Affinity purification of yeast cytochrome oxidase with biotinylated subunits 4, 5, or 6.
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.
Glerum
D M
DM
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Tzagoloff
A
A
D000483:Alleles; D001360:Avidin; D019921:Biotinylation; D002846:Chromatography, Affinity; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D001360:Avidin; D003576:Electron Transport Complex IV