...Kevin Lopez 03/18/13 (008945285) Succinate Dehydrogenase Enzyme Activity in Mitochondrial Fraction Succinate dehydrogenase (SDH) is an enzyme that catalysis the oxidation of succinate to fumarate in the citric cycle. During this step, electrons removed from succinate directly reduce an electron acceptor, known as FADH2; however, for this laboratory experiment an artificial electron acceptor, dichlororphenolindophenol (DCPIP), was utilized. By inhibiting complex IV of the electron transport chain with sodium azide, the electrons from succinate reduce DCPIP and alter its color from blue to colorless. Therefore the color of the reaction can be measured using a spectrophotometer, and a decrease in A600 indicates enzyme activity that corresponds with the concentration of succinate found in the tube samples (i.e. higher succinate concentration = higher enzyme activity; vice-versa). In addition, by plotting a Michaelis-Menten graph, the maximum velocity of the reaction (Vmax) and the concentration of the substrate that results in ½ Vmax (Km). The first step in this experiment was the creation of twelve tube samples that contained a mixture of reaction buffer (3.2 mL), DCPIP (0.5 mL), and succinate (varying from 0-0.5 mL). Of the twelve, tube 3 included the addition of 0.2 mL Malonate, which served as a competitive inhibitor and tube 4 was denatured affecting enzyme activity. These will be discussed later. Upon completion of all twelve mixtures, their absorbance values were...
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...Summary 1 Succinate Dehydrogenase Enzyme Activity In Cellular Fractures (Lab 5) The nuclear, mitochondrial and cytoplasm fractions of cauliflower inflorescence tissue was isolated . The concentration of the proteins in the fractions was then measured, denatured and reduced by SDS-PAGE. The purpose of this lab was to measure the activity of the succinate dehydrogenase (SDH) enzyme. I predict that the presence of heat will denature the enzyme. I also predict that out of the different cellular fractions, the mitochondrial fractions will have the highest activity. In order to measure the activity of the SDH enzyme, 12 tubes were with varying combinations of sodium azide, which inhibits the 4th complex of the electron transport chain, DCPIP (dichlorophenolindophenol), an artificial electron acceptor, malonate, a competitive inhibitor, and SDH, which deprotonizes FADH2 into FAD+. This results in a reduction of DCPIP, once DCPIP is reduced, it becomes colorless. The spectrophotometer was utilized to measure the color of the reaction at an absorbance of 600nm, which was determined to be the absorbance of the enzyme activity. The absorbance of the 12 tubes were taken and recorded a intervals of 0,5,10 and 20 min, then the change from each of the intervals from 0 min was recorded. The absorbencies were then graphed in the form of a Michaelis Menten Plot. Prior to have an absorbance read, Tube 4 was the only tube heated in a hot water bath. All of the tubes were put in a chilled bath...
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...Metabolism III: Oxidative Cellular Respiration Introduction Oxidative cellular respiration is composed of series metabolic processes that convert broken down food molecules into usable energy in the form of adenosine tri-phosphate (ATP). The process follows oxidation (catabolic) and reduction (anabolic) pathways. Processes involved are glycolysis, Krebs or tricarboxylic acid (TCA) cycle, and the electron transport chain. One step in the TCA cycle is the enzyme-catalyzed conversion of succinate to fumarate in a redox reaction. In intact cells succinate loses hydrogen ions and electrons to FAD to form fumarate. This step in the TCA cycle will be used to study the rate of cellular respiration under different conditions. (Patriquin, M. Rand, T. 2012). Since DPIP is a reducing dye it will absorb the hydrogen ions and electrons from the redox reaction of the TCA cycle between succinate and fumarate producing a discoloration of the dye. The discoloration is measured in percent transmittance of light over 30minutes at 5 minute intervals. The change in dye color is the associated with cellular respiration activity, and will be used to record the cellular respiration rate in mitochondria isolated from pulverized lima beans (Phaseolus lunatus) and subsequent effects of different substrate concentration, pH, and metabolic inhibitors . If the difference of light percent transmission produced by (DPIP) can be recorded over time associated with the cellular respiration rate then...
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...citrate synythetase producing the 6 carbon molecule Citrate. Citrate once formed then undergoes an isomerisation reaction in the presence of aconitase, turning first the citrate into an intermediate known as cis-aconitase and then into isocitrate. The 6 carbon Isocitrate is then irreversibly catabolised via an oxidative decarboxylation by the enzyme Isocitrate dehydrogenase which is like PDH as it is a multi enzyme complex. The intermediate oxal succinate is formed which is quickly converted to the 5 carbon Alpha ketoglutarate, releasing a CO2 molecule and a reduced NAD for the electron transport chain. Another oxidative carboxylation occurs due to the enzyme alpha ketoglutaraten and the Coenzyme COA-SH, this time turning alpha keto glutarate into four carbon Succinyl COA causing a CO2 and a reduced NAD to be released. Succinate synthase then causes the release of GTP a denominator of ATP which is readily converted in to the ATP during the only example of substrate level phosphorylation. Succinate is formed, in the next reaction the Succinate undergoes an oxidation reaction in the...
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...Innovagen Restal Innovagen Restal contains doxylamine succinate as the active ingredient, and comes in 25mg tablets. Doxylamine succinate is an antihistamine that provides temporary relief for symptoms such as sneezing, a runny nose and watery eyes that accompany hay fever, allergies, and colds. It works by blocking acetylcholine and histamine, which are natural substances produced by the body. Since it produces a sedative effect on people, adults at times, use it to relieve occasional insomnia or sleeplessness. For this reason, Innovagen Restal is in the company's product section of Sleep Aides and Stress Reduction. Administration and Dosage Information Take Innovagen Restal tablets orally. If stomach upset is a problem, a person can take this medication with milk or food instead of just water. The body absorbs doxylamine succinate...
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...Finally, two of the carbon atoms are removed as CO2, then the four carbon molecule regenerates. The Kreb’s Cycle yields 1 ATP, 1 FADH2, and 3 NADH per Acetyl CoA that enters the cycle. Since two Acetyl CoA enter the cycle, it yields a total 2 ATP, 2 FADH2, and 6 NADH by the end. (Lerner, 2008) The Kreb’s Cycle occurs in the mitochondria of the cell, and we ran an experiment during which we observed cellular respiration rates. We placed a mixture of mitochondrial suspension from ground up Lima beans, a buffer, DCPIP, and succinate of different proportions into labeled cuvettes. DCPIP is a blue chemical used to observe and measure the rates of cellular respiration, as it loses its color as cellular respiration occurs. For one set of three trials, we will keep the amount of mitochondrial suspension at .3 mL. For our experimental trial, we will increase the mitochondrial suspension for each cuvette. Our hypothesis is that if we increase the amount of mitochondrial suspension, then more cellular respiration would occur. If...
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...elements (IREs), which are stem-loop structures found in the 5' UTR of ferritin mRNA, and in the 3' UTR of transferrin receptor mRNA. When the protein binds to IRE, it results in repression of translation of ferritin mRNA, and inhibition of degradation of the otherwise rapidly degraded transferrin receptor mRNA thus increasing the formation of aconitase. During iron overload, the aconitase mRNA detaches from the IRE thus suppressing the translation of aconitase. Drosophila succinate dehydrogenase mRNA (DSD mRNA): the protein formed is succinate dehydrogenase which forms part of complex II of the Electron Transport Chain [ETC]. It performs an important role in linking the TCA to the ETC. During an iron overload, the DSD mRNA receptor attaches to the IRE which causing the transcription of Drosophila succinate dehydrogenase that will increase ETC reactions in complex II. In a state of iron deficiency, DSD mRNA receptor mRNA detaches from the IRE and thus suppresses the formation of the protein Drosophila succinate...
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...Week 1 – HOW LIFE WORKS: Biology – Science of How Life Works; study of life; attempt to understand processes of life. How life works - many levels. Three levels: -Molecular mechanisms within the cell -Integrated actions of many cells within an organ or body -Interactions among different organisms in nature Life Processes – Microscopic and macroscopic levels: All methods of scientific study use the Scientific Method Scientific Method – Systematic approach to finding out how things work. (i.e., structured and disciplined way to study things) Deliberate way of asking questions about things we don’t yet understand. Start with observation – Sensing the world around us with our 5 senses or with augmented abilities such as scientific instruments; – seeing a particular event or occurrence; Scientists then make proposed, tentative explanations < - - i.e., hypothesis (a predictive guess) and plan out detailed experiments or detailed observations to see if the tentative explanation is correct (i.e., true). Test that tentative explanation [pic] Make some observations of the image above. Ideas: -What motivates this behavior? -Is the bird feeding? -Does the bird choose this flower because of its color? -Does the flower benefit from the bird’s behavior? Observations made and the questions they raise allow us to propose tentative explanations, or hypotheses. ...
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...Metabolism Western Governors University Aston Portis 1. All enzymes are catalyst because they cause a chemical reaction to take place faster than it would on its own. It also does not get used during the reaction (Hudon-Miller, 2012). 2. (Hudon-Miller, 2012). 3. (Hudon-Miller, 2012). 4. Fructose is component of sucrose, normal table sugar, along with glucose. Whereas glucose is able to immediately enter into glycolysis, fructose is not. Fructose is broken down via fructokinase into fructose- 1-phosphate. Fructose – 1-phospate then gets converted into DHAP+ glyceraldehyde via aldolase B. DHAP+ glyceraldehyde is used in glycolysis to produce pyruvate that goes into the citric acid cycle to produce ATP (Hudon-Miller, 2012). 5. In aldolase B deficiency the substrate fructose 1- phosphate is unable to produce DHAP+ glyceraldehyde but the fructose is still getting phosphorylated by fructokinase. This causes a buildup of fructose- 1-phosphate. It is not being used in glycolysis or gluconeogenesis. There is a buildup of phosphate which causes the phosphate to get stuck and the free phosphate to be depleted by because of so much is being used by the fructose 1-phosphate. Since our phosphate levels were depleted it slows production of ATP, phosphate is needed in the electron transport chain. Since ATP production has slowed and liver cells are low on energy liver damage can occur which can ultimately lead to liver failure. Fructose-1-phosphate buildup...
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...Introduction : Today the problem of waste pollutants has increased due to increasing number of industries as well as urbanization. Water is polluted in large amount due to these waste pollutants. These waste products can be from different sources food waste, industrial waste, agricultural waste, urban waste etc. Waste pollutants which are obtained from these sources are usually organic materials. The process of anaerobic digestion aims on breakdown of these waste materials. "It is a complex biochemical process of biologically mediated reactions, by a consortium of microorganisms to convert organic acids to methane and carbon dioxide." The process stabilizes the waste by reduction in its mass, reduction in odour and reduction in number of pathogens present in it. The process helps in reduction of landfill gas in the atmosphere. It produces energy in the form of biogas. Biogas contains methane and carbon dioxide. Digestate or waste produced after the process is over is rich in nutrients and can be used as fertilizers. General equation of the whole process is The process : The process was divided into three stages namely Hydrolysis, acid formation and gas formation (Lawrence and McCarty 1967). Stage of acid formation can be termed as non-methanogenic phase and gas- formation as methanogenic phase. A large number of bacterial population is involved in these processes. Different species of microorganisms which are used in anaerobic digestion vary according to the...
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...eating too much spicy food. However, the palpitations have become progressively more prominent over the last 4-5 days and therefore the patient thought he should visit the doctor. The patient states that he has had no recent medication changes and has been on the same medication regimen for about 2-3 years now. The patient also stated that he felt similar chest pounding about 4 months ago but it seemed to go away really quick and decided he did not need to do anything about it. PMH: Meds: DM Type 2 ASA 81 mg PO daily HF (LVEF ~35%) (13 years ago) Furosemide 20 mg PO daily HTN KCL 10 mEq PO daily s/p MI (16 years ago) Lisinopril 20 mg PO daily Hypercholesterolemia Atorvastatin 40 mg PO daily Gout Metoprolol succinate XL 25 mg PO daily Glipizide XL 20 mg PO daily Allopurinol 150 mg PO daily Allergies: NKDA SH: Lives with wife; employed as a construction foreman; quit drinking alcohol after HF diagnosis; quit tobacco after MI; tries to stay active and does moderate exercise at the gym at least 3 times/week. Plays golf every other weekend. FH: non-contributory Today’s PCP Visit: ROS: Denies fever, chills, N/V, weight gain, fatigue or SOB (although admits to having some SOB with strenuous or heavy exercise; + chest palpitations PE: BP 132/84 HR 140 RR 13 Temp 37.3 Ht 6’2” Wt 87 kg Gen: A &O x 3; well-nourished male in no apparent distress Lungs: CTA CV: Irregularly irregular rhythm Abdomen: Soft, NT, ND, +BS Extremities:...
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...Contaminated water contains biological microorganisms such as bacteria, fungi, and viruses. Silver nanoparticles are used conventionally as an antimicrobial compound to exhibit multi-interactional activity to fight bacteria. Inevitably, low concentration of silver compounds will remain in the environment. For example values of accumulated silver nanoparticles for surface water, American soil, sludge, , Sewage Treatment Plant (STP) effluent, STP sludge, and sediment were reported 0.088 µg/L/year, 6.6 µg/kg/year, 526 µg/kg/year, , 16.40 µg/L/year, 1.29 mg/kg/year, and 153 µg/kg/year respectively. The residue of silver nanoparticles could cause the evolution in the bacteria in order to adapt to novel environments. The consequence of exposing bacteria...
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...History Back in the 1840s, the presence of granule-like structures within muscle cells and other cell types were being recognized by several scientists. (Ernster and Schatz 1981) In 1890, Richard Altmann, who was a cytologist, used a dye technique to detect the granules and termed them as “bioblasts.” He speculated that they were the basic units of cellular activity. (Ernster and Schatz 1981) It was in 1898 when Carl Benda gave these bioblasts a new the Greek name “mitochondria” meaning thread granules. Discovery of the mitochondrion however, cannot be limited to just a few people. Over decades of time, many contributions have been made in relation to the properties and functions of the mitochondria. (Ernster and Schatz 1981) This organelle is the “power house” of the eukaryotic cell and is located in the cytoplasm. The mitochondrion requires transcription of several genes associated with the organelle along with translocation, targeting and assembly of proteins. (Hood and Joseph 2004) Mitochondria’s main function is to convert energy into forms that can be used by the cell. Along with generating fuel for the cell’s activities, the mitochondrion functions in a range of other processes including, cell signaling, cell division, cell growth, and cell death. Structure The mitochondrion can have different overall structures depending on the cell type. Most mitochondria appear as rod-like shaped organelles although sometimes they can appear like a branched interconnected tubular...
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...Action of enzymes as catalysts in biochemical processes * Enzymes acts as catalyst and increase the rate of all the chemical reactions. * Enzymes are also described by two properties like all other catalysts. It composed of two main functions. * The first function is that, they increase the rate of chemical reactions by without consumed themselves or undergo any change or alteration in the reaction. . ( Zemitec et,al 2008). * The second function is, they increase reaction rates without changing the chemical equilibrium between the reactants and products. ( Bhagavan et,al 2006). * The reaction between two substrates are catalyzed by enzymes. The enzyme brings a template upon which the two substrates are combined together in the proper position and make them to react each other. Deficiency in aldolase B and hereditary fructose intolerance * Hereditary fructose intolerance is a condition I which affects a humans ability to digest the fructose sugar. The incidence of hereditary fructose intolerance is 1to 2 in 20000 to 30000 individuals in a year worldwide. .( John .R.H 1996) * Hereditary fructose intolerance can be caused by mutations in the ALDOB gene. The ALDOB gene is responsible for making the aldolase B enzyme. The aldolase B enzyme is primarily seen in the Liver. This helps for the fructose metabolism. This enzyme is responsible for the further step in the metabolism of fructose, which breaks down the molecule fructose-1-phosphate into other...
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...Role of Enzymes ¨. Catalysts that help a molecule breakdown faster. ¨. Catalysts speed up or slow down a biochemical reaction by increasing or decreasing the activation energy. Does not change in reaction. ¨. Enzymes bind to a substrate to form the enzyme substrate complex. When bound the enzyme has a specific job it was designed to do. Once the job is done, the substrate has become a product, and the enzyme moves along to the next substrate. ¨. If enzymes were not involved, the reactions would take too long and breakdown would not occur properly. (Enzyme Characteristics) Enzymes ¨. More specifically, enzymes are substrate specific. ¨. Once they find a match the enzyme takes the substrate and creates a product out of it. The product then continues on the pathways to be a substrate for a different enzyme. ¨. If the temperature is up, enzymes work faster. ¨. If the temperature is down, enzymes work slower. ¨. No work occurs if the temperature is too hot. ¨. Aldolase B is an enzyme that breaks fructose down. (Enzyme Characteristics) Aldolase B & HFI ¨. Aldolase B turns Fructose-1-Phospate to DHAP andglyceraldehyde. These two substances then enter glycolysis to make ATP for the body. ¨. If Aldolase B is lacking, fructose can not be broken down, potentially causing Hereditary Fructose Intolerance. ¨. In this process, there is a protein that is missing...
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