Succinate Dehydrogenase Enzyme In Inner Mitochondrial Membrane Biology Essay

Succinate Dehydrogenase Enzyme In Inner Mitochondrial Membrane Biology Essay Succinate dehydrogenase (SDH) is an enzyme found in the inner mitochondrial membrane, which makes it an easy target to isolate when studying the citric acid cycle. This enzyme is responsible for catalyzing the oxidation of succinate into fumarate and can be used as a marker enzyme during the isolation of mitochondria through differential centrifugation. The isolated mitochondria can be treated with a sodium azide reagent to inhibit the mitochondrion transport of electron in the cell extract. To measure the activity of the enzyme, an artificial electron acceptor (2, 6-dichlorophenolindphenol, DCIP) is used to accept two electrons. Upon receiving electrons, the oxidized DCIP is reduced and the color of the mixture changes from blue to colorless. Spectrophotometry at the 600nm range can then be used to quantify this color change, and give an indication of the mitochondrial content of a given sample. As the The findings show that the experiment mimics Michaelis-Menten kinetic properties Enzymes are regulators of metabolic pathways that lower the activation energy in order to catalyze the acceleration of biochemical reactions [1]. Most enzymes are characterized as showing Michaelis-Menten (M-M) kinetic properties. Simply, enzymes work by binding its substrate reversibly changing its conformation to form an enzyme-substrate complex, and then detach to form free enzyme and product. If there is low substrate concentration, there will be very little enzyme activity and the rate of the reaction will slow down. If there is high substrate concentration, the enzyme will be more active and the reaction will be faster. At a certain point, if the substrate concentration is saturated, the rate of the reaction will not increase [1]. Along with the substrate concentration, these dynamics can be characterized as the M-M constant (Km) and maximum velocity (Vmax). These factors determine the initial velocity of the biochemical reaction and contribute to the understanding of the M-M e quation (in fig.1) However, when a competitive inhibitor is present, the inhibitor can bind to the active site to prevent the normal substrate from binding and forming the product. Thus, both the inhibitor and substrate compete for the active site of the enzyme, which based on the M-M equation, allows the Vmax to stays constant and the Km to change [2]. In the experiment, we will examine activity of SDH, an important component of the citric acid cycle that is responsible for catalyzing the oxidation of succinate to fumarate in the inner membrane of the mitochondria. The enzymatic activity will be determined by mitochondria fractionation from isolated cells of cauliflower by the technique of differential centrifugation. Also, we will determine the effects of enzyme concentration and competitive inhibition on the initial velocity of the reaction by adding the malonate, a classic competitive inhibitor. We will measure the reaction by blocking the electron transport with sodium azide and monitoring the reduction of the DCIP that can be followed by the change in spectrophotometry absorbance reading at 600 nm over time Since the oxidized form of the dye is blue and the reduced form is colorless, the reaction can be reestablished based on the experiment (in fig.2),. Thus, we hypothesize that the reaction will follow M-M kinetics as the abs orbance will decrease when the malonate is added Methods In isolating mitochondria, we removed with a scalpel 20 g of cauliflower from the outer 2-3 mm surface. Then, we grinded the tissue with a pestle in a chilled mortar in 40 ml of ice-cold mannitol grinding buffer for 4 min. We filtered the suspension and squeeze the solution out through four layers of cheesecloth into three chilled 15 ml centrifuge tube. Then, we centrifuged the filtrate solution at 1000 x gravity for 10 min and decanted the supernatant into a chilled 50 ml centrifuge tube. After, we re-spun the filtrate solution at 10,000 x gravity for 30 min at 0-4 °C and discarded the supernatant in the sink leaving the pellet. Then, we added 7.0 ml of icecold mannitol assay buffer to the mitochondrial pellet and scraped and mixed the mitochondrial pellet from the wall of the centrifuge tube with a spatula and vortex thoroughly to re-suspend the pellet in the assay buffer. Until needed, we transferred the mitochondrial suspension to a test tube and stored it in an ice bath. In measuring the activity of SDH, we label 10 test tubes or cuvettes as shown in table 1. We heated 0.6 ml of the ice cold mitochondria suspension in a boiling water for 5 min and placed it in an ice bath to cool. Then, we added correct volumes of azide, DCIP, malonate, and succinate to all labeled test tubes indicated in the table, covered them with Parafilm and inverted to blend the solutions. After, we add specific volume of the mitochondrial suspension to blanks 1-4 and tubes 1-4. Using a spectrophotometer set at 600nm, we blanked and took the absorbance of tubes 1-4 every two minutes until 20 minutes after the first reading. Then, we repeated again by taking the absorbance using only test tubes 5-7 for every two minutes. Results The spectrophotometer results we obtained are presented in Table 2, and shown graphically in Figure 3-7. In Table 2, the first 4 test tubes and blanks we were only able to take 3 readings and the test tubes 5-7, we were able to take 4 readings. The greatest absorbance reading was obtained for test tube 4 at 2.363 abs., which is because malonate, the competitor inhibitor, is present along with the substrate, succinate. In Tube 6, one of the lowest absorbance readings because it is a negative control and does not have any cellular suspension. This is shown experimentally when the reaction mixture will remain the color blue because with the succinate there is no reaction between the marker enzyme and the DCIP. In Table 3, we calculated the change in absorbance from tube 1-4 for every 2 minutes. We also calculated the initial velocity by dividing the change in absorbance by the elapsed time. In Figure 3, the graph shows the initial velocity depends on the enzyme concentration. When the e nzyme concentration is high, it start to rapidly decrease the initial velocity because the ratio of substrate to enzyme will be abnormally low, which will decrease the formation of product. In Figure 4, the graph represent the data in Table 2, where the initial velocity measured by elapsed time. The second highest reading was found for Tube 2 (0.987), which was also in concordance with the class results. This sample contained the heaviest constituents of the cell (mostly nuclei), as well as any unbroken whole cells that may have remained after the mechanical grinding and initial centrifugation at 600x. We found Tube 8 to have the third highest absorbance reading (0.626) and Tube 4 with the lowest (0.483). However, the sample from Tube 8 should have had a lower absorbance value than Tube 4, as was seen in the average class results displayed in Table 1. Tube 8 should contain the majority of the mitochondria (as well as some lysosomes), and Tube 4 should have any residual mitochondria and smaller organelles that did not remain in the pellet after the 12,000x centrifugation. Discussion There are a number of reasons why our findings did not match up with the expected results. Although improbable, it is possible that the 12,000x centrifugation for 30 minutes was not properly carried out, perhaps because the samples were not maintained at a consistent temperature of 0-40C. It is more likely that the re-suspension of the pellet (Tube C) with the mannitol assay buffer was not performed effectively. The pellet clumps may not have been properly dispersed, and so even though more mitochondria may have been present in Tube 8 (as they should have been), they were not free to interact with the other reagents in solution. A third reason may be that too much DCIP was added to Tube 8 (relative to Tube 4), and so there was an excess of the blue DCIP reagent in that sample (and hence a higher absorbance reading due to a lower degree of color loss). Differential centrifugation, when done correctly, is a reasonably effective method for mitochondrial isolation, although separation is achieved based only on size differences of the cell components. When dealing with small organelles, a more appropriate method to use may be sucrose gradient centrifugation, which allows for separation based on size as well as shape, especially when dealing with crude cellular extracts such as cauliflower. Cited Resources Nelson, D.L., Cox, M.M. (2007) Lehninger: Principles of Biochemistry, Fifth Edition, Freeman, New York, NY Gilbert, H.F. (2000) Basic Concepts in Biochemistry, Second Edition, McGraw Hill, New York, NY Figure 1 Figure 2 SDH-FADH2 + DCIP(blue) > SDH-FAD + DCIP (colorless) + 2H+ Table 1 Cuvette Assay Medium Azide DCIP Malonate Succinate Mitochondrial Suspension Blank 1 3.7 mL 0.5 mL - 0.5 mL 0.3 mL 1 3.2 mL 0.5 mL 0.5 mL - 0.5 mL 0.3 mL Blank 2 3.1 mL 0.5 mL - - 0.5 mL 0.9 mL 2 2.6 mL 0.5 mL 0.5 mL - 0.5 mL 0.9 mL Blank 3 3.4 mL 0.5 mL - 0.5 mL 0.6 mL 3 2.9 mL 0.5 mL 0.5 mL 0.5 mL 0.6 mL 4 2.7 mL 0.5 mL 0.5 mL 0.2 mL 0.5 mL 0.6 mL 5 3.4 mL - 0.5 mL 0.5 mL 0.6 mL 6 3.4 mL 0.5 mL 0.5 mL 0.6 mL 7 2.9 mL 0.5 mL 0.5 mL 0.5 mL 0.6 mL Table 2 Trial 1 Trail 2 Trial 3 Trial 4 Blank 1 0.04 0.003 0.006 0.008 1 1.10 1.16 1.158 1.112 Blank 2 0.03 0.001 0.004 0.003 2 0.64 0.644 0.648 0.645 Blank 3 0.06 0.002 0.008 0.005 3 0.56 0.06 0.670 0.682 4 2.36 1.85 2.221 2.223 5 0.83 0.73 0.723 0.720 6 0.76 0.73 0.734 0.725 7 0.78 0.72 0.704 0.705 Table 3 Time (min:sec) Tubes 1-4 à ¢Ã‹â€ Ã¢â‚¬  Abs (nm) Initial Velocity (à ¢Ã‹â€ Ã¢â‚¬  Abs/min) Time (min:sec) Tubes 5-7 à ¢Ã‹â€ Ã¢â‚¬  Abs (nm) Initial Velocity (à ¢Ã‹â€ Ã¢â‚¬  Abs/min) 11:10 -.5505 -.0500 9:10 .1057 .0117 13:10 -.0008 -.00062 11:10 .032 .0029 15:10 .4989 .03326 13:10 .0617 .0047 17:10 .5062 .0298 15:10 .1161 .00774 19:10 -.0533 -.0028 17:10 .0338 .00199 21:20 -.0043 -.0002 19:10 .0784 .0041 23:10 -.1095 -.0048 21:10 .1195 .0057 25:10 .142 .00568 23:10 .0428 .0019 - 25:10 .0781 .0031 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

Indonesian Culture Essay

Praise and gratitude I prayed to Almighty God for the implementation of events on this day well. I also says thank you for your time and opportunity that has been in invite In this speech I want to convey about the importance of creativity in preserving the culture of Indonesia. As we know, the Indonesian culture stretched from Sabang to Marauke. Having dozens of cultural heritage that is rich in art. Many nations are amazed and want to claim the Indonesian culture as the culture of their nation. Therefore, it is then we as the younger generation is very good if we realize that culture is an irreplaceable treasure. Culture can not be created with ease, especially with a short time. It has a lot of effort to preserve the culture of Indonesia, but these measures were not the maximum. One of the legendary Indonesian culture is batik. Batik is one of the culture that has worldwide and at times lately, batik started getting attention from the younger generation. Batik day is a day where all parties were invited to use the batik. This is very good for the younger generation and continuity of batik culture itself. Creativity will flourish on its own batik with the number of requests. Modern fashion and in accordance with the age would make batik as a means of creativity for young designers. Make a spear batik fashion formal, semi-formal and informal. Devise an original batik fashion is very trendy but if the wear by every level of generation. Besides batik, there are also other fabrics that are not less beautiful like songket, ulos cloth and so forth. It is time we as a younger generation of cultivating the culture without losing its originality. Make batik as one of the fashion icons of cool and interesting to young people. Besides batik, Indonesian culture most often in the interest of the dance area. Indonesian dance is no less exotic and graceful in compare with other countries. Lots of foreign tourists coming to Indonesia just to see the original dance Indonesia. Have you ever witnessed a cultural festival or to welcome important guests who came to Indonesia. Art music and dance in performing very unusual. In my opinion the area of ​​dance should be introduced among the students and the students so that the scene of the contest are no less interesting with cheerleading competition. Padukanlah regional dance with modern dance, as one of the concepts of dance in one of the most popular events in Indonesia. Hilangkanlah connotation that the dance area is not slang. Berkreasilah, and teach local dances native to Indonesia as one of the dances are in the interest of the subject. Besides creativity, promotion factors also play an important role. Promote their Indonesian Arts to foreign countries with interesting ideas and unique. Thus Indonesian Arts gained international recognition and is not easy to duplicate. Do not ever get bored in the creative, cultural lestarikanlah Indonesia, quicken your identity as bangssa Indonesia. A few of my speech. Thank you for your attention.

Is Forgiving Student Loan Debt A Good Idea - 1009 Words

The education correspondent for Time magazine, undergraduate at the University of Washington, concentrating on journalism and political science, and graduate work at Northwestern University, specializing in new media, Kayla Webley, in her essay â€Å"Is Forgiving Student Loan Debt a Good Idea?† states Robert Applebaum’s solution for student loan debt is a â€Å"radical and wildly unfeasible solution† in both economically and politically. Applebaum’s proposal is to â€Å"provide a one-time bailout of student debt†¦as a way to stimulate the still-limping economy.† However, Webley counters the solution has to have â€Å"the purported benefited and fairness of a one-time student loan bailout.† In Webley’s essay, in the sixth paragraph she mentions the average debt total of the graduate students. Student who has six-figure debts is only one percent of the overall college graduate students, Webley said, â€Å"the average debt total at graduation is a much more reasonable—yet still significant—$27,500.† This evidence is irrelevant to her essay topic of stating a reason why there should not have a one-time bailout of student loan. This information is ineffective to the essay due to her point related to how much the student borrowed. With the amount of money that is vanished, it would not simply be back into the economy in a split second. If a one-time bailout of student loan debt is a legitimate thing to do, â€Å"someone who has $50,000 in debt forgiven isn’t likely to pump all those dollars back into theShow MoreRelatedIs Forgiving Student Loan Debt A Good Idea?1227 Words   |  5 Pagesnew media, Kayla Webley, in her essay â€Å"Is Forgiving Student Loan Debt a Good Idea?† states Robert Applebaum’s solution for student loan debt is a â€Å"radical and wildly unfeasible solution both economically and politically. Applebaum’s proposal is to â€Å"provide a one-time bailout of student debt†¦as a way to stimulate the still-limping economy.† However, Webley counters that the solution has to have â€Å"the purported benefited and fairness of a one-time student loan bailout.† In Webley’s essay, in the sixthRead MoreIs Forgiving Student Loan Debt A Good Idea?970 Words   |  4 PagesMONEY In the article â€Å"Is forgiving student loan debt a good idea† by Kayla Webley, a writer for Time, Webley feels that from a human standpoint forgiving student debt holds some appeal (2). Kayla Webley refers to Robert Applebaum who started a petition in 2009 with a petition of nearly 670,000 signatures. The comments from persons posting the petition are quoted as â€Å"guessing this will never happen but it can’t hurt to sign on† (1). 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Student loans are a massive predicament in theRead MoreEssay on Student Loan Debt Should be Forgiven1256 Words   |  6 PagesDoes the amount of student loan debt have an effect on the economy? If so would forgiving student loan debt help lower the national debt or would it just increase it? According to Mary Claire Fischer, a writer for Kiplinger’s Personal Finance magazine, â€Å"two-thirds of stu dents who receive bachelor’s degrees leave college with an average debt of twenty-six thousand dollars† (Fischer). This means that the average student debt has doubled since 2007 (Ross 24). The total student loan debt is $1.2 trillionRead MoreStudent Loan Loans Should Not Be Forgiven938 Words   |  4 Pages Should student loan borrowers be forgiven for their debt? 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Student Loan debt has become a national problem with no solution. Many students are borrowing more money to keep up with the rising cost of tuition in universities, leaving themselves with thousands of debt after graduation. Students after gaining this debt, have to find jobs to support it which can come

Biography of Moses, Leader of the Abrahamic Religions

Moses, if he existed, likely lived in Egypt during the dynastic New Kingdom, and he was an early leader of the Hebrews and one of the most important figures in Judaism. He is a significant patriarch of all the Abrahamic religions, those who use the Torah, Christian Old Testament, or Quran as sacred texts. Fast Facts: Moses Known For: Patriarch of the Torah, Christian Old Testament, and QuranBorn: Land of Goshen, New Kingdom, EgyptParents: Yocheved and AmramDied: Mount Nebo, MoabSpouse(s): Adoniah or Tharbis, an Ethiopian princess; Tzipporah the MidianiteChildren: From Tzipporah, Gershom and Eliezer. Early Life If there was a historical man named Moses, he would most likely have been born in Egypt (the Land of Goshen) during the reign of Ramses II (ruled 1279–1213 BCE), the pharaoh of the New Kingdoms 19th dynasty. According to the Torah, Moses was the youngest of three children born to Yocheved (sometimes spelled Jochebed) and Avram. Yocheved was the daughter of Levi; she married Avram, a grandson of Levi, which means Yocheved was also Avrams aunt. Moses siblings were Aaron (the founder of the Hebraic priestly dynasty) and Miriam (an important prophetess). Pharaohs Curse Not much else is available on Avram or Yocheved in the Torah itself, but Midrashim records—ancient rabbinical commentaries on the Torah—say that Yocheved was 130 years old when Moses was born and that Avram divorced Yocheved while she was pregnant, so that their son Moses would escape the pharaohs decree. According to Exodus, the pharaoh of Egypt decreed that all Hebrew boy babies were to be drowned at birth. Yocheved hid her newborn son for 3 months and then placed her baby in a wicker basket in the Nile River reeds. The baby cried and was rescued by one of the pharaohs daughters, who kept the baby. This legend is similar to one in the Mesopotamian story of Gilgamesh, when the Sumerian king Sargon I was placed in a reed basket and floated down the Euphrates river. In the Court of the Pharaoh Moses sister, the prophetess Miriam, knew what would occur and was watching when the daughter of the pharaoh took the baby. Miriam came forward to ask the princess if she would like a Hebrew wet nurse for the infant. When the princess agreed, Miriam fetched Yocheved. Moses grew up in the palace as an adopted son of the pharaohs daughter (identified the Midrash as Queen Bithia), but he went to see his own people when he grew up, and as an adult he may have been a governor working for Ramses II. During Ramses IIs reign, Ethiopia was an Egyptian province with an Egyptian governor named Mesui, who some scholars suggest was Moses. While in Ethiopia, Moses married an Ethiopian princess named Tharbis or Adonais. When he witnessed an overseer beating a Hebrew, Moses struck the Egyptian and killed him, with the beaten Hebrew as a witness. The pharaoh learned that Moses was the murderer and ordered his execution. Moses fled to the land of Midian, where he married Tzipporah, daughter of Jethro. Their sons were Gershom and Eliezer. A Burning Bush In the land of Midian, Moses was tending a flock of sheep for his father-in-law when he saw a bush that was burning but not being consumed by the flames. He approached the bush and first an angel and then God (or more properly Yahweh) himself spoke to him, telling him that he must return to Egypt and shepherd the Israelites out to Canaan, their promised land of milk and honey. Moses was convinced when Yahweh changed his staff to a snake, then gave him a new staff with which to lead his people. Moses returned to Egypt to seek the release of the Hebrews and to bring them to Canaan, but when he approached the pharaoh, Ramses refused to release the Hebrews. In retaliation, Yahweh imposed a series of 10 plagues, the last being the killing the firstborn of every Egyptian. Only after suffering through the beginning of the tenth plague did the pharaoh relent, telling Moses he could take the Hebrews out of Egypt. However, after Moses and the Hebrews left, the pharaoh reversed his decision and had his men follow them. When they reached the Red Sea, Moses used his staff to part the waters and allow the Israelites to pass through the seabed. The Egyptian soldiers also entered the dry seabed, but once the Israelites had safely crossed Moses lifted his arms: the sea closed, and the Egyptian army was drowned. The Biblical Exodus During the 40-year journey of the Hebrews from Egypt to Canaan, Moses went to Mount Sinai to fast and commune with Yahweh for 40 days. There, he received the 10 Commandments from Yahweh. While Moses was gone, his followers including Aaron became nervous that he would not return and built a golden calf. Moses told Yahweh that his followers had begun to leave and Yahweh wanted to kill them, but Moses dissuaded him. But, when Moses saw the actual calf and altar he was so angry he hurled and shattered the two tablets holding the 10 Commandments; Moses made two more tablets and Yahweh inscribed them again. When the people complained they needed food in the desert, Yahweh fed the Israelites with manna, a substance white like coriander seed and tasted like wafers made with honey that rained down from the heavens, and quail. Death Near the end of the 40 years, Yahweh informed Moses that only the new generation of Israelites would enter Canaan, and for that reason, Moses would never see the Promised Land. Moses climbed Mt. Abarim and saw Canaan on the horizon, but that was as close as he would come. Moses chose Joshua as the successor, and, at the ripe old age of 120, Moses climbed Mt. Nebo and died. Who was Moses? Much of this tale is legendary and full of miracles, the stuff of ancient religion. But the role of Moses in the Bible, to Jews, Christians, and Moslems, is rich and complex beyond the miracles. He is seen by all three as the leader of the Israelite people who shepherded them out of Egypt. He is the embodiment of Mosaic law—the one who interceded with Yahweh on behalf of his people, and the one who acted as a judge on behalf of the sacred. He was a teacher and the founder of the cult and sanctuary of the ancient Hebraic religion. The last four books of the Torah—Exodus, Leviticus, Numbers, and Deuteronomy—are primarily dedicated to the life and activities of Moses and his people. Exodus starts with the birth of Moses and Deuteronomy ends with his death and burial by Yahweh. Early interpretations of that circumstance suggested that Moses himself wrote the books of the Torah (or received them direct from Yahweh). Modern biblical scholars mostly agree that the five books were redacted from four independently written documents written long after Moses would have died. The Ptolemaic-era Egyptian historian Manetho mentions Moses—again, long after Mosess death. There are other late historical references in the writings of the Roman historians Josephus, Philo, Apion, Strabo, Tacitus, and Porphyry. His story is told in the Bible in the book of Exodus and the ancient commentaries on the biblical text known as the midrashim. As Musa, he is also is a significant prophet in the Quran. Biblical scholar J. Van Seters, said it best, The quest for the historical Moses is a futile exercise. He now belongs only to legend. Sources Feldman, Louis H. Josephus Portrait of Moses. The Jewish Quarterly Review 82.3/4 (1992): 285–328.Josephus Portrait of Moses: Part Two. The Jewish Quarterly Review 83.1/2 (1992): 7–50.Nigosian, S. A. Moses as They Saw Him. Vetus Testamentum 43.3 (1993): 339–50.Robinson, Marilynne. Moses. Salmagundi 121/122 (1999): 23-46.RÃ ¶mer, Thomas. Moses Outside the Torah and the Construction of a Diaspora Identity. The Journal of Hebrew Scriptures 8.15 (2008): 1–12.Van Seters, John. Moses. The Encyclopedia of Religion. Ed. Eliade, Mircea. New York: Macmillan, 1987. 116.Wineman, Aryeh. Between Person and Metaphor: Moses in the Hasidic Homily-Literature. Hebrew Studies 59 (2018): 209–20.