Graduate Thesis Or Dissertation
 

Evidence for an intermediate related to oxidative phosphorylation in mitochondria from blowflies, Phormia regina

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  • A large number of experiments were done to detect and identify one or more intermediates involved in coupling ATP synthesis to electron transport. Using mitochondria from blowflies, addition of ADP after a period of incubation with Pi³² was found to give rise to a temporarily rapid esterification (the "ATP jump"), inhibited by antimycin A, DNP, oligomycin and others. This briefly rapid esterification may indicate accumulation of an intermediate containing P³², before the addition of ADP. The supernatant fluid from blowfly mitochondria, incubated with Pi³² and substrate, was found to contain material which reacted upon addition of ADP to give increased acid-stable P³² ester, which by all tests to date appears to be ATP³². Inclusion of oligomycin during incubation of the mitochondria appears to increase the amount of acid-stable P³² ester in the extracts, but abolishes the change on adding ADP; oligomycin only partially inhibits the ADP effect when added to the extracts. Extracts from mitochondria incubated with DNP (and Pi³²) gave no detectable reaction upon addition of ADP. Addition of ADP and Pi³² to extracts from blowfly mitochondria, previously incubated with substrate (without Pi³²), was also found to give acid-stable P³² ester, suggesting that part (at least) of the "intermediate" may not be phosphorylated before this addition. The amount of P³² ester measured was reduced by addition of DNP and by omission of substrate during the initial incubation. The nature of the two intermediates suggested by these results, and the possibility that they may be identical, can be determined only by additional research.
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