The characterization and localization of DCCD binding to cytochrome b from the b c(1) complex of yeast mitochondria.
Item
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Title
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The characterization and localization of DCCD binding to cytochrome b from the b c(1) complex of yeast mitochondria.
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Identifier
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AAI9136795
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identifier
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9136795
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Creator
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DeLoskey, Richard James.
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Contributor
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Adviser: Diana S. Beattie
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Date
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1990
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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, Cell
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Abstract
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Dicyclohexylcarbodiimide (DCCD) binds to cytochrome b resulting in the inhibition of H{dollar}\sp+{dollar}-translocation in yeast {dollar}b{dollar}-{dollar}c\sb1{dollar} complex without significantly inhibiting electron transfer activity.;The approach to determining which carboxy amino acid of cytochrome b binds DCCD was to cleave cytochrome b into peptide fragments, purify these peptides, and determine the amino acid sequence of the peptide fragments with bound ({dollar}\sp{14}{dollar}C) DCCD. Finally the amino acid sequencing results could be compared with the sequence of cytochrome b deduced from the DNA sequence.;Three methods of isolating cytochrome b from {dollar}b{dollar}-{dollar}c\sb1{dollar} complex were used: preparative 12.5% SDS PAGE, molecular sieve HPLC, and chromatography on phenyl-sepharose CL-4B. The latter two methods did not resolve cytochrome b from all the other subunits of the {dollar}b{dollar}-{dollar}c\sb1{dollar} complex. In contrast, cytochrome b could be resolved from the other {dollar}b{dollar}-{dollar}c\sb1{dollar} complex subunits by preparative 12.5% SDS PAGE.;Cytochrome b labeled with ({dollar}\sp{14}{dollar}C) DCCD extracted from preparative gels was cleaved at methionine residues using CNBr. Two methods were used to purify the resultant fragments of cytochrome b: C{dollar}\sb{18}{dollar} reverse phase HPLC and SDS-urea (SUDS) PAGE.;C{dollar}\sb{18}{dollar} reverse phase chromatography did not resolve the radioactive CNBr fragments of cytochrome b. Also the recovery of total radioactive counts loaded onto the column was at best 41%. When CNBr fragments of ({dollar}\sp{14}{dollar}C) DCCD labeled cytochrome b were chromatographed by SUDS PAGE, two radioactive bands migrating with apparent molecular weights of 10.7 kDa and 5.2 kDa were observed.;The effects of different {dollar}b{dollar}-{dollar}c\sb1{dollar} complex inhibitors and ubiquinone analogues have on the binding of DCCD to cytochrome b were investigated. Antimycin, HQNO, BAL, DBH{dollar}\sb2{dollar}, and Q{dollar}\sb6{dollar}H{dollar}\sb2{dollar} clearly inhibit ({dollar}\sp{14}{dollar}C) DCCD binding to cytochrome b. n-HNQ, EFA, DNFB, and DQH{dollar}\sb2{dollar} all stimulate ({dollar}\sp{14}{dollar}C) DCCD binding. Myxothiazol may result in a slight inhibition of DCCD binding.
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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.
