Succinate Dehydrogenase

Succinate dehydrogenase is an enzyme found in the mitochondrial inner membrane. The enzyme catalyzes the reaction of oxidizing its substrate, succinate, into fumarate via the removal of hydrogen ions from succinate. This oxidation is vital in the Krebs cycle.

In our experiment we will use differential centrifugation to isolate the mitochondria of cauliflower and then assay SDH activity using a fixed time assay. We will then measure protein content in our fractions and calculate specific activity and total activity of our fractions.

Understanding the activity of enzymes in different muscle types can aid greatly in obtaining more information about other processes such as metabolism of the tissues (Anderson et al., 2012). There are many different methods in order to achieve this information based in two different major categories but the most convenient method is one called continuous assay. This process includes the use of a spectrophotometer to continuously monitor the assay. This method allows for an easy way to calculate the initial rate of reaction as one can establish time points easily. In the lab performed the continuous method was used in order to determine the measurement of the activity enzyme succinate dehydrogenase (SDH). SDH is most commonly found in mitochondria. It is present in many different muscle tissues including the heart, red, and white tissue. In the following lab the enzyme was tested and measured in these three muscle types in the Oncorhynchus mykiss in order to determine which type contained the highest and the lowest activity. This enzyme is involved in multiple processes such as involved in both the Krebs cycle and the electron transport chain (ETC) (Smith, 2014). Its role in

Cytoplasmic streaming was measured 4 times after flushing the cells with pond water, at 5 minute intervals

Mitochondria are small organelles found in eukaryotic cells which respire aerobically. They are responsible for generating energy from food to ‘power the cell’. They contain their own DNA, reproducing by dividing in 2. As they closely resemble bacteria, it gave the idea that they were derived from bacteria (which were engulfed by ancestors of the eukaryotes we know today). This idea has since been confirmed from further investigations, and it is now widely accepted. (Alberts et al., 2010a)

Each mitochondrion has a double-layered membrane like the cell membrane, however the inner layer is folder which produces ‘shelves’ which are known as cristae, this is where the end stages of glucose oxidation are located. The energy that has been released is stored until required by a ‘chemical battery’ called adenosine triphosphate.

Homogenates were provided made from liver, kidney, or heart in a 1:20 ratio with sucrose-phosphate buffer that was stored at -70° C. The tissue I tested was the liver homogenate. To qualitatively analyze proteins of the homogenate, 5µl of original liver homogenate was combined with 2 µl of protein gel sample buffer and heated in a water bath at 80° C for 10 minutes. After centrifuging the sample for 5 seconds, the contents were loaded onto a polyacrylamide protein gel set at 100V for Electrophoresis and stained Coomassie blue for an hour and results were observed the next class period a week later (Clendening).

The first hypothesis was unsupported as Tube 4 (positive control) had the most succinate (.2mL) but ended with the lowest % transmittance out of the three sample we were examining (Tubes 2-4). We believe this discrepancy to be due to the variability of the mitochondrial solution. If the Tube 4 mitochondrial suspension had less concentrated enzyme than the solution for Tube 3 and Tube 2 then Tube 4 would be biologically incapable of matching their rate of reduction. It's worth mentioning that Tube 2 and 3 had a %T change of 23.1% and 23.2% respectively while Tube 4 exhibited a change of only 20.4%; further supporting the idea that Tube 4, despite its increase in succinate concentration, wa lacking in other enzymes/proteins necessary for the

It achieves this my measuring the mitochondrial enzyme activity and as such is used to determine the cytotoxicity of potential medical drugs. It measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT starts as yellow but as it enters the cells and passes into the mitochondria where it is reduced and changes in colour to a dark purple formazan product. The cells are then solubilised with an organic solvent and released, solubilised formazan reagent is measured using a spectrometer (Cree, 2011). Some errors that may occur while using this assay are that uneven evaporation of culture fluid in the wells may cause erroneous results. The MTT solution is stable for 6 months when stored at 0°C but at 2-8°C for more than 2 weeks may cause decomposition and erroneous results may occur. Microbial contamination will contribute to cleavage of MTT and the formation of MTT formazan, causing erroneous results (Sigma Aldrich,

The goal of this experiment is to successfully stage and observe redox reactions in mitochondrial respiration and fermentation, to better understand these processes. As for hypothesizing, prior to the lab I thought that glucose at 37 degrees would be the greatest yield of CO2 and that as time went on, the transmittance readings of the reduced DPIP would decrease. Neither was entirely correct. Sucrose at 37 degrees seemed to yield the most CO2 and the results for the transmittance readings of the reduced DPIP varied by sample.

The mitochondria has been known as the powerhouse of the cell. What does that even mean? Well, what it means that the mitochondria does all of the cell energy conversion. It takes nutrients from the cell and transforms it into viable ATP. ATP, molecule adenosine triphosphate, is the energy that cells can use. The process in turning nutrients into ATP is called ATP Synthase. The first part of ATP synthase is an ending of cellular respiration. The mitochondria plays a small but large role in the cell. The structure of the mitochondria plays a huge part of cellular respiration. Mitochondrial structure has two membranes an inner and an outer. Inside the inner membrane you have the matrix and the cristae. The first part of cellular respiration is glycolysis, it is made outside of the mitochondria in a gel like fluid called the cytoplasm. Next, is the citric acid cycle, also known as the Krebs cycle, named after the German researcher Hans Krebs, goes in through the outer membrane. Enzyme Acetyl CoA enters and combines the two carbon groups with another four carbon groups. The result is six carbon molecules citrate, which are acidic. The next part in the Krebs cycle is that the hydrogen atoms are stripped and produce NADH molecules. The final Krebs step is; ADP is transferred to ATP the succinate is oxidized forming another four carbon molecule. The two hydrogen carbons react and their electrons transform from FAD to FADH2. The Krebs cycle makes only about 4 ATP and in the

The second experiment dealing with cellular respiration had 4 tubes, one with 4.6 mL phosphate buffer, 0.3 mL mitochondrial suspension, and 0.1 mL succinate, which was the blank. The second had 4.4 mL phosphate buffer, 0.3 mL DPIP, 0.3 mL mitochondrial suspension, 0 mL succinate. Third tube had 4.3 mL phosphate buffer, 0.3 mL DPIP, 0.3 mL mitochondrial suspension, 0.1 mL succinate. The fourth and final tube had 4.2 mL phosphate, 0.3 mL DPIP, 0.3 mL mitochondrial suspension, 0.2 mL succinate. All of which the blank was used to zero the spectrophotometer and the remaining three were placed in the machine every 5 minutes for 30

The molecule is an important part of the inner part of the mitochondria in which the actual production of the energy occurs.

C1.2.2. Optimization of analysis of movies of mitochondrial transport: In terms of acquiring the data for the large scale screen, we need to address important questions in terms of plate design and statistical analysis that build on our previous experience with such problems 18,44-47. In our initial studies described in C1.1.4, we assessed the effect of disrupting microtubules on mitochondrial transport by screening the plate before and after drug application. We did this because of the statistical power associated with paired measurements and because we were unsure how much well-to-well variation would impact the statistical analysis. Overall, we found that this provided an excellent means of measuring changes in mitochondrial transport. Having refined

Mitochondrion is an importance structure that lies in the cytoplasm area. Mitochondrion is the plural word for mitochondria, which is the key organelle that converts energy from one form to another. Mitochondria changes the chemical energy stored in food into compounds that are more convenient for the cell to use. The mitochondrion contains two special membranes. The outer membrane surrounds the organelle, and the inner membrane has many folds that increase the surface area of the mitochondrion.