Destabilizing effects of fructose-1,6-bisphosphate on membrane bilayers.
Destabilizing effects of fructose-1,6-bisphosphate on membrane bilayers.
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1,6-二磷酸果糖对膜双层的不稳定作用。
DOI：
10.1007/s11745-002-0975-2
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发表时间：
2002
期刊：
影响因子：
1.9
通讯作者：
Chien,Sufan
中科院分区：
文献类型：
--
作者：
Ehringer,WilliamD;Su,Susan;Chiangb,Benjamin;Stillwell,William;Chien,Sufan
果糖-1，6-二磷酸（FBP）是一种高能糖酵解中间体，可降低缺血的影响;它已成功用于器官灌注和保存。细胞如何利用外部FBP来增加能量产生以及分子穿过膜双层的机制尚不清楚。本研究检查了FBP对膜双层渗透性、膜流动性、磷脂堆积和膜电位的影响，以确定FBP如何穿过膜双层。制备由卵磷脂（Egg PC）组成的大单层囊泡，并在30°C下与加标14 C-FBP的50 mM FBP孵育。DBP的摄取是显著的（P<0.05），并依赖于脂质浓度，表明FBP影响膜，双层通透性。加入10 mM钙后，脂囊对FBP的摄取显著降低（P<0.05）。添加5或50 mM的FBP导致Egg PC羧基荧光素渗漏显著增加（P<0.05）。我们假设，FBP的膜透化作用可能是由于膜双层的不稳定性。制备了由二棕榈酰pC（DPPC）组成的含有二苯基-1，3，5-己三烯（DPH）或三甲基氨-DPH（TMA-DPH）的小单层囊泡，并检查了FBP对荧光标记的荧光各向异性（FA）的影响。DBP使DPPC液晶态下DPH的FA显著降低（P<0.05），对DPPC液晶态下TMA-DPH的FA无影响，但使DPPC凝胶态下TMA-DPH的FA增加。根据DPPC/DPH或TMA‐DPH的相变测量，我们计算了相变的斜率，作为DPPC分子协同性的指标。DBP显著降低斜率，表明脂肪酰基链相互作用降低（P<0.05）。添加50 mM FBP导致mercury 540的液晶/凝胶态荧光比率显著降低（P<0.05），表明头基堆积增加。为了确定这些变化对细胞膜的影响，我们用膜电位探针3，3 ′-二丙基硫代碳菁碘化物（DiSC 3）标记人内皮细胞，然后加入FBP。FBP导致DiSC 3荧光显著、剂量依赖性降低，表明膜去极化。我们认为，FBP通过减少脂肪酰基链相互作用使膜双层不稳定，导致膜渗透性显著增加，使FBP扩散到细胞中，在那里它可以用作糖酵解中间体。
Fructose‐1,6‐bisphosphate (FBP) is a high‐energy glycolytic intermediate that decreases the effects of ischemia; it has been used successfully in organ perfusion and preservation. How the cells utilize external FBP to increase energy production and the mechanism by which the molecule crosses the membrane bilayer are unclear. This study examined the effects of FBP on membrane bilayer permeability, membrane fluidity, phospholipid packing, and membrane potential to determine how FBP crosses the membrane bilayer. Large unilamellar vesicles composed of egg phosphatidylcholine (Egg PC) were made and incubated with 50 mM FBP spiked with14C‐FBP at 30°C. Uptake of FBP was significant (P<0.05) and dependent on the lipid concentration, suggesting that FBP affects membrane, bilayer permeability. With added calcium (10 mM), FBP uptake by lipid vesicles decreased significantly (P<0.05). Addition of either 5 or 50 mM FBP led to a significant increase (P<0.05) in Egg PC carboxyfluorescein leakage. We hypothesized that the membrane‐permeabilizing effects of FBP may be due to a destabilization of the membrane bilayer. Small unilamellar vesicles composed of dipalmitoyl pC (DPPC) were made containing either diphenyl‐1,3,5‐hexatriene (DPH) or trimethylammmonia‐DPH (TMA‐DPH) and the effects of FBP on the fluorescence anisotropy (FA) of the fluorescent labels examined. FBP caused a significant decrease in the FA of DPH in the liquid crystalline state of DPPC (P<0.05), had no effect on FA of TMA‐DPH in the liquid crystalline state of DPPC, but increased the FA of TMA‐DPH in the gel state of DPPC. From phase transition measurements with DPPC/DPH or TMA‐DPH, we calculated the slope of the phase transition as an indicator of the cooperativity of the DPPC molecules. FBP significantly decreased the slope, suggesting a decrease in fatty acyl chain interaction (P<0.05). The addition of 50 mM FBP caused a significant decrease (P<0.05) in the liquid crystalline/gel state fluorescence ratio of merocyanine 540, indicating increased head‐group packing. To determine what effects these changes would have on cellular membranes, we labeled human endothelial cells with the membrane potential probe 3,3′‐dipropylthiacarbocyanine iodide (DiSC3) and then added FBP. FBP caused a significant, dose‐dependent decrease in DiSC3fluorescence, indicating membrane depolarization. We suggest that FBP destabilizes membrane bilayers by decreasing fatty acyl chain interaction, leading to significant increases in membrane permeability that allow FBP to diffuse into the cell where it can be used as a glycolytic intermediate.