Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.

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An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.