13. ATP synthase was first identified by dissociation and reconstitution studies abundant knoblike protruding structures were observed on the matrix side of the inner mitochondrial membrane by EM (Racker in 1960) The inside-out submitochondrial particles with the knobs are capable of both electron transferring and aTP synthesis When the protruding Fi part was removed by agitation, electron transferring could still occur, but neither proton gradient nor ATP are produced
13. ATP synthase was first identified by dissociation and reconstitution studies • Abundant knoblike protruding structures were observed on the matrix side of the inner mitochondrial membrane by EM (Racker in 1960). • The inside-out submitochondrial particles with the “knobs” are capable of both electron transferring and ATP synthesis. • When the protruding F1 part was removed by agitation, electron transferring could still occur, but neither proton gradient nor ATP are produced
ATP synthesis reappeared when FI was reconstituted back(the solubilized F, alone can catalyze ATP hydrolysis, thus was originally named as F,ATPase) f was the first essential factor identified for oxidative phosphorylation
• ATP synthesis reappeared when F1 was reconstituted back (the solubilized F1 alone can catalyze ATP hydrolysis, thus was originally named as F1ATPase). • F1 was the first essential factor identified for oxidative phosphorylation
燃了
b Intact mitochondrion membrane esicles, the inner membrane is inverted the vesicle contain the electron carriers, are able to carry out el transfer to O but nnot phosphorylate ADP reconstitution The reconstituted to electron transfer
14. Isotope exchange experiments revealed that the G for AtP synthesis on purified F, is close to zero. When solubilized f(act as a ATPase)was incubated with aTP in the presence of O-labeled H,O, three or four 8O atoms were incorporated into the Pi, indicating that aTP formation/hydrolysis are readily reversible and multiple rounds of aTP formation and hydrolysis occurred on the enzyme Measurement of Kd values: ATP has a much higher affinity than adp to the enzyme(10-12 M vs 10-5 M) The proton gradient was thus proposed to drive the release of ATP from the enzyme surface
14. Isotope exchange experiments revealed that the G`0 for ATP synthesis on purified F1 is close to zero! • When solubilized F1 (act as a ATPase) was incubated with ATP in the presence of 18O-labeled H2O, three or four 18O atoms were incorporated into the Pi , indicating that ATP formation/hydrolysis are readily reversible and multiple rounds of ATP formation and hydrolysis occurred on the enzyme! • Measurement of Kd values: ATP has a much higher affinity than ADP to the enzyme (10-12 M vs 10–5 M). • The proton gradient was thus proposed to drive the release of ATP from the enzyme surface