Cholesterol Superlattice Modulates the Activity of Cholesterol Oxidase in Lipid Membranes

Here, the interplay between membrane cholesterol lateral organization and the activity of membrane surface-acting enzymes was addressed using soil bacteria cholesterol oxidase (COD) as a model. Specifically, the effect of the membrane cholesterol mole fraction on the initial rate of cholesterol oxidation catalyzed by COD was investigated at 37 °C using cholesterol/1-palmitoyl-2-oleoyl-L-α-phosphatidylcholine (POPC) large unilamellar vesicles (LUVs, ∼800 nm in diameter). In the three concentration ranges examined (18.8-21.2, 23.6-26.3, and 32.2-34.5 mol % cholesterol), the initial activity of COD changed with cholesterol mole fraction in a biphasic manner, exhibiting a local maximum at 19.7, 25.0, and 33.4 mol %. Within the experimental errors, these mole fractions agree with the critical cholesterol mole fractions (C_r) (20.0, 25.0, and 33.3) theoretically predicted for maximal superlattice formation. The activity variation with cholesterol content was correlated well with the area of regular distribution (A_reg) in the plane of the membrane as determined by nystatin fluorescence. A similar biphasic change in COD activity was detected at the critical sterol mole fraction 20 mol % in dehydroergosterol (DHE)/POPC LUVs (∼168 nm in diameter). These results indicate that the activity of COD is regulated by the extent of sterol superlattice for both sterols (DHE and cholesterol) and for a wide range of vesicle sizes (∼168-800 nm). The present work on COD and the previous study on phospholipase A₂ (sPLA₂) [Liu and Chong (1999) Biochemistry 38, 3867-3873] suggest that the activities of some surface-acting enzymes may be regulated by the extent of sterol superlattice in the membrane in a substrate-dependent manner. When the substrate is a sterol, as it is with COD, the enzyme activity reaches a local maximum at C_r. When phospholipid is the substrate, the minimum activity is at C_r, as is the case with sPLA₂. Both phenomena are in accordance with the sterol superlattice model and manifest the functional importance of membrane cholesterol content.