Pajot-Augy E
Unité de Recherche en Développement Concerté INRA-INSERM (U.310), Institut de Biologie Physico-chimique, Paris, France.
Cryobiology. 1993 Jun;30(3):286-98. doi: 10.1006/cryo.1993.1027.
Organic cryosolvents essential for cryopreservation of living cells have a colligative effect on water properties, but also affect cellular structures such as the membrane, actin, or tubulin cytoskeleton. The effects of cryosolvents on actin and its binding proteins are starting to be well investigated. In parallel, tubulin assembly characteristics were investigated comparatively, with 0-30% 1,2-propanediol, dimethyl sulfoxide, or glycerol, and with or without microtubule-associated proteins, at 37 or 4 degrees C. Tubulin association was monitored by spectrometry and sedimentation, providing the concentration in free protein, cold-depolymerizable microtubules, and cold-resistant associations. At 37 degrees C, 1,2-propanediol and dimethyl sulfoxide induce a similar association level and cold stability of the assemblies. Glycerol yields a lower level of tubulin association. Cold stability of the assemblies requires the presence of solvent, the amount of which is modulated by microtubule-associated proteins (MAPs): 15% 1,2-propanediol or dimethyl sulfoxide, decreasing down to 10% with MAPs, or 10% glycerol with MAPs only. At 4 degrees C, some cold-stable association is promoted by 1,2-propanediol or dimethyl sulfoxide above 10-15%, in the presence or absence of MAPs, but not with glycerol. In addition, protein content of the various fractions obtained with MAPs and 30% solvent was examined by densitometry of electrophoresis gels. Cold-labile associations obtained at 37 degrees C with 1,2-propanediol or dimethyl sulfoxide are lacking in tubulin and enriched in tau proteins relative to control or glycerol. Associations formed at 37 degrees C and stable to subsequent cold treatment, or at 4 degrees C, regardless of the solvent, present a large tubulin content, as well as few tau proteins and high-molecular-weight MAPs.
用于活细胞冷冻保存的有机冷冻溶剂对水的性质有依数性影响，但也会影响细胞结构，如细胞膜、肌动蛋白或微管蛋白细胞骨架。冷冻溶剂对肌动蛋白及其结合蛋白的影响已开始得到充分研究。与此同时，研究了在37℃或4℃下，0 - 30%的1,2 - 丙二醇、二甲基亚砜或甘油，以及有无微管相关蛋白时微管蛋白的组装特性。通过光谱法和沉降法监测微管蛋白的缔合，得出游离蛋白、冷可解聚微管和抗冷缔合的浓度。在37℃时，1,2 - 丙二醇和二甲基亚砜诱导出相似的缔合水平和组装体的冷稳定性。甘油产生的微管蛋白缔合水平较低。组装体的冷稳定性需要有溶剂存在，其含量受微管相关蛋白（MAPs）调节：15%的1,2 - 丙二醇或二甲基亚砜，有MAPs时降至10%，或仅在有MAPs时为10%的甘油。在4℃时，1,2 - 丙二醇或二甲基亚砜在10 - 15%以上时，无论有无MAPs，都会促进一些冷稳定缔合，但甘油不会。此外，通过电泳凝胶的光密度测定法检测了用MAPs和30%溶剂获得的各种组分的蛋白质含量。相对于对照或甘油，在37℃用1,2 - 丙二醇或二甲基亚砜获得的冷不稳定缔合缺乏微管蛋白，而富含tau蛋白。在37℃形成并对随后的冷处理稳定的缔合，或在4℃形成的缔合，无论使用何种溶剂，都含有大量微管蛋白，以及少量tau蛋白和高分子量MAPs。
