Question

why was it necessary to isolate mitochondria to study and also use engineered vesicles that either did or did not contain atp synthetase?

by the first ATP s r aasing the enzyme to function as an ATPase and result in ATP ATP synthase would likely drive the ATP synh reverse direction, s Original Paper The oniginal experiments on pli and ATP formation were donc on chloroplasts The chemiosmotic generation of ATP is simila to mitochondria, but occurs across tihe thylašoid membrane which is analogous to the inner tmitochondrial membeane. experiments have been repeated in mitochondria The Jagendorf, A. T and E. Uribe 1966, ATP formation caused by acid-base transition of spinach chloroplasts Procecdings of the Nattional Acaiemy of Scriences 35: 170 177. Working with Data Two Experiments Demonstrate the Chemiosmotic Mechanism (For additional links on this topic, refer to the Chapter9 Experiment Links) Eniversity of Calformia Sonia C Son rcs Anmation Mvis uf ATP Synthase Introduction Previous rescarch into the force htm INVESTIGATING LIFE Molermlar amd Cellwar Learning Conter. Virnmal Cell Arnimation Colection:C Processes: ATP Synthase Gradient: The Movie synthesis in mitochondra led to the postulation Analyze the Data In the experiment, chloroplasts were isolated at pi 8 and then incuhated with ADe phosphate, and Mg ions at cither plI 7 or gli 3.8. ATP formation was measurod in two ways: The first employed the enzyme, luciferase, which catalyzes the formation of a luminescent molecule if ATP is present The second employed molybdate to measure ch directly. Table 1 shows the data from the paper Precisely how this mechanism uses ATP symthase to couple proton diffusion to ATP synthesis was demonstrated two insightful TABLE1 ATP FORMATION DUE TO AcID-BASE TRANSITION Luciferase Aasy P-Molybdate Extraction - Cpm 2200 Complete In the first experiment -ADP 45 48 50 mitochondria were placed in a medium with a low H' concentration until equilibrium was reached The Completa, in this esse, indicstes an acid stage at pH 3.8 with 10 mM suecinate and 3 X 10- M DOMU ee refer to toe yield of RTP ftetraeting the contropass broughi only to plH 7 before at pH 8.0 with compopente se indiested in Methode section. et t mamoles ATP per mg eblorophyll. iald of ATP after sabiracig the costrol of ehloroplasts brought only to pII 7 before itóchondna were then moved to a medium with a high H" concentration. The H' ions moved down the concentration gradient and into the mitochondria, driving the syathesis of ATP. In the sccond experiment, a profon pump was added to an artif and used to pump H" into the vesicle, generating a gradient ATP synthase was then inserted into the vesicke membrane As the H' diffused out of the vesicle, ATP synthase drove the synthesis of ATP Together, the results of these two experiments provided evidence that the chemiosmotic mechanism uses ATP synthase to couple proton diff to ATP syathesis It is important to realize that whilc ATP synthase normally functions to drive the synthesis of ATP, it is also capable of functioning in the reverse direction, leading to ATP hydrolysis and the formation of ADP and P. It is the H' gradient, as well the pherphorylation stage. was added to an artificial lipid vesicle Question 1 What is the significance of the difference between the Total and "Net" columns? DO NOT give the numerical difTerences. What variable does this control control for? Question 2 Why did ATP production go down in the absence of PO.? Question 3 In the described experiments, why was it necessary to, in the first case, isolate mitochondria to study, and in the second case, use engineered vesicles that either did or did not contain ATP synthetase? Why couldn't the researchers simply look at whole cells low concentration of ATP in the mitochondrial mairis, that drives the reaction toward ATP production Consider, then, what would happen in the second experiment ifa second ATP synthase, oriented in the opposite direction of the one originally insertod in the membrane, was added. In this scenario, the conditions for the second ATP synthase would differ from that for the original ATP symhase. The second ATP synthase would in the second experiment if a to demonstrate the chemiosmotic mechanism? lack a H' concentration gradient, thereby preventing ATP production Further, the ATP Question 4 Why couldn't the rescarchers perform their experiments at pH 12? What would you predict you would see in the luciferase assay had they performed their experiments at a pH of 12?

Answer 1

atp in the mitochondria is produced by chemiosmosis, wich involves a gradient of H+ ions, mitochondria membrane is packed with AtpSynthase an enzymatic complex wich helps in production of ATP by using the proton gradient or chemiosmosis, wich involves production of ATP. A concentration gradient drives H+ ions into Atp synthase and it gets rotated to release the protons into matrix,wich helps in production of ATP. but an equilibrium of Concenteration gradient is always maintained by the electron transport chain wich pumps the H+ ions Outside by using Nadh dehydrogenase complex,cytochrome b-c complex and the cytochromeoxidase complex wich pump the H+ ions outside so that a concentration gradient is maintained and Atp production doesnt stop.

That was the exact mechanism involved in production of ATP in Normal cells,Now Scientists have isolated mitochondria to study how exactly Atp is produced so they have used engineered vesicles to conclude if Atp Synthase is involved in ATP Production. so that a normal cell and an engineered vesicle could show a difference in atp production can conclude the atp production mechanism.