Graduate Thesis Or Dissertation
 

Mammalian heart cytochrome oxidase

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  • Preparation of a cytochrome c-cytochrome oxidase complex was achieved, using crystalline beef cytochrome c and purified cytochrome oxidase from beef heart muscle, or from the Keilin-Hartree preparation. Succinate oxidase was reconstituted with the complex. A mixture of cytochrome oxidase, solubilized with nonionic detergent, and excess cytochrome c was exposed to sonic irradiation. The cytochrome c-cytochrome oxidase complex was isolated using gel filtration of the mixture. The ratio of the concentration of cytochrome c to cytochrome oxidase in the complex was increased with the length of sonic treatment. The complex with the maximum ratio of one was separated after a 45-60 minute period of sonic irradiation. Differential sedimentation and chromatography on Sephadex showed the complex to be an integrated entity of two components. The complex could be formed with cytochrome oxidase and either intact cytochrome c or guanidinated cytochrome c. However, both acetylated cytochrome c and succinylated cytochrome c completely failed to form any kind of complex with cytochrome oxidase. These results indicate that the interacting force between the two components is mainly electrostatic, Absolute and difference absorption spectra of the carbon monoxide compound of the complex showed a unique, distinct maximum at 415 mμ. On the other hand, a "free mixture" of both components exhibited no absorption peak at the same wave length in the presence of CO. The formation of this CO compound was prevented by sodium cholate. After separation by means of cation exchange resin, neither component of the complex showed the absorption maximum at 415 mμ in the presence of CO. From these and other observations, the possibility of a conformational change in the protein moiety of the cytochromes as a result of the interaction was hypothesized in reference to the electron transport mechanism Infrared spectrum of the complex showed no superposition of absorption bands of the two components but showed a unique band at 950 and at 1050 cm⁻¹. The physiological activity of the complex was verified by the functional reconstitution of succinate oxidase with soluble succinate dehydrogenase, the cytochrome b-c₁ particle, and the complex, The reconstituted succinate oxidase was inhibited by the usual respiratory inhibitors in the same manner as the Keilin-Hartree heart muscle preparation, the carbon monoxide inhibition of the reconstituted succinate oxidase was reversed by light. The behavior of the complex in the oxidation of ascorbate was the same as that of the heart muscle preparation. Addition of a catalytic amount of tetramethyl-p-phenylenediamine dramatically stimulated ascorbate oxidation. The cytochrome oxidase (cytochrome a plus cytochrome a₃) was functionally and structurally reconstituted from cytochrome a and intact heme a. About sixty percent of the original cytochrome oxidase activity was recovered from an incubation mixture of cytochrome a, ascorbate-EDTA, cytochrome c and hematin a. Hematin a was prepared from cytochrome oxidase or directly from heart muscle mince by means of metalation of purified porphyrin a. After removal of excessive hematin a by Sephadex chromatography, the reconstituted oxidase showed spectral properties almost identical to those of the intact preparation; a slight difference was found at the α-band.
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file details KuboyamaMorio1965.pdf 2017-08-09 Público Baixar