Cell Fractionation

Objectives

• learn the techniques of tissue homogenization and different centrifugation
• perform enzyme assays on marker enzymes

Introduction and Background Information

Organelles are membrane-enclosed vesicles inside all eukaryotic cells that function in a variety of important cellular processes. In this lab, we will isolate various subcellular fractions from rat liver by a technique termed differential centrifugation. We will then assay these fractions for the presence of specific organelles using enzyme assays. In this experiment, we will learn the technique of differential centrifugation, and enzyme assays for succinate dehydrogenase and acid phosphatase. In the next lab, you will apply this knowledge to design experiments for optimizing the yields of certain organelles.

Cellular Fractionation

The arrangement of macromolecules within a cell is as important to cellular function as their catalytic activities. Cellular compartmentalization provides efficiency by bringing together related compounds that can interfere with each other (i.e. lysosomal hydrolytic enzymes). Cellular compartmentalization is accomplished in part by various subcellular organelles. In this lab module, we will isolate several subcellular organelle fractions from liver cells, and will examine their various properties. The method we will use to separate the various organelles utilizes differential centrifugation to isolate components of different densities. With this technique, the heaviest or most dense organelles, nuclei pellet in less time and with less force than is required to pellet lighter organelles such as mitochondria. First, a cell homogenate is made by rupturing the cell membranes in the tissue. The homogenate is then centrifuged for a short period of time to remove cell debris and nuclei.

The supernatant is then transferred to another tube and centrifuged longer to pellet the lighter mitochondria.

For this type of fractionation, which tissue we use, and the method of homogenization are dictated by the biological system. Homogeneous cell populations from cell culture are well suited for cell fractionation. Some tissues, like those in the liver also have one cell type that predominates, so are also well suited. Most chlorophyll-free plant tissues are acceptable for preparing mitochondria, but recently-harvested plant tissues are usually required, making their use uncertain during winter months. Once a cell type is chose (we will work with liver), it is important to obtain the organelles in a biochemically active, morphologically whole state. Homogenizers are used to break open the cells without damaging the organelles. Homogenizers have a precise clearance between the glass tube and pestle, which breaks the cell membrane leaving the smaller organelle membranes intact. The homogenization buffer is a solution which often includes sucrose to partially dehydrate the organelles, keeping them intact.

No technique used to isolate organelles is perfect. It is very difficult to get pure unbroken preparations of any organelle. Techniques providing optimal isolation of one organelle may completely rupture another organelle. Thus methods are often used to measure the contamination of one organelle fraction by another. This can be done by analyzing each organelle fraction for organelle-specific marker enzymes.

Theory: Cellular Fractionation

The arrangement of macromolecules within a cell is as important to cellular function as their catalytic activities. Compartmentalization provides efficiency by bringing related compounds together (i.e. the enzymes of the mitochondrial electron transport chain), or by separating those that would interfere with each other (i.e. lysosomal hydrolytic enzymes). In this experiment, several subcellular fractions from liver cells will be prepared, and various properties of these organelles examined. The activity of each fraction is influence by a variety of factors. They are affected by the diet, age, sex, and extent of fasting of the host animal, and also by the experimental method used to obtain the fraction. The method we will use to separate the cell organelles utilizes differential centrifugation to isolate components of different densities. The particular fractionation procedure we will use was designed to maximize the activity of mitochondria (they are extremely fragile) while partially sacrificing the yield and resolution of some of the more stable fractions.

We will evaluate the purity of our subcellular fractions by analyzing each fraction for various marker enzyme activities. It is well documented that some enzymes are located specifically within certain cell fractions. For example, succinate dehydrogenase is specific to mitochondria; glucose-6-phosphatase is specific to microsomes; acid phosphatase is specific to lysosomes; and lactate dehydrogenase is specific to the cytoplasm.

* Although the nucleus contains many specific enzymes, they are not easily assayed in our lab.

Theory: Assay of Mitochondria:

Mitochondria are the eukaryotic subcellular organelles which contain the enzymes of the citric acid cycle, the electron transport chain, and oxidative phosphorylation. The organelle is composed of an inner and outer membrane. The organelle is composed of an inner and outer membrane. The outer membrane is particularly permeable, allowing most molecules with molecular weights up to 10,000 daltons to pass into the intermembrane space. This outer membrane contains a curious mixture of enzymes involved in such diverse activities as the oxidation of epinephrine, the degradation of tryptophan, and the elongation of fatty acids. The inner membrane, however, is very impermeable and allows only small uncharged molecules (such as water and pyruvic acid) to penetrate to the matrix. The enzymes for the electron transport chain and oxidative phosphorylation are embedded in the inner side (matrix side) of the inner membrane. With the exception of succinate dehydrogenase, which is also located within the inner membrane, all the enzymes of the citric acid cycle are located within the matrix.

Mitochondria can be prepared from a variety of eukaryotic tissues (both plant and animal) by differential centrifugation. The presence of mitrochondria in an isolated subcellular fraction can be verified by assaying for mitochondrial-specific enzymes. In these assays, care must be taken to choose low-molecular=weight non-ionic substrates and dyes which are capable of penetrating both the outer and inner mitrochodrial membranes. Mitochondrial-specific properties which are easily assayed include: 1) the ability to take up oxygen, 2) the ability to reduce dye (dichlorophenal-indophenol), and 3) the ability to oxidize cytochrome C (this requires an active cytochrome oxidase). Succinate is a commonly used substrate, and its oxidation to fumarate requires an active succinate dehydrogenase, which is located within the inner membrane. Normally, in the absence of exogenous dyes, the reaction occurs with the concurrent reduction of FAD:

However, during an assay for succinate dehydrogenase, an excess concentration of a dye (usually dichlorophenal-indophenol, (CDPIP) is used to observe its reduction. The reduction of the dye decreases its absorbance at 600 nm: it turns from blue to clear. Moreover, the concentration of substrate (succinate) is also used in excess to prevent the reverse reaction: the reduction of fumarate with the simultaneous oxidation of the dye:

HOOC-CH2-CH2-COOH	DCPIP	→	DCPIP-H2 +	HOOC-CH=CH-COOH
(Succinate) (excess)	(dye-blue) (excess)	Succinate Dehydrogenase	(dye-colorless)	(Fumarate)

In the present experiment, we will use the succinate/DCPIP oxidation-reduction assay to rest various liver subcellular fractions for the presence of active succinate dehydrogenase. We will also observe the change in the activity of the succinate dehydrogenase as an electron-transport chain enzyme (cytochrome oxidase) is inhibited by KCN. KCN blocks cytochrome oxidase for the completion of the reaction, thus the rate of DCPIP reduction increases, and the slope of the decreasing spectrophotometric OD curve becomes steeper. In general, mitochondrial activity can be directly inhibited by three classes of chemical agents. The first class, represented by the antibiotic oligomycin, inhibits both oxidative phosphorylation and respiration. The second class, represented by m-chlorocarbonylcyanide phenylhydrazone (CCP), inhibits only oxidative phosphorylation. The third class, represented by rotenone and cyanide, are site-specific respiratory inhibitors.

Protocol for Isolation of Liver Cell Fractions:

Work rapidly or the fractions will lose enzymatic activity. Conduct all procedures on ice. Once the mitochondria have been isolate, two members of each lab group should proceed with the assay of Succinate Dehydrogenase while the other member continues to repurify the nuclear fraction and isolate the microsomes.

Tissue Preparation:

1. Obtain one of the beakers containing the liver tissues (prepared by the Instructor). The weight of the liver is written on the side of the beaker, record it. _______________
2. Using scissors, mince the tissue.
3. Decant and discard the pink solution, including the floating material.
4. Optional: Tissue Wash: This step removes any enzymes released into the buffer as a result of physical tissue damage. These released enzymes could have been solubilized into the final Homogenization Buffer, where they could register as free cytoplasmic enzymes in our subsequent assay.
   1. Submerge the liver tissue in fresh Homogenization Buffer.
   2. Continue to mince and rinse the tissue until the Buffer is nearly colorless (2-4 times).

Tissue Homogenization:

5. Transfer about one-half (~2 gm) of the tissue to a glass dounce homogenizer on ice. Do not use a tight-fitting ground-glass homogenizer in this step since the organelle membranes may disrupt partially.
6. Add 18 ml of ice-cold Homogenization Buffer to the homogenizer. Note: the Calcium in the buffer is used here to stabilize the organelle membranes.
7. Homogenize 3-5 strokes until a homogeneous homogenate is made. Keep the homogenizer on ice. Caution: Excessive grinding or heating can damage and inactivate subcellular fractions.
8. Optional: Homogenize with a teflon Potter-Elvehjem homogenizer to ensure total tissue disruption.
9. Transfer the homogenate into a plastic 50-ml JA-20 tube on ice.
10. Repeat steps 5-9 (combining all homogenates) until all the minced liver tissue has been homogenized.

Isolation of Nuclei:

11. Centrifuge the JA-20 tube from step 10 at 700 x g (2.5K for the JA-20 rotor), for 10 min. at 0ºC. The pellet contains the cell debris and nuclei. The supernatant contains all the lighter cellular fractions.
12. Without disturbing the crude nuclear pellet, carefully decant the supernatant into another JA-20 tube on ice. Since the mitrochondria are to be assayed today, and time is short, one lab group member should immediately proceed to “Isolation of the Mitochondria”, another member should proceed with isolation of the nuclei, and the third member should proceed to "Protocol for Succinate Dehydrogenase Assay".
13. To continue with the nuclear isolation: Optional: Nuclear Wash:
    1. Add 10 ml of ice-cold Homogenization Buffer to your crude nuclear pellet.
    2. Use a teflon or dounce homogenizer to resuspend the pellet in the buffer.
    3. Place two layers of cheesecloth in a funnel over at JA-20 tube. Filter the 10 ml suspension through the cheesecloth.
    4. Rinse the debris in the filter with another 10 ml of Homogenization Buffer collecting the filtrate in the same JA-20 tube. Discard the filter with its cellular debris.
    5. Centrifuge the combined filtrates (20 ml) at 1500 x g (3.5K in the JA-20 rotor) for 10 min at 0ºC. The pellet contains the washed nuclear fraction. Without disturbing the nuclear pellet, discard the supernatant.
14. Add 5.0 ml of ice-cold Homogenization Buffer to the nuclear pellet.
15. Use a dounce or teflon homogenizer to resuspend the pellet in the buffer.
16. Using a pipette, aliquot the nuclear suspension into two glass test tubes. Measure the total volume as you aliquot it, and record it below and in the "Result Summary Table". Total volume of nuclei = __________ ml.
17. Label the two glass tubes "Nuclei", and store them at -20 °C. Caution: Glass tubes should not be greater than half full, or they may crack upon freezing.

Isolation of Mitochondria

18. Obtain the supernatant from step 12 in a JA-20 tube on ice.
19. Centrifuge at 5,000 x g (for the JA-20 this is 6.5K) for 15 min. at 0ºC. If you are pressed for time: centrifuge at 8.0K for 10 min at 0ºC. The pellet contains the crude mitochondrial fraction. The supernatant contains microsomes, membrane fragments, ribosomes, cytoplasmic enzymes, etc.
20. Without disturbing the mitochondrial pellet, carefully decant the post-mitochondrial supernatant (and the pink partially sedimenting layer) into a graduated cylinder. Measure and record its volume. Total original volume of post-mitochondrial supernatant = ________ ml.
21. Pour the supernatant into a clean JA-20 tube on ice. Swirl the tube to mix, and aliquot 4.0 ml to a test tube on ice for use as a "Negative Control" in the Succinate Dehydrogenase assay.
22. With the remainder of the supernatant, the lab group member who isolated the nuclei should proceed to "Isolation of the Microsomes".
23. To continue with mitochondrial isolation: Optional: Mitochondrial Wash:
    1. Add 20 ml of ice-cold Homogenization Buffer to the crude mitochondrial pellet.
    2. Using a dounce or teflon homogenizer, resuspend the pellet in the buffer.
    3. Pour the suspension back into the JA-20 tube, add another 20 ml of buffer, mix, and centrifuge at 234,000 x g (this is 14K for the JA-20 rotor) for 10 min. at 0ºC. The pellet contains the washed mitochondria.
    4. Without disturbing the mitochondrial pellet, decant and discard the supernatant.
24. Add 5.0 ml of ice-cold Homogenization Buffer to the mitochondrial pellet.
25. Using a dounce or teflon homogenizer, resuspend the pellet in the buffer.
26. Using a 10 ml pipette, measure the total volume of mitochondria and record it below and in the "Result Summary Table". Total volume of mitochondria = __________ ml.
27. Using a pipette, aliquot 2.0 ml of the mitochondrial suspension into a test tube on ice for the Succinate Dehydrogenase assay. Aliquot the remainder of the suspension into two glass test tubes on ice. Label the tubes "Mitochondria" and store them at -20°C.
28. Proceed immediately to the "Succinate Dehydrogenase Assay", which should already be set up by one member of your lab group.
29. Obtain the supernatant from step #22 in a JA-20 tube on ice.

Isolation of Microsomes: (Skip for this Lab)

30. Using a pipette, fill two Type-40 Ultracentrifuge bottles with supernatant, determining the exact volume as you transfer.

Protocol for Succinate Dehydrogenase Assay: Final assay volume = 6.0 ml.

1. The power for the Spec-20 should have been on for at least 15 minutes prior to use to allow it to warm up. This stabilizes the OD readings. Using the top knob on the Spec 20, adjust the wavelength to exactly 600 nm.
2. Using 10 ml test tubes which fit directly into the Spec-20, prepare the following four tubes on ice:



Solution	Tube 1	Tube 2	Tube 3	Tube 4
Volume (ml) of 0.1 M Succinate pH 7.5:	1.00	1.00	1.00	1.00
Volume (ml) of 20X Phosphate-Buffered-Saline pH 7.5:	0.25	0.25	0.25	0.25
Volume (ml) of 5.0 mM KCN (not the 0.1 M Stock) *:	----	1.00	----	1.00
Volume (ml) of Distilled Water:	2.75	1.75	2.75	1.75
Volume (ml) of Mitochondrial Suspension from Step #27:	1.00	1.00	----	----
Volume (ml) of Post-Mitochondrial Supernatant from Step #21:	----	----	1.00	1.00

* Caution: Poison! Do not mouth pipette this solution!

3. Parafilm or stopper the four tubes, and invert them several times to gently mix the contents. Caution: vigorous vortexing can denature enzymes. Incubate on ice for 30 seconds to allow the substrate and inhibitor to penetrate the mitochondria.
4. Perform the following to one tube at a time:
   1. To tube #1, add 1.0 ml of dye (0.5 mM DCPIP). The dye is added only after the inhibitor has had a chance to act.
   2. Gently mix.
   3. Equilibrate the tube to 25ºC by immersing it in tap water for a few seconds. This step avoids condensation on the cuvette during the assay.
   4. Zero the spectrophotometer: Without a tube in the sample slot, use the front-left knob to place the needle exactly at 0% transmittance (infinite absorbance).
   5. Place tube #1 in the Spec-20, leave it in place, close the Spec-20 cover, use the front-right knob to place the needle at 0.800 absorbance (the needle will be slowly moving to the right due to the activity of the mitrochondria). Immediately start timing, and measure and record the absorbance at 600 nm every 30 seconds for 6 min.
5. Repeat step 4 for tubes 2-4.

Results:

1. Using one sheet of standard (non-logarithmic) graph paper, plot the OD 600 on the vertical axis versus time (minutes) on the horizontal axis for each of the four tubes. Place all four curves on one graph.
2. Using the starting and ending OD values, calculate the OD/min. for each tube. Note: The plots are not usually linear, thus exact slopes are difficult to determine.
3. Perform this step after Lab 3: Divide the OD/min values for tubes 1 and 3 (no inhibitor) by the #mg protein in the mitochondrial fraction to obtain the specific activity (change in OD/min/mg).

Discussion:

1. Based on a comparison of the OD/min values, or the OD end-points for tubes 1 and 2, explain the effect of KCN on mitochondrial Succinate Dehydrogenase.
2. What is the mechanism of action of potassium cyanide?
3. What other inhibitors of mitochondrial activity could we have used in this experiment?
4. Based on a comparison of the OD/min values, or the OD end-points for tubes #1 and #3, how clean was your mitochondrial pellet? If some contamination of the post-mitochondrial supernatant with Succinate Dehydrogenase was observed, what could have caused it?

Prelab Questions - Use a resource of your choice - Basic Biochemistry, Cell Biology or Biology Text

1. List 4 cell organelles and the functions and processes which occur in them.
2. In this lab experiment - we are isolating mitochondria. What is the structure of this organelle (subcellular particle)? What metabolic chemical reactions occur in mitochondria?
3. What is an enzyme marker?