
TOTAL HEXOSES [μg/mg protein]
4
HEXOSAMINES [μg/mg protein
1
Phospholipids
329.5± 21.9
319.5±31.7
2
Total cholesterol
287.5± 39.8
251.5±29.6
3
Free cholesterol
108.6± 18.9
100.5±16.5
4
Triglycedrides
Sucrase %
Isomaltase %
A
Brush border in Sheep
0 – 0.1
0 - 0.2
C
Cytosol in Sheep
0.5-0.8
0.5-1
97.38± 14.5
95.7± 9.1
77.94± 15.2
Lipid Fraction [μg/mg protein] in BBM
68.1± 17.5
89.4± 12.7
Distribution of Sucrase and Isomaltose in Sheep Small Intestine
Fraction
86.5± 10.2
Source of support: Nil, Conflict of interest: None Declared
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