Cell Fractionation
Cell Fractionation: Isolation of Mitochondria from Cauliflower and Determination of Specific Enzyme Kinetics
Introduction
Mitochondria is an organelle found in eukaryotic cells that play a role in biochemical processes such as respiration and energy production. Mitochondria even play an important role in apoptosis, or programmed cell death. This is achieved by disruption of electron transport, oxidative phosphorylation, and ATP production or even the release of proteins that trigger activation of caspase family proteases and alteration of cellular reduction-oxidation (redox) potential (Reed and Green, 1998). Succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) are enzymes that play a role in the Citric Acid Cycle portion of cellular respiration and are investigated further in this experiment. The purpose of this experiment was to prepare whole as well as broken preparation of mitochondria and compare the enzymatic activity of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH). This experiment will answer the question of whether enzymatic activity will be greater in intact or broken mitochondria as well as in SDH or MDH. It is hypothesized that enzymatic activity will be higher in intact mitochondria because the intact mitochondria contains both SDH and MDH, where the broken mitochondria only contains MDH. Because the broken mitochondria only contain one enzyme complex, there should be less enzymatic activity. It is hypothesized that the MDH activity will be greater in broken mitochondria rather than intact because in the broken mitochondria, the membrane is removed. Because the membrane is removed, the MDH won’t have to diffuse through said membrane to reach the proteins, thus causing activity to occur faster. It is also hypothesized that SDH activity will be greater in the intact mitochondria rather than the broken mitochondria because the SDH is embedded in the membrane of the mitochondria. This would mean that there shouldn’t be any
Introduction
Mitochondria is an organelle found in eukaryotic cells that play a role in biochemical processes such as respiration and energy production. Mitochondria even play an important role in apoptosis, or programmed cell death. This is achieved by disruption of electron transport, oxidative phosphorylation, and ATP production or even the release of proteins that trigger activation of caspase family proteases and alteration of cellular reduction-oxidation (redox) potential (Reed and Green, 1998). Succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) are enzymes that play a role in the Citric Acid Cycle portion of cellular respiration and are investigated further in this experiment. The purpose of this experiment was to prepare whole as well as broken preparation of mitochondria and compare the enzymatic activity of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH). This experiment will answer the question of whether enzymatic activity will be greater in intact or broken mitochondria as well as in SDH or MDH. It is hypothesized that enzymatic activity will be higher in intact mitochondria because the intact mitochondria contains both SDH and MDH, where the broken mitochondria only contains MDH. Because the broken mitochondria only contain one enzyme complex, there should be less enzymatic activity. It is hypothesized that the MDH activity will be greater in broken mitochondria rather than intact because in the broken mitochondria, the membrane is removed. Because the membrane is removed, the MDH won’t have to diffuse through said membrane to reach the proteins, thus causing activity to occur faster. It is also hypothesized that SDH activity will be greater in the intact mitochondria rather than the broken mitochondria because the SDH is embedded in the membrane of the mitochondria. This would mean that there shouldn’t be any
References: Green, Douglas R., and John C. Reed. "Mitochondria and Apoptosis." Science 281 (1998): 1309-312. Science.com. Science, 28 Aug. 1998. Web. 2 Dec. 2012. Neufeld, Gaylen J., and Zhenqiang, Li. "Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology." SciVerse 128.2 (2001): 325-38. Science Direct. Web. 2 Dec. 2012. <http://www.sciencedirect.com/science/article/pii/S1096495900003304>.