BIOL 109
Laboratory Four
A study of lactose intolerance to further understand the
function of enzymes.
Introduction
Lactose intolerance, also called Lactase deficiency and hypolactasia, is the
inability to digest lactose, a sugar found in milk and some dairy products. Lactose
intolerant individuals have insufficient levels of lactase—the enzyme that metabolizes
lactose—in their digestive system.
Lactose (Figure 1) is a disaccharide sugar that is found most notably in milk and
is formed from galactose and glucose. Lactose makes up around 2~8% of milk (by
weight), although the amount varies among species and individuals. It is extracted from
sweet or sour whey. The name comes from lac or lactis, the Latin word for milk, plus the
-ose ending used to name sugars. It has a formula of C12H22O11.
Figure 1 The structure of Lactose. Lactose is derived from the condensation of galactose and glucose,
which form a β-1→4 glycosidic linkage. Its systematic name is β-D-galactopyranosyl-(1→4)-Dglucose.
Infant mammals nurse on their mothers to drink milk, which is rich in lactose. The
intestinal villi secrete the enzyme called lactase (β-D-galactosidase) to digest it. This
enzyme cleaves the lactose molecule into its two subunits, the simple sugars glucose and
galactose, which can be absorbed. Since lactose occurs mostly in milk, in most mammals
the production of lactase gradually decreases with maturity due to a lack of constant
consumption.
Many people with ancestry in Europe, West Asia, India, and parts of East Africa
maintain lactase production into adulthood. In many of these areas, milk from mammals
such as cattle, goats, and sheep is used as a large source of food. Hence, it was in these
regions that genes for lifelong lactase production first evolved. The genes of lactose
tolerance have evolved independently in various ethnic groups. By descent, more than
70% of western Europeans can drink milk as adults, compared with less than 30% of
people from areas of Africa, eastern and south-eastern Asia and Oceania.
What are the symptoms?
Symptoms of lactose intolerance can be mild to severe, depending on how much lactase
your body makes. Symptoms usually begin 30 minutes to 2 hours after you eat or drink
milk products. If you have lactose intolerance, your symptoms may include:
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Bloating.
Pain or cramps.
Gurgling or rumbling sounds in your belly.
Gas.
Loose stools or diarrhea.
Throwing up.
Many people who have gas, belly pain, bloating, and diarrhea suspect they may be
lactose-intolerant. The best way to check this is to avoid eating all milk and dairy
products to see if your symptoms go away. If they do, then you can try adding small
amounts of milk products to see if your symptoms come back.
If you feel sick after drinking a glass of milk one time, you probably do not have
lactose intolerance. But if you feel sick every time you have milk, ice cream, or another
dairy product, you may have lactose intolerance. Sometimes people who have never had
problems with milk or dairy products suddenly have lactose intolerance. This is more
common as you get older.
How is it treated?
There is no cure for lactose intolerance, but you can treat your symptoms by
either using dietary supplemental digestive enzymes that help digest lactose, or by
limiting or avoiding milk products. Some people use milk with reduced lactose, or they
substitute soy milk and soy cheese for milk and milk products. Some people who are
lactose-intolerant can eat yogurt without problems, especially yogurt with live cultures..
In time, most people with lactose intolerance get to know their bodies well enough to
avoid symptoms.
One of the biggest concerns for people who are lactose-intolerant is making sure
they get enough of the nutrients found in milk products, especially calcium. Calcium is
most important for children, teens, pregnant women, and women after menopause. There
are many nondairy foods that contain calcium, including:
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Broccoli, okra, kale, collards, and turnip greens.
Canned sardines, tuna, and salmon.
Calcium-fortified juices and cereals.
Calcium-fortified soy products such as soy milk, tofu, and soybeans.
Almonds.
The Lac operon
Escherichia coli (E. coli); is a rod-shaped bacterium that is commonly found in the
lower intestine of warm-blooded organisms. This bacterium is part of the normal flora of
the gut, and can benefit their hosts by producing vitamin K2, and by preventing the
establishment of pathogenic bacteria within the intestine, as well as aiding in our digestive
processes.
Figure 2: The Lac operon in detail. β -galactosidase is coded for by the gene LacZ, Lactose Permease is
coded for by the gene LacY, and Thiogalactoside transacetylase is coded for by the gene LacA. The
catabolite activator protein (CAP), the Promoter (P), and the binding site for the repressor (O) are also
shown.
Jacob and Monod (1961) were the first scientists to fully understand the
transcriptionally regulated steps of the lactose metabolism system in E. Coli. When the
bacterium is in an environment that contains lactose it should turn on the enzymes that
are required for lactose degradation. The genes required for this are:
β-galactosidase: This enzyme hydrolyzes the bond between the two sugars,
glucose and galactose. It is coded for by the gene LacZ.
Lactose Permease: This enzyme spans the cell membrane and brings lactose into
the cell from the outside environment. The membrane is otherwise essentially
impermeable to lactose. It is coded for by the gene LacY.
Thiogalactoside transacetylase: The function of this enzyme is not known. It is
coded for by the gene LacA.
The Operator (LacO) is the binding site for repressor. The Promoter (LacP) is the
binding site for RNA polymerase, and the Repressor (LacI) gene encodes the lac
repressor protein, which binds to DNA at the operator and blocks binding of RNA
polymerase at the promoter (P). Finally, the CAP region is the binding site for
cAMP/CAP complex.
It would seem that the cell would want to turn these genes on when there is
lactose around and off when lactose is absent. However, the story is more complicated
than that! A bacterium's prime source of food is glucose, since it does not have to be
modified to enter the respiratory pathway. So if both glucose and lactose are around, the
bacterium wants to turn off lactose metabolism in favor of glucose metabolism. There
are sites upstream of the Lac genes that respond to glucose concentration.
When lactose is present, it acts as an inducer of the operon. Lactose can
SLOWLY enter the cell and bind to the Lac repressor, inducing a conformational change
that allows the repressor to fall off the DNA. Now the RNA polymerase is free to move
along the DNA and RNA can be made from the three genes. Lactose can now be
metabolized.
When the inducer (lactose) is removed: The repressor returns to its original
conformation and binds to the DNA, so that RNA polymerase can no longer get past the
promoter. No RNA and no protein is made. RNA polymerase can still bind to the
promoter though it is unable to move past it.
When the cell is ready to use the operon, RNA polymerase is already there and
waiting to begin transcription; the promoter doesn't have to wait for the enzyme to bind.
We could say that the operon is primed for transcription upon the addition of lactose.
When levels of glucose (a catabolite) in the cell are high, a cyclic AMP is inhibited from
forming, and as glucose levels drop, more cAMP forms. This cAMP binds to a protein
called CAP (catabolite activator protein), which is then activated to bind to the CAP
binding site. This activates transcription, perhaps by increasing the affinity of the site for
RNA polymerase.
This phenomenon is called catabolite repression, a misnomer since it involves
activation, but understandable since it seemed that the presence of glucose repressed all
the other sugar metabolism operons. A very good animation of the Lac operon can be
found at: http://www.youtube.com/watch?v=oBwtxdI1zvk
Really, - Legume intolerance?!!!!!
So, lactose intolerance involves the inability to produce the enzyme
β-galactosidase which breaks the β-linkage in lactose to produce glucose and galactose.
A very lesser known player in sugar dietary intolerance is the enzyme
α-galactosidase which breaks the α-linkage in melibiose which also produces glucose
and galactose. The disaccharide melibiose is mostly found in legumes such as such as
vanilla and radish. Well-known legumes include: alfalfa, clover, peas, beans, lentils,
lupins, mesquite, carob, soy, and peanuts.
Figure 3. The structure of melibiose. Melibiose is a reducing disaccharide formed by an alpha-1,6
linkage between galactose and glucose (D-Gal-α(1→6)-D-Glc).
The inability to produce the enzyme α-galactosidase to break down melibiose
leads to exactly the same symptoms as lactose intolerance
The operon which governs the activity of the production of the enzyme
α-galactosidase works in the same manner as the Lac operon shown in figure 2.
α-, β- what?
So, what is all this about alpha (α) and beta (β) linkages and
α- and β- galactosidase?
Note the position of the hydroxyl group (red or green) on the
anomeric carbon relative to the CH2OH group bound to carbon 5 in
figure 4.
If the linkage is on the opposite (green) side of the OH group
an α-glycosidic linkage (the bonds joining the simple sugar together)
is formed. If the linkage is on the same (red) side of the OH group
a β- glycosidic linkage is formed.
An α-glycosidase will ONLY break an α-linkage and a
β- glycosidase will ONLY break a β-linkage.
Figure 4: The α
and β anomers
of glucose
Experimental Plan:
This experiment, derived from the work of Reinking et al. (1994) and Preszler
(2000), uses enzymes found in two dietary supplements to evaluate the lock and key
model of enzyme specificity. The first dietary supplement, Lactaid® contains
β-galactosidase which breaks the β−linkage in lactose to produce glucose and galactose;
in contrast, Beano® contains α-galactosidase which breaks the α-linkage in melibiose
which also produces glucose and galactose.
Yeast is also included in each solution to produce a bioassay that generates
carbon dioxide from glucose if the first reaction has occurred. Now, there are four
things to remember and fully understand for this experiment:
♦ Yeast cannot metabolize lactose
♦ Yeast cannot metabolize melibiose
♦ Dietary supplements do not contain any ingredients that can be metabolized by yeast.
♦ Yeast can metabolize glucose
Therefore, if the enzyme treatment of lactose and melibiose allows yeast to use one of the
breakdown products - glucose – then the yeast will be able to undergo cellular
respiration. If this is the case then CO2 will be produced in the experimental system, and
liquid will be displaced.
There is a direct correlation between the amount of liquid displaced and the
amount of CO2 produced. This makes this system ideal for the study of the specific
nature of enzymes.
Enzyme preparations:
To prepare the Lactase (β -galactosidase) enzyme solution, grind two caplets of
Lactaid® to a fine powder using a mortar and pestle to produce a fine powder. Place the
powder in a 100 ml beaker and add a few drops of distilled water. Stir and continue
adding small amounts of water until you have a smooth paste. Top up to the 50ml mark
and store on ice until needed.
To prepare the α-galactosidase enzyme solution, grind three caplets of Beano®
to a fine powder using a mortar and pestle to produce a fine powder. Place the powder in
a 100 ml beaker and add a few drops of distilled water. Stir and continue adding small
amounts of water until you have a smooth paste. Top up to the 50ml mark and store on
ice until needed.
Figure 5: Basic experimental set up
Sugar solutions:
Each sugar solution used will be a 2.5% solution (2.5 grams in 100 ml) in distilled
water, and at a pH of 7.0. The sugars to be used are as follows:
• Lactose.
• Melibiose.
• Glucose.
• Galactose.
Experimental set up:
We will follow the following experimental set up:
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Sugar
(4ml)
Lactose
Melibiose
Lactose
Melibiose
Lactose
Melibiose
Glucose
Glucose
Galactose
Galactose
None
None
Enzyme
(2ml)
Lactaid
Lactaid
Beano
Beano
None
None
Lactaid
None
Lactaid
None
Lactaid
Beano
Yeast
(10 ml)
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
Each sample will be incubated for 25 mins and any water displacement will be measured.
Finally:
Then, yet again, write up a fantastic lab report. Points which must be included in the
report:
• What is your hypothesis?
• Define the terms catalyst, assay, substrate, and active site
• What are the controls?
• What does each experimental sample prove about the function of enzymes?
• What does this experiment explain to you about lactose intolerance?
References
Jacob, F., and Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of
proteins. Journal of Molecular Biology, 3 (3), 318–356.
Preszler, R., W. (2000). The use of writing in investigative biology laboratories. In tested
studies for laboratory teaching (ed, Karcher, S. J.), 21, 492-496.
Reinking, L.N., J.L. Reinking, and K.G. Miller. (1994). Fermentation, respiration and
enzyme specificity: a simple device and key experiments with yeast. American Biology
Teacher, 56: 164-168.