Reference no: EM131029177
3.4 EXPERIMENT 3 (PART 1): CELL FRACTIONATION
Background:
Initial metabolic experiments are generally carried out in unfractionated cell-free homogenates. However, localising a metabolic pathway within a specific cell compartment requires fractionating the homogenate to separate the intracellular organelles. This separation into intracellular compartments is usually achieved through differential centrifugation (a series of centrifugation steps of increasing Relative Centrifugal Force [RCF; also called g force]). Lysis of the cells is generally via chemical and/or mechanical means. The disrupting agent employed needs to rupture the plasma membrane; however maintenance of morphologically intact organelles is also important. The organelles can then be pelleted by centrifugation at different speeds for different lengths of time.
The eukaryotic cell contains various membrane-bound structures that effectively partition specific intracellular components from the surrounding cytosol. These structures include the nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and chloroplasts (in plant cells). The endoplasmic reticulum is an extensive membranous compartment within the cell, sections of which are rich in ribosomes. Disruption of the cell yields a suspension of organelles and vesicles in the cytosol. ‘Microsomes’ is the name given to the disrupted endoplasmic reticulum membranes. Differential centrifugation of the mixture yields cell fractions enriched in nuclei, mitochondria and microsomes. Larger/heavier organelles will pellet at lower spin speeds. The smaller the organelle, the longer and faster the centrifugation needs to be to pellet the organelle.
You will homogenise rat liver and apply differential centrifugation to the homogenate. You will use high-speed centrifugation (10,000xg) to yield a pellet consisting mostly of cell debris and nuclear material. Ultra-speed centrifugation (100,000xg) will provide a pellet consisting predominantly of microsomes (called the microsomal fraction). The supernatant fraction from the ultra-speed centrifugation contains proteins largely derived from the cell cytosol. Two site-specific proteins will be analysed in this practical; cytochrome P-450 and glutathione-S-transferase. Both molecules play major roles in detoxifying xenobiotics (organic molecules that are foreign to the body). Cytochrome P-450 is localised in the smooth endoplasmic reticulum of the liver and small intestine, and represents a marker protein for the microsomal fraction. If Cytochrome P-450 is detected in the samples, then microsomes are present, if it is not detected then cytochrome P-450 may not be in the sample (or it may have been degraded during preparation). Glutathione-S-transferase is a soluble protein and is a marker for the cytosol (supernatant fraction).
A note regarding the preparation of animals for this experiment: In this experiment, rats have been fed for 3 days with pentobarbitone-laced jelly (sodium phenobarbitone at the level of 88 mg/kg rat weight is incorporated into 12 mL of fruit-flavoured jelly). The pentobarbitone supplement serves to raise the level of liver cell Cytochrome P-450, one of the key compartment markers under investigation, above basal levels (which are usually low). This increase in activity is part of the detoxification response of the body. The term ‘inducible enzyme’ is used to refer to enzymes that require prolonged exposure to an inducer (e.g. pentobarbitone) before enzyme levels are raised in response to the drug. The level of this inducible enzyme rises significantly in response to the drug, to levels readily measurable by the technique employed in this practical.
Laboratory Organisation:
Part 1: Preparation of the microsomal and the supernatant fractions.
Students will be presented with a Podcast on preparing the liver tissue fractions for use in this practical. The liver from one rat (~10 grams) will provides sufficient starting material for your group work. The dissection of the rat will be demonstrated in the Podcast.
Part 2: The microsomal and supernatant fractions will be assayed for cytochrome P450 content using spectrophotometric analysis.
Protein content of the samples will also be determined using the Lowry method and the specific activity of this enzyme in the fractions will be calculated.
Part 3: The microsomal and supernatant fractions will also be assayed for glutathione-S-transferase activity by enzyme activity assay using the substrate 1-chloro-2, 4-dinitrobenzene [CDNB]. Protein content of the samples will again be determined using the Lowry method and the specific activity of this enzyme in the fractions will be calculated.
In the first week of experimentation, half of the class will perform Part 2 (cytochrome P-450 analysis) while the other half of the class will perform Part 3 (glutathione-S-transferase analysis).
The following week, the groups will swap tasks to complete the remaining section of the experiment; be it Part 2 or Part 3.
Reagents
Perfusion buffer: Tris 20 mmol/L, EDTA 3 mmol/L, KCl 1.15%, pH 7.0 at RT / 7.4 at 4°C. Store on ice at 0-4°C.
Storage buffer: as for Perfusion buffer but with the inclusion of 20% (v/v) Glycerol. (Glycerol helps preserve protein material stored at very low temperature). Store on ice at 0-4°C.
Method
All materials must remain ice-cold throughout the proceedings: elevated temperature will significantly diminish enzyme activity.
1. Place a small beaker (~100 mL size), perfusion buffer, and storage buffer on ice.
2. The rats will be dissected and the entire liver will be excised and removed by trained laboratory staff. Using a pair of surgical scissors, an incision will be made along the midline of the lower abdomen and a cut will then be made along the midline towards the rib cage. Cutting laterally from the midline may be necessary. This will expose the liver; the large lobular red organ.
3. Once received, immediately transfer the liver sample to a pre-chilled beaker and add 12 mL of ice-cold perfusion buffer.
Use a pair of curved scissors to cut the liver into small pieces. This procedure will release a large portion of the entrapped hepatic blood cells. Red blood cells will interfere with future analysis of the fractions. Therefore, if the suspension is significantly red in colour (due to red blood cells), then carefully drain off the liquid (do not lose the tissue pieces), add another 12 mL of ice-cold perfusion buffer and observe the colour.
Repeat the above procedure until the suspension is not very red in colour (confirm this with a demonstrator). Then, carefully drain off the liquid, taking care not to lose any of the tissue pieces.
Add 12 mL of storage buffer to the tissue.
4. Homogenise the liver suspension using the motor-driven Teflon pestle and glass tube homogeniser set up in the cold room. A demonstrator will help you operate this equipment.
5. Transfer the homogenate to a Sorvall centrifuge tube and centrifuge at 10,000xg (~10,000 revolutions per minute [rpm]) for 10 min at 4°C, on the Sorvall super T-21 using the SL-50T rotor. Tubes must be precisely balanced in the rotor (check with a demonstrator).
6. Transfer the supernatant from the above step to a centrifuge tube and centrifuge at 100,000 x g (~37,000 rpm) for 1 hour at 4°C on the Sorvall OTD-55B using the T-865.1 fixed angle rotor. Tubes must be balanced precisely in the rotor.
7. Gently push a Pasteur pipette through the fatty layer at the top of the tube. Remove 5-10 mL of supernatant and dispense it as 1 mL aliquots (approximate volumes only) into eppendorf tubes. Label these tubes SF (for Supernatant Fraction). Carefully drain off the remaining supernatant, taking care not to disrupt the pellet. Keep supernatant preparations on ice.
8. Using a Pasteur pipette, gently resuspend the pellet, initially with 1.5 mL of storage buffer. Note: The pelleted material comprises two layers: one gelatinous and the other ‘fluffy’. The fluffy layer contains the microsomes and is easily resuspended.
9. After this initial resuspending add an extra 5 mL of storage buffer and transfer the contents to a plastic capped tube. Rinse the ultra-crimp tube with a further 5 mL of Storage buffer and add this volume to the plastic capped tube. The total volume will now be 11.5 mL, equivalent to the original volume of centrifuged material. Invert gently the tube contents to mix. Aliquot 1 mL aliquots (approximate volumes only) into eppendorf tubes. Label these tubes MF (for Microsomal Fraction). Keep these fractions on ice.
10. The Microsomal Fractions (MF) and Supernatant Fractions (SF) will be stored at -80°C until required for further analysis (Parts 2 and 3).
As you perform each assay, you should be thinking about the expected results. Different enzymes and molecules are found in different cellular compartments. Where do you think cytochrome P-450 will be found: the microsomal or supernatant fraction? What about glutathione S-transferase, will it predominate in the microsomal or in the supernatant fraction? Hopefully, you will not be relying on 50-50 odds to get the correct answer.