Graduate Thesis Or Dissertation
 

Evidence for a soluble intermediate of oxidative phosphorylation isolated from cabbage mitochondria

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  • An intermediate of oxidative phosphorylation was apparently solubilized from cabbage mitochondria. The method of solubilization used primarily was extraction of a mitochondrial acetone powder with 0.1 M glycylglycine, pH 7.4, at 4° C. The intermediate betrayed its presence by causing the transfer of Pi³² to ADP with the resultant formation of ATP³², or G-6-P³² when the hexokinase trap was used. The formation of ATP³² or G-6-P³² was determined as N. E. P³²(non-extractable P³²) by the isobutanol-benzene extraction procedure. The transfer reaction used for assay took place in a completely soluble system, and both ADP and extract were required. The historical development of an isolation procedure and of a suitable assay system were described. Initial attempts to detect an intermediate were directed at introducing the radioactive label of Pi³² while the mitochondria were still intact, or during the extraction of the acetone powder. At these stages of the work, the assay was also conducted during the extraction of the acetone powder by inclusion of the acceptor system (ADP, hexokinase and glucose) and Mg⁺⁺ in the extraction medium. Most of the significant facts were obtained through the use of a clarified extract which had not been exposed to Pi³² previously. Although it was possible to centrifuge the crude extract at 105,000 X g for 90 min. without sedimenting the component necessary for the transfer reaction, this high speed centrifugation was not employed on a routine basis. The alleged intermediate was apparently proteinaceous as indicated by its response to heat, aging, dialysis, inhibitors and trichloroacetic acid (TCA). The transfer activity of the extract was lost upon incubation at 50° C. for five min., upon aging at 4° C., upon the addition of TCA, and in the presence of 2 X 10⁻⁵ M p-chloromercuribenzoic acid (PCMB). Although non-dialyzable, the extract was inactivated by dialysis, probably due to the loss of an essential small molecule such as NAD⁺ or NADP⁺. Both NAD⁺ and NADP⁺ stimulated the transfer activity in crude extracts and in extracts which had undergone the centrifugation at 105,000 X g. The action of these coenzymes was attributed to their possible role in promoting the labeling of the intermediate from Pi³². In addition to these two activators, the rate of the transfer reaction was markedly dependent upon the Pi concentration, up to 1 X 10⁻³ M Pi. The yield of intermediate from slightly less than two kilo-grams of cabbage was usually 50-100 millimicromoles under favorable assay conditions. The probability that this represents less than the maximum yield was considered. The transfer activity was rather severely inhibited by 1 X 10⁻³ M arsenate and partially inhibited by 1 X 10⁻⁴ M dinitrophenol (DNP); it was not sensitive to oligomycin. ATP, AMP, and GDP did not substitute for ADP nor did NADH substitute for NAD⁺. The intermediate appeared to be labile when kept in an aqueous system in the presence of Mg⁺⁺. Problems posed by such items as the impurity of the Pi³² and by side reactions were discussed. The extract also contained succinic thiokinase exchange activity. Most of the available evidence, including a preliminary ammonium sulfate fractionation, would indicate that this exchange was due to a separate protein. On the basis of experimental evidence obtained in this study, a sequence for the operation of the transfer reaction in the assay system was proposed. It was suggested that both the non-phosphorylated (A≈X) and the phosphorylated (X≈P) forms of an intermediate of oxidative phosphorylation were present in the extract. It was further speculated that NAD⁺ and/or NADP⁺ functioned as the A of A≈X and were active in promoting the labeling of the X≈P form with Pi³². In view of no ready alternate explanation for the transfer activity of the extract, it was suggested that this activity merits consideration as the expression of an intermediate of oxidative phosphorylation.
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file details JohnsonMorrisA1966_Redacted.pdf 2017-08-09 Pubblico Scaricare