PURIFICATION AND CHARACTERIZATION OF CYTOCHROME B AND ITS ASSOCIATED PROTEINS FROM YEAST MITOCHONDRIA.
Item
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Title
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PURIFICATION AND CHARACTERIZATION OF CYTOCHROME B AND ITS ASSOCIATED PROTEINS FROM YEAST MITOCHONDRIA.
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Identifier
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AAI8319761
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identifier
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8319761
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Creator
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DOMENICO, THOMAS JOHN.
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Contributor
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Diana S. Beattie
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Date
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1983
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Animal Physiology
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Abstract
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Historically, two forms of cytochrome b with molecular weights of 32,000 and 29,000 have been purified from yeast mitochondria in our laboratory. Both purified proteins were found to be antigenically, cytochrome b.;These proteins were examined for changes in concentration affected by proteolytic digestion during purification. No discernible difference in the relative concentration was recognized, using proteolytic inhibitors (PMSF, TPCK, TLCK, NEM, EDTA). New modifications were introduced into the purification scheme to optimize the amount of purified products collected while minimizing the conditions where proteolytic digestion can occur. Growth studies revealed that the relative amount of cytochrome b increased with the age of the growth culture or when cells were grown as obligate aerobes (lactate specifically increased the 32,000 protein band). These two purified forms were subjected to characterization studies. They were compared by amino acid analysis, N-terminal analysis, isoelectric focusing and by cyanogen bromide cleavage. The results of these characterizations on the primary structure of these proteins revealed that the 29,000 protein band consisted of more than one protein and that the majority of this protein isolated does not have sequence homology with the 32,000 protein. This characterization showed that the 32,000 protein band was a single polypeptide; it has a blocked N-terminal consistent with what has been found for the N, crassa cytochrome b; and it has an isoelectric point consistent with other reported values for yeast cytochrome b.;Immunotransfer studies and immuno-precipitation studies using a fresh anti-32,000 antibody showed specificity only for the cytochrome b polypeptide. Immuno-inhibition studies were done on mitoplasts, SMP and cholate solubilized mitochondria. The antibody was found to inhibit only the mitoplast preparations and that a maximum inhibition of only 20% could be obtained. From these immuno-inhibition studies, the site of antigenicity of cytochrome b was localized to the outer surface of the inner membrane.;A closely associated protein of cytochrome b which copurifies with it has a molecular weight of 53,000. This protein requires additional steps in the purification to obtain molecular weight purity due to its tight associations, even in the presence of SDS. An antibody was raised against the 53,000 molecular weight pure protein. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
