Abstract
Null mutants in COX4, COX5a, or COX6, which encode subunits 4, 5, and 6 of yeast cytochrome oxidase are blocked in assembly of the enzyme. The mutants are complemented by gene constructs expressing cytochrome oxidase subunits with a carboxyl terminal extension containing a biotinylation signal sequence. Spectra and enzyme activities of mitochondria from transformants expressing a biotinylated subunit indicate restoration of a functional cytochrome oxidase. Biotinylated cytochrome oxidase can be affinity-purified from mitochondrial extracts by fractionation on a monomeric avidin column. This method can be used to purify the enzyme from small amounts of starting material.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alleles
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Avidin
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Biotinylation
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Chromatography, Affinity
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Electron Transport Complex IV / chemistry
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Electron Transport Complex IV / genetics
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Electron Transport Complex IV / isolation & purification*
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Electron Transport Complex IV / metabolism
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / isolation & purification*
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Fungal Proteins / metabolism
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Mitochondria / enzymology
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Polymerase Chain Reaction
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification*
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / ultrastructure
Substances
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Fungal Proteins
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Recombinant Fusion Proteins
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Avidin
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Electron Transport Complex IV