| Abstract: | A novel method for the isolation of well-defined populations of inside-out vesicles from rat liver mitochondria is described. The technique utilizes specific immunoprecipitation of vesicles with accessible carbohydrate residues from a mixed population of inner membrane fragments using wheat germ agglutinin and anti-wheat germ agglutinin IgG. The unprecipitated fraction comprises 30--50% of the original population and exhibits little or no cytochrome c oxidase activity as estimated with exogenous cytochrome c as substrate. Addition of deoxycholate to promote membrane disruption results in an 8--10-fold increase in enzymic activity compared to only 1.5--2.0-fold stimulation in standard preparations of submitochondrial particles. It is concluded that the lectin affinity-purified membranes represent a sealed homogeneous (90--95% pure) population of inside-out inner membrane vesicles. |
