Summary Of Succinate Dehydrogenase Activity Of Mitochondria

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 contrast, there are four metabolic stages happened in cellular respiration, which are the glycolysis, the citric acid cycle, and the oxidative phosphorylation. Glycolysis occurs in the cytoplasm, in which catabolism is begun by breaking down glucose into two molecules of pyruvate. Two molecules of ATP are produced too. Some of they either enter the citric acid cycle (Krebs cycle) or the electron transport chain, or go into lactic acid cycle if there is not enough oxygen, which produces lactic acid. The citric acid cycle occurs in the mitochondrial matrix, which completes the breakdown of glucose by oxidizing a derivative of pyruvate into carbon dioxide. The citric acid cycle produced some more ATPs and other molecules called NADPH and FADPH. After this, electrons are passed to the electron transport chain through

There are two types of cellular respiration, aerobic and anaerobic. Aerobic respiration occurs when there is oxygen present and in the mitochondria (in eukaryotic cells) and the cytoplasm (in prokaryotic cells). Aerobic respiration requires oxygen; it proceeds through the Krebs cycle. The Krebs cycle is a cycle of producing carbon dioxide and water as waste products, and converting ADP to thirty-four ATPs. Anaerobic respiration is known as a process called fermentation. It occurs in the cytoplasm and molecules do not enter the mitochondria for further breakdown. This process helps to produce alcohol in yeast and plants, and lactate in animals. Only two ATPs are produced through this process. In yeast fermentation is used to make beer, wine, and whiskey.

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

Aerobic respiration happens only when oxygen is presented in the cell. Aerobic respiration starts with pyruvate crossing into the mitochondria. When it passes through, a Coenzyme A will attach to it producing Acetyl CoA, CO2, and NADH. Acetyl CoA will enter into the Krebs cycle. In the Krebs cycle Acetyl CoA will bound with Oxaloacetic Acid (OAA), a four carbon molecule, producing the six carbon molecule, Citric Acid. Citric Acid will reorganize into Isocitrate. This will lose a CO2 and make a NADH turning itself into alpha ketoglutarate, a five carbon molecule. Alpha ketoglutarate will turn into an unstable four carbon molecule, which attaches to CoA making succinyl CoA. During that process a CO2 and NADH is made. An ATP is made when CoA leaves and creates Succinate. This molecule is turned into Fumarate, creating two FADH2 in the process. Then Fumarate is turned into Malate then into OAA making two NADH. Only two ATP is produced in Krebs cycle but the resulting NADHs and FADH2s are passed through an electron transport chain and ATP synthase. When the molecules passes through that cycle a total of 28 ATP molecules are produced. In all aerobic respiration produces 32 ATP and waste products of H2O and

Cellular respiration is the series of metabolic process by which living cells produce energy through the oxidation of organic substances. Cellular respiration takes place in the mitochondria. Fermentation is the process by which complex organic compounds such as glucose, are broken down by the action of enzymes into simpler compounds without the use of oxygen. The significance of these pathways for organisms is to allow for an organism to be able to generate ATP. Some organism that undergo cellular respiration are bacteria and fungi. Some organism that undergo fermentation are yeast and muscle cells. In cellular respiration, glucose is oxidized and releases energy. In cellular respiration, glucose produces ATP and 3-carbon molecules of pyruvate. The pyruvate is then further broken down in the mitochondria where it becomes oxidized and releases CO2 (Upadhyaya 2014). In the fermentation process oxygen does not play a part. This process converts glucose into pyruvate and produces ATP. From there pyruvate breaks down into CO2 and acetaldehyde (Upadhyaya 2014) Monosaccharides are known as simple sugars and their main function is being the source of energy for organisms. Disaccharides are two monosaccharides joined by a covalent bond and their primary function is to provide food to monosaccharides. Some disaccharides

The Purpose of this experiment was to determine the importance of cellular respiration in the processes of Life. The objective of this experiment was to determine the rate of cellular respiration and how the presence of carbohydrates and pollutants will affect it. Our hypothesis was that an organism has larger rate of Cellular Respiration with the source of Carbohydrates as compared to the one that is without the Carbohydrates source and vice versa in

The third and final step in cellular respiration is the electron transport chain which takes place in the inner mitochondrion membrane. This process uses the high-energy electrons from the Krebs cycle to convert ADP into ATP. These high-energy electrons are first passed along the electron transport chain. Every time 2 electrons travel down this chain, their energy is used to transport hydrogen ions (H+) across the membrane. These H+ ions escape through channels into an ATP synthase. This causes it to spin, transforming the ADP into ATP. On average, each pair of high-energy electrons that moves down the electron

The two carbon molecule bonds four carbon molecule called oxaloacete forming a carbon molecule knew as citrate. The second step reaction is classified as oxidation/reductions reactions. This process is formed by two molecule of CO2 and one molecule of ATP. The cycle electrons reduce NAD and FAD, which join the H+ ions to form NADH and FADH2, this result to an extra NADH being formed during the transition. In the mitochondrion, four molecules of NADH and one molecule of FADH2 are produced for each molecule of pyruvate, two molecules of pyruyate enter the matrix for each molecule of oxidized glucose, as a result of these eight molecules of NADH+ two molecules are produced. Six molecules of NADH+, molecules of FADH2 and two molecules of ATP synthesize itself in Krebs cycle. As a result, no oxygen is used in the described reactions. During chimiosmosis, oxygen only plays a role in oxidative phosphorylation. The next step is the electron transport; the electrons are stored on NADH and FADH2 and are used to produce ATP. Electron transport chain is essential to make most ATP produced in cellular respiration. The NADH and FAD2 from the Krebs cycle drop their electrons at the beginning of the transport chain. When the electrons move along the electron transport chain, it gives power to pump the hydrogen along the membrane from the matrix into the intermediate space. This process forms a gradient concentration forcing the hydrogen through ATP syntheses attaching

Rat liver cells can be separated and analysed by Subcellular fractionation using homogenisation and then centrifugation to separate the cells into the subcellular fractions. When the fractions are separated succinate dehydrogenase is used to identify mitochondria. Succinate dehydrogenase is an enzyme that catalyses the oxidation of succinate to fumarate in the Krebs cycle and is usually on the mitochondria inner membrane and not found in other organelles in the cell(Rustin, Munnich, and Rötig, 2002). Care needs to be taken when separating the subcellular fractions as organelles can be damaged and can cause lysis giving incorrect results. Regulation of conditions like the temperature and the pH are important as incorrect conditions could cause the enzymes to denature. Also adding an isotonic buffer not water will help to avoid damage to cell organelles due to osmotic an imbalance (Greenawalt and Vasington, 1968). When it comes to measuring the SDH it is measured indirectly because the succinate dehydrogenase and the fumerate involved in the reaction are both colourless so it is impossible to measure any changes using a spectrophotometer. Instead the absorbance of formazan is measured at 490nm. The formazan is produced by reacting the FADH2 from the SDH reaction with INT which a tetrazolium salt. The formazan produced is red in colour and when dissolved into the ethyl acetate can be extracted and then read on the spectrophotometer (Smith and McFeters, 1997). As

Cellular respiration is a procedure that most living life forms experience to make and get chemical energy in the form of adenosine triphosphate (ATP). The energy is synthesized in three separate phases of cellular respiration: glycolysis, citrus extract cycle, and the electron transport chain. Glycolysis and the citric acid cycle are both anaerobic pathways because they do not bother with oxygen to form energy. The electron transport chain however, is aerobic due to its use of oxidative phosphorylation. Oxidative phosphorylation is the procedure in which ATP particles are created with the help of oxygen atoms (Campbell, 2009, p. 93). During which, organic food molecules are oxidized to synthesize ATP used to drive the metabolic reactions necessary to maintain the organism’s physical integrity and to support all its activities (Campbell, 2009, pp. 102-103).

The second pellet (P2) was centrifuged for 5 minutes at 2000 x g. The third pellet (P3) was centrifuged at 21,000 x g for 15 minutes. The supernatant from the third pellet formed the third supernatant (S3). After obtaining P1, P2, P3, and S3, the goal of part 2 of experiment 1 was to figure out which cell fraction has the highest amount of mitochondria. DCIP, which is an electron acceptor, was added to P1, P2, P3, and S3 to test for SDH, a biochemical marker for the presence of functional mitochondria (McAllister & Leicht p.71). DCIP changed from blue to colorless when electrons are being reduced, indicating a high SDH activity level and a large presence of mitochondria. The amount of color change is proportional to the amount of SDH enzyme and the amount of mitochondria in a cell fraction (McAllister & Leicht p.71). Blank cuvettes containing assay buffer, sodium azide, and a

Mitochondrial movements are then traced manually using the Segmented Line Tool and overlaid onto the stack file. Step 2 then produces a kymograph, using the Kymograph Builder Tool, for each selection. This is followed by manually tracing the mitochondrial movement on the kymographs. Step 3 then measures the mitochondria’s initial and final X (in pixels) and Y (in frames) coordinates using the Measurement Tool.

In the metabolic reactions, oxidation-reduction reactions are very essential for ATP synthesis. The electrons removed in the oxidation are transferred to two major electron carrier enzymes. The electrons are transported through protein complexes in present in the inner mitochondrial membrane. The complexes contain attached chemical groups which are capable of accepting or donating one or more electrons. The protein complexes are known as the electron transfer system (ETS). The ETS allow distribution of the free energy between reduced coenzymes and the O2. The ETS is associated with proton (H+) pumping from the mitochondrial matrix to intermembrane space of the mitochondria.