To understand the structural basis for the apparent strong interaction between cholesterol and sphingomyelin (SPM), we have synthesized an analogue of SPM, 3-deoxy-2-O-stearoyl-SPM, in which an ester-linked acyl chain replaces the amide-linked acyl chain at C-2 and a hydrogen replaces the hydroxy group at C-3. We have compared the behavior of this analogue with that of 3-deoxy-N-stearoyl-SPM in monolayers and vesicles, both as pure phospholipids and in mixtures with cholesterol. The force-area isotherm of 3-deoxy-2-O-stearoyl-SPM was similar to that of 3-deoxy-N-stearoyl-SPM. The surface potential across the pure SPM monolayer at the air-water interface was larger for 3-deoxy-2-O-stearoyl-SPM than for 3-deoxy-N-stearoyl-SPM (about 430 mV and 330 mV, respectively, at 50 Angstrom(2)). The overall dipole moment of 3-deoxy-2-O-stearoyl-SPM was almost constant at 570 mD (between a mean molecular area range of 45-85 Angstrom(2)), whereas that of 3-deoxy-N-stearoyl-SPM was about 420 mD. Cholesterol appeared to be equally miscible in both SPM monolayers, as determined from the condensing effect cholesterol had on the lateral packing of the two SPMs. The oxidation of monolayer cholesterol by cholesterol oxidase was also determined using both SPMs. The stoichiometry at which free cholesterol clusters disappeared in monolayers, when going from high to low cholesterol content, was 2:1 (mol sterol/mol SPM) for both SPMs. Cholesterol was oxidized faster in 3-deoxy-2-O-stearoyl-SPM mixed monolayers than in 3-deoxy-N-stearoyl-SPM mixed monolayers; a 5.4-fold difference in rate of oxidation susceptibility was found at an equimolar ratio of SPM to sterol at 25 degrees C. The rate and extent of enzyme-catalyzed oxidation of cholesterol incorporated into small unilamellar vesicles (SUVs) formed from 3-deoxy-2-O-stearoyl-SPM were higher than in SUVs prepared with 3-deoxy-N-stearoyl-SPM by factors of 4.8 and 2.6, respectively, at 30 degrees C. The results of this study indicate that the NH group of SPM is important in allowing a tight interaction between SPM and cholesterol in monolayers and SUVs; however, the amide linkage is not critical for the 2:1 stoichiometry typical of cholesterol oxidation in cholesterol/SPM monolayers.
|Status||Publicerad - 1994|