Influence of vesicle size and oxidase content on respiratory control in reconstituted cytochrome oxidase vesicles.

Abstract

Previous work has shown that the respiratory control or coupling exhibited by reconstituted cytochrome oxidase systems can be markedly sensitive to the lipid composition, the lipid to protein ratio, and the vesicle size. In this work we have attempted to ascertain which, if any, of these factors plays a definitive role in determining the observed coupling. Vesicles prepared from dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine (1:4) were fractionated by DEAE chromatography to obtain a population homogeneous with respect to their lipid to protein ratio. This subpopulation was then applied to a Sepharose 4B-CL column to separate the vesicles by size. Fractions eluted from the column were assayed for enzyme activity in the presence and absence of carbonyl cyanide ( trifluoromethoxy )phenylhydrazone plus valinomycin. The coupling ratio was found to be dependent upon vesicle size; the smaller the vesicles, the higher the ratio. This suggests that lipid composition per se does not determine coupling characteristics. Reducing vesicle size for a given lipid to protein ratio has two effects. First, the radius of curvature is increased and, second, the average number of oxidase molecules per vesicle is reduced. In order to identify which of these factors was responsible for the observed tighter coupling, the oxidase was reconstituted with dioleoylphosphatidylcholine into vesicles of a defined size and the number of oxidase molecules per vesicle varied. The highest coupling ratios were observed for vesicles containing on average only one oxidase dimer. As the fraction of vesicles containing more than one protein was increased, the coupling ratio rapidly declined.(ABSTRACT TRUNCATED AT 250 WORDS)