Purification of Mitochondria by Subcellular Fractionation of Mouse Brain

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This paper explores the hypothesis that there are varying quantities of mitochondria between mutant and healthy organisms. The experimental aim of this investigation was to create a method to obtain a subcellular fraction that was enriched in mitochondria. We initially designed an experiment for the purification of mitochondria from mouse brains using subcellular fractionation. Using differential centrifugation and marker enzymes (succinate dehydrogenase) we were able to obtain a subcellular fraction that was enriched in mitochondria. We compared the results from control and mutant mouse brains, through the analysis of succinate dehydrogenase, we found that Pellet 2 (P2) had the highest specific SDH activity that that there was no significant difference between the control and mutant except from the SDH activity of the Homogenate (H) and Pellet 2 (P2). From the data we had obtained we concluded that Pellet 2 (P2) is where all the SDH was, therefore the mutation doesn’t affect the protein content, but it does affect the SDH activity.

Most eukaryotic cells contain mitochondria, which is a membrane enclosed organelle that creates most of the cells energy in the form of Adenosine triphosphate (ATP). The inner mitochondrial membrane contains the electron transport chain. The electron transport chain is considered the molecular apparatus for energy production. The Electron transport chain consist of five protein complexes. In the chain, complex I, III and IV pump protons from the mitochondrial matrix to the intermembrane space. This generates a proton gradient which is essential for the synthesis of Adenosine triphosphate. Complex V is commonly referred to as ATP synthase, it acts as the final enzyme in the production of ATP via oxidative phosphorylation. ATP synthase uses the energy resulting from the proton gradient to drive the synthesis of Adenosine diphosphate and Phosphate to form Adenosine triphosphate. Additional functions of the mitochondria include programmed cell death (apoptosis), calcium signalling and cellular differentiation. Neuronal energy supplies are completely dependent on mitochondrial oxidative phosphorylation. Neurons have limited capacity to obtain energy through glycolysis when oxidative phosphorylation is compromised (Herrero-Mendez A, 2009) which makes them particularly vulnerable to mitochondrial dysfunction consequently it is a vital component of brain functioning.

To understand mitochondria in the brain it is necessary to purify mitochondria and carry out enzyme activity assays. We decided to use Sub cellular fractionation to isolate the mitochondria in the mouse brain tissue. this is a process used to separate the cellular components of a cell whilst preserving the individual components of each cell (Alberts B, 2002), the process allows each component of the cell to function normally thus preserving the integrity of the components of the cell. We used sub cellular fractionation alongside differential centrifugation to purify the mitochondria so we could carry out enzyme activity assays. The components of a cell were separated based on their density in a centrifuge according to the centrifugal force they experience [2]. The processes combined provided us with an enriched source of protein that has been purified. The fractions we obtained were assayed for protein using Bradfords reagent and Succinate Dehydrogenase (SDH) activity. Succinate Dehydrogenase is only present in mitochondria therefore the distribution of SDH will indicate the distribution of mitochondria in the fractions.

Six mouse brains were collected (three control and three mutant) and weighed using a bench top balance. Ice cold Buffer A (20 mL of 0.25 M sucrose, 10 mL of 5 mM Tris-HCl pH 7.5, 3 mL of 3 mM KCl and 0.6 mL of 0.3 mM EDTA) was added to each mouse brain (1:10 (w/v). we homogenised the mixture with 20 strokes of a Dounce homogeniser. 500 µL of the Homogenate was collected and kept aside for further assay (H). The rest of the Homogenate was transferred into two centrifuge tubes and centrifuged at 2,700 rpm for 10 minutes in a 10 x 20 mL MSE rotor at 4 oC. The supernatant was transferred into two clean centrifuge tubes, and the pellet (P1) was resuspended in 5mL of Buffer A. The Supernatant was then centrifuged at 9,300 rpm for 20 minutes in a 10 x 20 mL MSE rotor at 4 oC. the supernatant was collected (S) and the pellet (P2) was resuspended in 5mL of Buffer A.

Fractions (H), (P1), (P2) and (S) were assayed using Bradfords reagent (0.004% (w/v) Comassie Brilliant Blue G-250, 4.2% (v/v) phosphoric acid and 2.1% (v/v) ethanol.). The fractions were diluted as the following: 100 µL of homogenate (H) was diluted in the ratio of 1:50 with Buffer A, 200 µL of P1 was diluted in the ratio 1:20 with Buffer A, 200 µL of P2 was diluted in the ratio 1:50 with Buffer A and the supernatant (S) was not diluted. 50 µL of each fraction was transferred into a duplicate and 200 µL Bradford reagent was added. The diluted fractions were incubated for 5 minutes and then read at 650 nm in a spectrophotometer.

Fractions (H), (P1), (P2) and (S) were assayed using Succinate dehydrogenase (Sodium succinate (50 mM), Potassium phosphate buffer (pH 7.4, 65 mM), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) (50 mM). The fractions were diluted as the following: Homogenate (H) was diluted in the ratio 1:10 with Buffer A, (P1) was diluted in the ratio of 1:10 with Buffer A, (P2) was diluted in the ratio of 1:20 with Buffer A, Supernatant (S) was not diluted. 20 µL of each fraction was transferred into duplicates with 130 µL phosphate buffer, 50 µL INT and 50 µL sodium succinate for 15 min in a 30 oC water bath. The absorbance was read at 490 nm in a spectrophotometer.

Figure 1.  total protein content in the homogenate (H) and each fraction (P1, P2, S) for control and mutant mouse brains. Average total protein (mg) was higher in the Homogenate (H) compared to Pellet 1 (P1), Pellet 2 (P2) and S. The decrease of average total protein between the homogenate and pellet 1 and S was not significantly different (p ≥ .05). However, the decrease of average total protein between homogenate and pellet 2 was significantly different (p ≤ .05

). The total protein content was measured using a Bradfords assay. Error bars represent SEM of n = 3 mice.

Figure 2.  % recovery of protein in each fraction and the homogenate for control and mutant mouse brains. Average % recovery of protein was highest in the homogenate and decreased considerably in each fraction. The difference in % recovery between homogenate and pellet 1 (P1) and (S) was not significantly different (p ≥ .05) suggesting the difference was due to chance. However, the difference in average % recovery in protein between homogenate and pellet 2 was significant (p ≤ .05) suggesting that the difference is not likely to be due to chance. Error bars represent SEM of n = 3 mice.

Figure 3. specific activity of SDH in the homogenate and each fraction for control and mutant mouse brains. The specific SDH activity was highest in the homogenate (H) and pellet 2 (P2) and was considerably lower in pellet 1 (P1) and (S). The difference in SDH activity between homogenate (H) and pellet 2 (P2) was significantly different (p ≤ .05) suggesting that is it not likely to be a result of chance. However, the difference in SDH activity between homogenate (H) and Pellet 1 (P1) and (S) was not significantly different (p ≥ .05). The SDH activity was measured using an SDH assay. Error bars represent SEM of n = 3 mice.

From the data we obtained we found that Pellet 2 (P2) had the highest specific SDH activity and there was a significant difference in SDH activity between homogenate (H) and pellet 2 (P2), this led us to conclude that pellet 2 (P2) is where all the SDH was, therefore suggesting that the mutation doesn’t affect the protein content but it does affect the SDH activity. It is for this reason we believe that we have obtained a fraction enriched in mitochondria (pellet 2).

References

Alberts, B; Johnson, A. “Fractionation of Cells”. Molecular Biology of the Cell. 4th edition, 2002.

Herrero-Mendez A, Almeida A, Fernandez E, Maestre C, Moncada S, Bolanos JP. The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1. Nat Cell Biol. 2009;11:747-52.

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