BIOL 109 Laboratory Four A study of lactose intolerance to further understand the function of enzymes.

Transcription

1 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 C 12 H 22 O 11. 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.

2 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: 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

3 most important for children, teens, pregnant women, and women after menopause. There are many nondairy foods that contain calcium, including: 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.

4 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:

5 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 CH 2 OH 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

6 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 CO 2 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 CO 2 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.

7 Figure 5: Basic experimental set up

8 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 Sugar Enzyme Yeast (4ml) (2ml) (10 ml) 1 Lactose Lactaid yes 2 Melibiose Lactaid yes 3 Lactose Beano yes 4 Melibiose Beano yes 5 Lactose None yes 6 Melibiose None yes 7 Glucose Lactaid yes 8 Glucose None yes 9 Galactose Lactaid yes 10 Galactose None yes 11 None Lactaid yes 12 None Beano 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?

9 References Jacob, F., and Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology, 3 (3), Preszler, R., W. (2000). The use of writing in investigative biology laboratories. In tested studies for laboratory teaching (ed, Karcher, S. J.), 21, 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: