Vishnyakova E A, Ruuge A E, Golovina E A, Hoekstra F A, Tikhonov A N
Department of Biophysics, Faculty of Physics, M.V. Lomonosov Moscow State University, Russia.
Biochim Biophys Acta. 2000 Aug 25;1467(2):380-94. doi: 10.1016/s0005-2736(00)00234-0.
The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.
为深入了解胚细胞膜中脂类结构域的结构组织，利用传统电子顺磁共振（EPR）和饱和转移EPR（ST-EPR）光谱技术，研究了脂溶性自旋标记物与小麦胚轴的相互作用。使用在其多氧基不同位置标记的硬脂酸自旋标记物（n-SASL）及其甲基化衍生物（n-MeSASL）（n = 5、12和16），来探测胚轴细胞膜脂部分不同区域的有序性和分子流动性。在-50至+20摄氏度温度范围内测定的、与SASL烃链上多氧基位置相关的有序性和局部极性，是生物膜和模型脂膜的典型特征，但与种子油滴中的显著不同。ST-EPR光谱的位置分布图表明，即使在大多数膜脂处于固态（凝胶相）的低温下，沿脂烃链的柔韧性分布图依然存在。膜表面区域中SASL氮氧自由基的有序性明显高于膜深度处的。MeSASLs的多氧基比相应SASLs的有序性更低（即使在低温下），表明MeSASLs位于膜脂主体中而非蛋白质边界脂中。对EPR和ST-EPR光谱参数分布图的分析使我们得出结论，积累在胚轴中的绝大多数SASL和MeSASL分子位于细胞膜中而非油体内部。多氧基硬脂酸盐优先分配到膜中，这证明了EPR自旋标记技术在原位研究不同水合水平种子中膜行为的潜力。
