Studies on the incorporation of membrane proteins into liposomes: --effect of "boundary lipids" on reconstitution of pig heart mitochondrial cytochrome oxidase into liposomes.

The reactivation of delipidated cytochrome oxidase depends on the reformation of "annular lipids", which is tightly bounded to the enzyme molecule. In the restoration of oxidase activity, the efficiencies of phospholipids with different polar head groups decrease in this order: PS greater than DPG greater than PI greater than PA greater than PG greater than PC, PE and in the case of phosphatidylcholines with different acyl chain the order is DOPC greater than LPC greater than PC greater than DPPC, DSPC. Therefore both the polar head group and the acyl chain of phospholipids must be considered in the reactivation process. The existence and the specificity of "annular lipids" obviously influence the incorporation of cytochrome oxidase into liposomes. When acidic phospholipids are used as "annular lipids", the effectiveness of reconstitution decreases in this order: PI greater than PS greater than DPG greater than PA, PG. Divalent metallic cations would facilitate the cytochrome oxidase reconstitution, but their effects depend on the composition of "annular lipids". Using dialysis method Ca2+ and Mg2+ could facilitate the incorporation into liposomes of the enzymes having PS or DOPC as their "boundary lipids". A comparison between the effects of different metallic cations on incorporation of cytochrome oxidase also shows that, with PI as "annular lipids", the effectiveness of different cations on incorporation by incubation method decreases in this order: Ca2+ greater than Mg2+ greater than Mn2+, Sr2+ greater than La3+. Apparently, the effect of metallic cations on incorporation cannot be interpreted by considering only the neutralization of the negative charged groups on membrane protein and lipids.