AFFINITY PURIFICATION OF YEAST CYTOCHROME-OXIDASE WITH BIOTINYLATED SUBUNIT-4, SUBUNIT-5, OR SUBUNIT-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. T he mutants are complemented by gene constructs expressing cytochrome o xidase subunits with a carboxyl terminal extension containing a biotin ylation signal sequence. Spectra and enzyme activities of mitochondria from transformants expressing a biotinylated subunit indicate restora tion of a functional cytochrome oxidase. Biotinylated cytochrome oxida se can be affinity-purified from mitochondrial extracts by fractionati on on a monomeric avidin column. This method can be used to purify the enzyme from small amounts of starting material. (C) 1998 Academic Pre ss.