Isolation of a sealed homogeneous population of inner membrane fragments with inverted orientation from rat liver mitochondria using specific lectin immunoprecipitation.
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.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D003840:Deoxycholic Acid / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism; D007074:Immunoglobulin G; D007163:Immunosorbent Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D037102:Lectins / Q000276:immunology; D008930:Mitochondria, Liver / Q000032:analysis*; D051381:Rats
D007074:Immunoglobulin G; D037102:Lectins; D003840:Deoxycholic Acid; D003576:Electron Transport Complex IV