Schroeder F, Kier A B, Sweet W D
Department of Pharmacology and Medicinal Chemistry, College of Pharmacy, University of Cincinnati Medical Center, Ohio 45267-0004.
Arch Biochem Biophys. 1990 Jan;276(1):55-64. doi: 10.1016/0003-9861(90)90009-n.
The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospholipids by culture medium supplementation, decreased the limiting anisotrophy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H2O2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbilayer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20-28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72-76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.
研究了多不饱和脂肪酸和脂质过氧化对LM成纤维细胞质膜单叶胆固醇分布和结构有序性的影响。细胞表面（内侧）叶比细胞外表面（外侧）叶更坚硬，且含有更多的胆固醇。通过相位和调制荧光测量结合三硝基苯基基团的叶特异性猝灭，确定了每个叶中二苯基己三烯（DPH）运动的静态（极限各向异性）和动态（旋转弛豫时间）结构成分。通过在培养基中添加多不饱和脂肪酸将其掺入膜磷脂中，降低了细胞表面叶而非细胞外表面叶中DPH的极限各向异性，从而消除了跨膜流动性差异。Fe(II)+H2O2引发的过氧化作用导致质膜细胞外表面叶变硬（极限各向异性和旋转弛豫时间增加），无论膜中含有饱和脂肪酸和单不饱和脂肪酸，还是富含亚油酸或亚麻酸。DPH报告的细胞表面叶的结构未受影响。通过脱氢麦角固醇荧光的叶特异性猝灭测量的质膜跨膜胆固醇分布表明，20-28%的胆固醇位于细胞外表面叶。对LM成纤维细胞补充多不饱和脂肪酸导致质膜跨膜胆固醇分布完全逆转（细胞外表面叶占72-76%）。在对照质膜中，膜叶之间的胆固醇跨膜分布完全抵抗交联剂暴露和过氧化作用的改变，在补充亚油酸或亚麻酸的膜中，也完全抵抗过氧化作用的改变。
