Fluorescence probes as monitors of surface membrane fluidity gradients in murine fibroblasts.
Fluorescence probes as monitors of surface membrane fluidity gradients in murine fibroblasts.
复制标题
荧光探针作为小鼠成纤维细胞表面膜流动性梯度的监测器。
DOI：
10.1111/j.1432-1033.1980.tb07205.x
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
F. Schroeder
中科院分区：
文献类型：
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作者：
F. Schroeder
荧光探针分子与猝灭剂联合使用，研究哺乳动物细胞表面膜可能表现出垂直不对称的物理化学性质。两种不同的方法表明，小鼠成纤维细胞表面膜的外单层比内单层流动性更强，第一，反式巴比酸的荧光氨基葡萄糖衍生物不能穿透完整的LM细胞(转化的小鼠成纤维细胞的一株)或来自这些细胞的吞噬小体。这种探针分子很容易被三硝基苯甘氨酸等非穿透性猝灭剂接触到，这表明它存在于暴露的膜单分子层中。在完整的LM细胞中，氨基葡萄糖反式氨基葡萄糖酸盐的荧光偏振度P为0.218±0.009，而在吞噬乳胶珠膜中的荧光偏振度为0.248 0.006。其次，以反式-巴豆酸、1，6-二苯基-1，3，5-己三烯和N-苯基-L-萘胺为膜探针，对分离的质膜囊泡进行了荧光检测。它们在质膜上的荧光可以通过将三硝基苯基共价连接到膜表面暴露的NH2组分上或通过加入水溶性非穿透猝灭剂三硝基苯甘氨酸来猝灭。三硝基苯氨基在415 nm处有最大吸收，因此可以化学猝灭反式-石蜡酸脂、1，6-二苯基-1，3，5-己三烯和N-苯基-1-萘胺的荧光，它们的最大荧光发射波长在415 nm附近。在这两种方法中，当只对膜外单层氨基进行三硝基苯基化时，膜上反式-羟基苯甲酸酯的吸收校正荧光发射和相对荧光效率分别降低了45±5%和44±6%。两种猝灭方法均不改变反式-石油酸酯的荧光寿命。相反，当质膜两侧具有共价连接的三硝基苯基时，这些参数减小了90%-95%。1，6-二苯基-1，3，5-己三烯和N-苯基-1-萘胺也得到了类似的结果。膜内单层硬度(P=0.364±0.005)大于全膜(P=0.323±0.005)。1，6-二苯基-L，3，5-己三烯也得到了类似的结果。然而，N-苯基-1-萘胺位于双层膜的极性界面附近，在这两种方法猝灭时，极化没有差别。因此，在没有猝灭剂的情况下，或在有猝灭剂存在的情况下，用反式-甲氨酸氨基葡萄糖和1，6-二苯基-1，3，5-己三烯获得的结果表明，在LM细胞质膜中可能存在某些物理化学参数的垂直不对称或梯度。初步数据表明，这种不对称性可以通过脂极性头部基团的操作来改变。
Fluorescence probe molecules were utilized in conjunction with quenching agents to investigate the possibility that mammalian cell surface membranes may display vertical asymmetry of physico-chemical properties. Two different approaches indicated that the outer monolayer of a murine fibroblast surface membrane was more fluid than the inner monolayer, First, a fluorescent glucosamine derivative of trans-parinaric acid did not penetrate intact LM cells (a strain of transformed murine fibroblasts) or phagosomes derived from these cells. This probe molecule was easily accessible to nonpenetrating quenching agents such as trinitrophenyl-glycine, indicating that it resided in the exposed membrane monolayer. The fluorescence polarization, P, of glucosamine trans-parinarate in intact LM cells was 0.218 ± 0.009 while in phagocytosed latex bead membranes the polarization was 0.248 0.006. Second, trans-parinaric acid, 1,6-diphenyl-1,3,5-hexatriene, and N-phenyl-l-naphthylamine were used as fluorescent membrane probes in isolated plasma membrane vesicles. Their fluorescence in the plasma membrane was quenched by either covalently linking trinitrophenyl groups to exposed NH2 constituents on the membrane surface or by adding the water-soluble non-penetrating quenching agent, trinitrophenyl-glycine. Trinitrophenyl-amino groups have an absorption maximum at 415 nm and can therefore chemically quench the fluorescence of trans-parinarate, 1,6-diphenyl-1,3,5-hexatriene, and N-phenyl-1-naphthylamine which have fluorescence emission maxima near 415 nm. With both methods, when only the outer monolayer amino groups were trinitrophenylated, the absorption-corrected fluorescence emission and the relative fluorescence efficiency of trans-parinarate in the plasma membrane were decreased by 45 ± 5% and 44 ± 6% respectively. Neither quenching method altered the fluorescence lifetime of trans-parinarate. In contrast, when both sides of the plasma membrane had covalently linked trinitrophenyl groups, these parameters were diminished by 90–95%. Similar results were obtained with 1,6-diphenyl-1,3,5-hexatriene and N-phenyl-1-naphthylamine. Polarization measurements of trans-parinarate indicated that the inside monolayer of the membrane was more rigid (P= 0.364 ± 0.005) than the whole membrane (P= 0.323 ± 0.005). Similar results were obtained with 1,6-diphenyl-l,3,5-hexatriene. However, N-phenyl-1-naphthylamine which resides near the polar interface of the bilayer showed no difference in polarization upon quenching by either method. Thus, results obtained with glucosamine trans-parinarate in the absence of quenching agents or with trans-parinarate and 1,6-diphenyl-1,3,5-hexatriene in the presence of quenching agents indicated that a vertical asymmetry or gradient of certain physicochemical parameters may exist in LM cell plasma membranes. Preliminary data indicated that this asymmetry could be altered by lipid polar head group manipulation.