Oxaloacetate, found in the mitochondrion, is a four-carbon molecule. In this project we performed a Bradford Assay and an SDH (Succinate Dehydrogenase) Assay. The goal of this project was to work with enzymes to gain a better understanding of how enzymes function and how they are impacted with the introduction of a variety of factors.
A significant part of the experiment was determining the protein concentration of the prepared mitochondrial fraction which will be further explained. The reason for that is by increasing enzyme concentration present in a sample, it can increase the enzyme's activity and that is what we are studying. We conducted a Bradford Assay to measure the protein concentration of the mitochondrial liver cell fractions. …show more content…
It is a part of the respiratory electron transfer chain and the citric acid cycle. During the citric acid cycle SDH oxidizes succinate into fumarate (Hagerhall 1997). SDH in Eukaryotic cells has four subunits encoded by the nuclear genome. SDH catalytic activity is regulated by post-translational phosphorylation, acetylation, and active site inhibition. It is also regulated by the Krebs cycle intermediates including oxaloacetate, which is what our group is studying. Succinate stimulates the dissociation of oxaloacetate from SDH which activates the enzyme (Gutman 1975). Cow liver was our source of SDH in this project. Liver has a high concentration of mitochondria to accommodate its great demand for ATP. We fractionated bovine liver cells to obtain separated mitochondria which had a higher concentration of SDH. SDH is found solely in the mitochondria so it is useful to perform an SDH assay on all separated fractions to view the relative SDH activity. So if other components of the cell are found with high concentration of SDH it is evident that it was polluted with mitochondria which shows the type of error you make. In our SDH Assay, we manipulated SDH activity by using reagents such as: potassium phosphate (maintains optimum range for SDH and is a buffer to control pH or reaction), azide (blocks electron transport chain to block the build up of FADH2), buffered sucrose (keeps mitochondria from bursting),
A significant part of the experiment was determining the protein concentration of the prepared mitochondrial fraction which will be further explained. The reason for that is by increasing enzyme concentration present in a sample, it can increase the enzyme's activity and that is what we are studying. We conducted a Bradford Assay to measure the protein concentration of the mitochondrial liver cell fractions. …show more content…
It is a part of the respiratory electron transfer chain and the citric acid cycle. During the citric acid cycle SDH oxidizes succinate into fumarate (Hagerhall 1997). SDH in Eukaryotic cells has four subunits encoded by the nuclear genome. SDH catalytic activity is regulated by post-translational phosphorylation, acetylation, and active site inhibition. It is also regulated by the Krebs cycle intermediates including oxaloacetate, which is what our group is studying. Succinate stimulates the dissociation of oxaloacetate from SDH which activates the enzyme (Gutman 1975). Cow liver was our source of SDH in this project. Liver has a high concentration of mitochondria to accommodate its great demand for ATP. We fractionated bovine liver cells to obtain separated mitochondria which had a higher concentration of SDH. SDH is found solely in the mitochondria so it is useful to perform an SDH assay on all separated fractions to view the relative SDH activity. So if other components of the cell are found with high concentration of SDH it is evident that it was polluted with mitochondria which shows the type of error you make. In our SDH Assay, we manipulated SDH activity by using reagents such as: potassium phosphate (maintains optimum range for SDH and is a buffer to control pH or reaction), azide (blocks electron transport chain to block the build up of FADH2), buffered sucrose (keeps mitochondria from bursting),