DifferentialCentrifugationfortheLocalizationofEnzymesandtheExaminationofEnzymeFunction

A common means of determining the location of an enzyme is through the process of differential centrifugation . This method utilizes the principal that different organelles and subcellular components can be separated from one another based upon their relative mass and density . The first step in this process requires homogenization , through which the contents of the cells become accessible. Progressing through successive centrifugation s at increasing speeds and times, various fractions are collected. The organelles and subcellular components, located in either the supernatant or the pellet after each centrifugation, can then be assayed for the presence of the enzyme of interest ( Figure 1 ).

In Part A of this lab, you will prepare a yeast homogenate and then use the process of differential centrifugation for the step-wise isolation of nuclei and mitochondria. The resulting fractions will then be assayed for the presence of alkaline phosphatase (AP) . In alkaline conditions, AP can dephosphorylate p-nitro phenyl phosphate (pnpp) to form p-nitro phenol (pnp) . The removal of this terminal phosphate group from pnpp results in an increase in absorbance when measured at a wavelength of 410 nm . Thus, this assay will allow the determination of the intracellular localization of AP by measuring the increase of absorbance over time in your various fractions, and determining which contain high levels of enzyme activity.

Figure 1: A schematic diagram illustrating the sequential centrifugations required to isolate nuclei, mitochondria, and microsomes from yeast cells. Yeast homogenate Nuclear pellet Mitochondrial pellet Microsomal pelletsupernatantPost-nuclear supernatant In Part B, we will use a similar process to isolate mitochondria from white mushrooms. We will use the mitochondria to analyze the function of succinate dehydrogenase (SDH) (See Figure 2 ). Unlike the other enzymes of the citric acid cycle, SDH and its coenzyme flavin adenine dinucleotide (FAD) are tightly bound to the inner mitochondrial membrane as complex II . SDH catalyzes the oxidation of succinate to fumarate in a reaction that generates two protons and two electrons. Fumarate is then a further reactant in the citric acid cycle , while FADH 2 carries the protons and electrons to ubiquinone , a process which can be blocked by including azide . 2,6-dichlorophenolindophenol (DCIP) is an artificial electron acceptor which will go from blue to colorless when it is reduced, allowing us to monitor the change in absorbance at 600nm in order to determine the amount of SDH activity.

This assay will allow us to examine the effect of increasing the amount of enzyme present within a reaction. Recall that the rate and direction of all chemical reactions are determined by the relative amounts of reactants and products, and in the case of enzymatic reactions, is also determined by the availability of the enzyme’s active site. The role of enzyme availability on reaction rates will also be examined by determining the effect of a competitive inhibitor ( malonate ). Malonate has a very similar molecular structure to succinate and can bind to the active site of SDH, thereby competing with the normal substrate ( succinate ) for the active site of the enzyme. However, malonate cannot be oxidized (or dehydrogenated), which results in an alteration in the rate of DCIP reduction. You will determine the maximum rate of the reaction by relating the oxidation of succinate by succinate dehydrogenase to a decrease in absorbance resulting from the reduction of DCIP.

Figure 2: Summary of the DCIP assay for succinate dehydrogenase activity. The transfer of electrons to DCIP instead of ubiquinone is further enhanced by adding azide , which effectively blocks the entire ETC. Malonate competes for the active site of SDH, but is unable to be oxidized, resulting in fewer electrons being transferred to DCIP . Adapted from: Aerobic respiration in the mitochondrial matrix. (Iwasa, J and Marshall, W (2016) Karp’s Cell and Molecular Biology, 8 th edition. Wiley. P. 194) 2Succinate Fumarate SDHDCIP Malonate Azide PROCEDURES Part A. Differential Centrifugation for the Intracellular Localization of Alkaline Phosphatase Prior to the lab a fresh yeast culture was centrifuged at 800 x g for 10 minutes at room temperature to sediment the yeast cells. You will find this sample on ice on your lab bench.

i. Preparation of Intracellular Fractions 1. Carefully decant the supernatant (the liquid culture medium) into the 500 ml Erlenmeyer flask on your bench (labeled “Yeast Cell Bio”). All yeast-derived waste must be collected into this flask for proper disposal at the end of the lab period.

2. Add 5 ml of yeast homogenizing solution to the pellet and resuspend by swirling. You may need to use a glass rod to dislodge the pellet.

3. Transfer the yeast slurry to a 50 ml centrifuge tube . Using the 10 ml scoop provided, add 10 ml of clean glass beads to the tube .

4. Cap the tube. Vortex the tube at top speed for 30 seconds . Place the tube on ice for 30 seconds . Repeat this sequence (vortex-ice/vortex-ice) until the total vortex time is 6 minutes (ie. vortex 12 times ) . Use the timer on your bench to ensure appropriate timing.

5. Add another 15 ml of homogenizing solution to the homogenate/glass bead mixture, and mix the contents well by swirling the tube.

6. Pour the entire contents of the tube through 4 layers of cheesecloth, and collect the filtrate in a small beaker . The glass beads will be collected in the cheesecloth and the yeast homogenate will drain into the beaker. Rinse the tube with another 5-10 ml of homogenizing solution, again pour through the cheesecloth, and then squeeze as much fluid as you can from the cheesecloth into the beaker. The yeast homogenate in the beaker is a mixture of the cytoplasmic contents of the broken cells, the fragments of cell walls, and unbroken cells.

IMPORTANT: Save the glass beads by washing them out of the cheesecloth into a new beaker. Run tap water into the beaker until the yeast residue is removed, and then rinse them with distilled water. Add them to the beaker labeled “USED GLASS BEADS”..

7. Pour the yeast homogenate into a graduated cylinder, and note its exact volume. Remove a 1 ml sample and place it in one of the small test tubes with a black lid. L abel it "yeast homogenate" and place it on ice until you are ready to do the assays.

8. Pour the homogenate into a 50 ml centrifuge tube . Place this tube on ice until ready to c entrifuge at 800 x g for 15 minutes at 4 o C to pellet the nuclei.

3 9. When the centrifugation is complete, remove the tube from the centrifuge rotor. Carefully decant the supernatant into a clean, graduated cylinder . Measure the volume EXACTLY and record it. Remove a 1 ml sample and place it in a glass test tube labeled "post-nuclear supernatant" , and place the tube on ice until a group member is ready to do the assay. Pour the remainder of the supernatant into a clean 50-ml centrifuge tube for the next spin (step 11).

10. The pellet that you now have is the nuclear pellet . Resuspend the pellet in 30 ml of homogenizing solution . Again, record the EXACT volume using a graduated cylinder . Remove a 1 ml sample to a glass tube labeled "nuclear pellet" . Place the tube on ice until a group member is ready to do the assay.

11. Balance your centrifuge tube containing the post-nuclear supernatant and spin at 10,000 x g for 10 minutes, at 4 o C . When the centrifugation from this spin is done, carefully pour the supernatant into a graduated cylinder. Measure and record the EXACT volume . This is the post-mitochondrial supernatant . Remove a 1 ml sample and place it in a labelled glass tube. Keep it on ice . 12. The pellet from this spin is the mitochondrial pellet . Add 5 ml of homogenizing solution to the pellet, resuspend it, record the EXACT volume , and place a 1 ml sample into a labelled glass tube. Keep this sample on ice until a group member is ready to do the assay. 13. At this point, you should have collected a total of FIVE fractions to assay for enzyme activity. These are:

1) yeast homogenate 2) post-nuclear supernatant 3) nuclear pellet 4) post-mitochondrial supernatant 5) mitochondrial pellet.

ii. Assay for Alkaline Phosphatase Activity The entire assay is carried out in a cuvette. An increase in absorbance at 410 nm indicates that alkaline phosphatase enzyme activity is present. You should assay your fractions as they are collected . Do not wait until all 5 fractions are collected.

1. Prepare 11 cuvettes containing 2.5 mls pnpp assay mix and 0.4 mls distilled water each. One cuvette will act as a blank, the other ten will be the experimental cuvettes.

2. Ensure your spectrophotometer is set to read absorbance at 410 nm and blank (zero) the machine.

3. Place one of the cuvettes you have prepared into the machine. WITHOUT REMOVING THE CUVETTE, add 100 microliters of the yeast homogenate you prepared in step 7 of Part i (use the micropipet provided). Stir the contents with the tip. Close the lid and record the absorbance value. This is considered time “zero”.

4 4. Without removing the cuvette or opening the lid , let the reaction continue for 3 minutes and again record the absorbance. 5. Obtain a replicate reading for this fraction by repeating steps 3 & 4, using a fresh cuvette and a fresh sample.

6. Repeat steps 3-5 for each of the other four yeast fractions.

iii. Interpretation of the data In the case of AP activity, as the amount of pnp produced increases, there will be an increase in absorbance. We can observe a correlation between the change in absorbance over time and the amount of enzyme activity. The activity of enzymes is measured in units , with one unit of enzyme activity being defined as the amount of enzyme activity which results in the generation of 1 micromole of product in 1 minute. Thus, to determine the number of enzyme units present in 100 microliters of each fraction, use the following procedure:

1. Determine the increase in absorbance that has occurred in 3 minutes for each replicate, average them, and then divide this value by 3 to obtain the increase in absorbance/minute.

2. Divide this change in absorbance by 0.01 (which is the change in absorbance due to the production of 1 micromole of pnp). This value is the number of enzyme units in 100 microliters of sample that you used from each yeast fraction. 3. Use the data tables on Nexus, to complete your analysis. This will be discussed further during the lab tutorial.

Continue to Part B on the following page! 5 Part B. Examination of Succinate Dehydrogenase Function i. Isolation of White Mushroom Mitochondria IMPORTANT: All reagents, tubes and containers should be kept cold throughout this isolation. Prior to the lab, 40 g of mushrooms were weighed and cut into pieces for you.

1. P lace the mushroom pieces into a chilled mortar . Add 5 grams of cold sand (as an abrasive) and 20 ml of cold isolation buffer . Grind the tissue vigorously for about 2 minutes , until the mixture becomes a smooth paste, about the consistency of applesauce. Keep the mortar on ice the whole time you are grinding!

2. Add an additional 20 ml of cold isolation buffer to the cell paste and grind for 2 minutes more.

3. Pour the slurry through four layers of cheesecloth (you may have to do this in stages), and collect the filtrate in a clean, cold 100 ml beaker, kept on ice. Wash the mortar with 5 ml of cold isolation buffer , and pour this wash through the cheesecloth as well. Squeeze as much “juice” out of the cheesecloth as you can.

4. Pour the extract into a clean 50 ml centrifuge tube. Balance your tube with the tube of the group across from you at your bench. Centrifuge the tubes at 600 x g for 10 minutes at 4 o C .

5. Remove the tubes from the centrifuge (carefully!) and pour the supernatant into a clean 50 ml centrifuge tube . Discard the pellet, which contains nuclei, unbroken cells and other debris.

6. Balance your tube and centrifuge the supernatant at 12,000 x g for 30 minutes at 4 o C .

7. Remove the tube from the centrifuge. Examine it carefully. You should see a distinct mitochondrial pellet at the bottom of the tube. Discard the supernatant by carefully pouring it into a waste beaker.

8. To the mitochondrial pellet , add 8 ml of assay mix . Resuspend the pellet with a brief vortex at medium speed. Do this in 5-second bursts, and inspect your progress. When the pellet has been resuspended, and seems to be uniformly dispersed, you can stop. Place the tube on ice. This is your mitochondrial pellet for the succinate dehydrogenase assay 9. With a clean pipet, remove 1 ml of the mitochondrial pellet suspension and place it in a clean glass tube. Attach a lid, loosely , to the tube. Place this tube in a beaker of boiling water for 3 minutes . Remove the tube and place it on ice. This tube contains the “ boiled pellet ” for use in your succinate dehydrogenase assay.

6 ii. DCIP Standard Curve The succinate dehydrogenase assay you are going to do monitors the loss of absorbance (from blue to colourless) of the dye, 2,6-dichlorophenolindophenol (DCIP) as it is reduced by accepting electrons from FADH 2 . Thus, you need a standard curve of DCIP dilutions to allow quantification of the assay.

1. Place five cuvettes in a test tube rack, labeled 1-5.

2. Prepare dilutions of the 50 micromolar stock solution of DCIP as in the table below:

Cuvette Assay Medium DCIP Concentration (  M) 1 5 ml --- 0 2 4 ml 1 ml 10 3 3 ml 2 ml 20 4 2 ml 3 ml 30 5 1 ml 4 ml 40 3. Cut off a 3 cm strip of Parafilm® from the roll provided. Invert each of the cuvettes several times to mix the contents, using Parafilm® as a stopper each time.

4. Using cuvette 1 as the blank , read the absorbance, at 600 nm , of cuvettes 2-5.

5. You will use these values to prepare a DCIP standard curve as described in Appendix A .

iii. Succinate Dehydrogenase Assay 1. Prepare the cuvettes for your assay as shown in the following table. Add the buffer, succinate, DCIP, azide, and malonate in that order, but do not add mitochondria or boiled pellet yet! Cuvette Buffer Succinate DCIP Azide Malonate Mitochondrial Pellet Boiled Pellet 1 (Blank) 5.2 ml 0.5 ml -- 0.5 ml -- 0.3 ml -- 2 3.2 ml 0.5 ml 2.0 ml 0.5 ml -- 0.3 ml -- 3 (Blank) 4.6 ml 0.5 ml -- 0.5 ml -- 0.9 ml -- 4 2.6 ml 0.5 ml 2.0 ml 0.5 ml -- 0.9 ml -- 5 (Blank) 4.9 ml 0.5 ml -- 0.5 ml -- 0.6 ml -- 6 2.9 ml 0.5 ml 2.0 ml 0.5 ml -- 0.6 ml -- 7 2.7 ml 0.5 ml 2.0 ml 0.5 ml 0.2 ml 0.6 ml -- 8 3.4 ml 0.5 ml 2.0 ml -- -- 0.6 ml -- 9 3.4 ml -- 2.0 ml 0.5 ml -- 0.6 ml -- 10 2.9 ml 0.5 ml 2.0 ml 0.5 ml -- -- 0.6 ml 7 2. Gently swirl the centrifuge tube containing the mitochondrial pellet obtained from Part A . SET A TIMER , then with a clean pipet add 0.3 ml of the mitochondrial suspension to cuvettes 1 and 2 ONLY. Using Parafilm® to cover the cuvettes, invert the cuvettes to mix. Immediately read and record the absorbance value of cuvette 2 at 600 nm, using cuvette 1 as a blank. 3. Replace cuvettes 1 and 2 in the rack at room temperature. You will repeat your readings every 5 minutes for 30 minutes. Use a timer! Use cuvette 1 to blank the machine each time before reading cuvette 2 and entering the absorbance on the chart. Remix the cuvettes prior to each reading by inverting. 4. Once you have completed your time zero reading for cuvette 2, gently swirl the centrifuge tube containing the mitochondria. Then, with a clean pipet, add 0.9 ml of the mitochondrial suspension to cuvettes 3 and 4 ONLY. Mix by inverting, blank with cuvette 3 and immediately read the absorbance of cuvette 4 at 600 nm. You will again be reading the absorbance every 5 minutes, so remember to set a timer for these samples as well. Don’t forget to remix the contents at each time point or to blank the spectrophotometer with cuvette 3 each time.

5. Repeat this process for cuvettes 5-10. Cuvette 5 is the blank, and must be used at the beginning of this series at each time point (ie. you do not need to blank before each cuvette, just before reading cuvette 6). Cuvettes 5-9 will have 0.6 ml of the mitochondrial pellet added to each, while cuvette 10 will contain 0.6ml of the boiled pellet . Remember to invert to remix at each time point.

iv Data analysis Data for this experiment will be posted on Nexus. How to perform the calculations and interpret the results will be discussed in the lab tutorial.

Prior to the Lab 3 Tutorial: There are no additional videos or appendices required for this tutorial. However, you may wish to review Appendix B: Spectrophotometry and Standard Curves .

ASSIGNMENT Your assignment for Lab 3 will include data analysis, figure and table preparation, and answering questions related to this lab. The specific details of this assignment can be found on Nexus in the Lab 3 folder, and is due on October 22 nd by 11:59 PM.

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