Yeagle P L, Albert A D, Boesze-Battaglia K, Young J, Frye J
Department of Biochemistry, University at Buffalo (SUNY) School of Medicine 14214.
Biophys J. 1990 Mar;57(3):413-24. doi: 10.1016/S0006-3495(90)82558-3.
Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.
利用甾醇类似物胆甾三烯醇的时间分辨荧光各向异性以及对[¹³C₄]标记胆固醇的¹³C核磁共振（NMR）自旋晶格弛豫时间（T1c）测量，来确定表征未超声处理的磷脂多层脂质体中胆固醇主要运动模式的相关时间。发现两种运动模式很重要：（a）旋转扩散和（b）轴向扩散或“摆动”时指向矢方向的时间依赖性。根据胆甾三烯醇在卵磷脂（PC）双层中的时间分辨荧光各向异性衰减，计算出摆动相关时间τ⊥的值为0.9×10⁻⁹秒，以及表征相同运动的序参数S⊥的值为0.45秒。τ⊥和S⊥对膜的温度和胆固醇含量相对不敏感。[¹³C₄]标记胆固醇的T1c测量未提供轴向扩散相关时间τ∥的定量测定。来自脂质烃链的T1c表明τ⊥的值与胆固醇的相似。胆甾三烯醇的稳态各向异性测量和时间分辨各向异性测量被用于探究多种纯脂质和混合脂质多层脂质体中的甾醇行为。这些荧光测量结果表明，脂质头部基团和脂质烃链都对膜中甾醇环境的确定有贡献。特别是，观察到了磷脂酰乙醇胺（PE）头部基团和脂质烃链中多个不饱和键的影响。然而，虽然稳态各向异性对这些因素敏感，但时间分辨荧光分析表明，τ⊥不受膜脂质组成的强烈影响。PE的存在可能会增加S⊥。胆甾三烯醇的稳态各向异性测量和时间分辨各向异性测量都被用于探究三种生物膜中的甾醇行为：牛视杆外段（ROS）盘膜、人红细胞质膜和轻兔肌肌质网膜。在ROS盘膜中，S⊥的值略高于PC膜中的值，这可能反映了PE的影响。观察到的显著差异在于τ⊥的值。在ROS盘膜和红细胞膜中，τ⊥是磷脂双层中τ⊥的三分之一到五分之一。这一结果可能揭示了膜蛋白对甾醇行为的影响。
