PHOSPHATIDYLSERINE DECARBOXYLASE - GENERATION OF ASYMMETRIC VESICLES AND DETERMINATION OF THE TRANSBILAYER DISTRIBUTION OF FLUORESCENT PHOSPHATIDYLSERINE IN MODEL MEMBRANE SYSTEMS
PHOSPHATIDYLSERINE DECARBOXYLASE - GENERATION OF ASYMMETRIC VESICLES AND DETERMINATION OF THE TRANSBILAYER DISTRIBUTION OF FLUORESCENT PHOSPHATIDYLSERINE IN MODEL MEMBRANE SYSTEMS
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DOI：
10.1016/0005-2736(86)90237-3
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发表时间：
1986-11-17
期刊：
影响因子：
--
通讯作者：
SCHROIT, AJ
中科院分区：
文献类型：
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作者：
DENKINS, YM;SCHROIT, AJ
在模型系统中使用含有磷脂酰丝氨酸（NBD-PS）的荧光类似物的大单层囊泡（LUV），以确定采用磷脂酰丝氨酸脱羧酶（PS-脱羧酶）产生不对称囊泡的可行性，并确定PS的跨双层分布。用超声裂解大肠杆菌JA 200 pLC 8-47制备的PS脱羧酶在无洗涤剂缓冲液中稳定，并催化NBD-PS转化为NBD-磷脂酰乙醇胺（NBD-PE）。PS-脱羧酶是能够脱羧几乎所有的NBD-PS存在于外叶的LUV含有对称或不对称（仅外部）分布的NBD-PS，但不是NBD-PS存在于内小叶的囊泡。PS-脱羧酶的能力，以脱羧只有NBD-PS位于外小叶的囊泡进行了独立验证的共振能量转移（NBD-PS和（丽丝胺）罗丹明B-标记的磷脂酰乙醇胺）和衍生与三硝基苯磺酸（TNBS）。这些技术揭示了所形成的可交换池（外小叶上的NBD-PS的级分）和Tnp-（NBD-PS）的相应级分与PS-脱羧酶可用于产生不对称囊泡的程度相当（即，PS内，PE外），并确定PS在模型膜中的双分子层内分布。
Large unilamellar vesicles (LUV) that contained a fluorescent analog of phosphatidylserine (NBD-PS) were used in model systems to determine the feasibility of employing phosphatidylserine decarboxylase (PS-decarboxylase) to generate asymmetric vesicles and to determine the transbilayer distribution of PS. PS-decarboxylase prepared by sonication of Escherichia coli JA 200 pLC 8-47 was found to be stable in detergent-free buffers and catalyzed the conversion of NBD-PS to NBD-phosphatidylethanolamine (NBD-PE). PS-decarboxylase was capable of decarboxylating virtually all of the NBD-PS present in the outer leaflet of LUV containing a symmetric or asymmetric (outside only) distribution of NBD-PS, but not NBD-PS present in the inner leaflet of the vesicles. The ability of PS-decarboxylase to decarboxylate only NBD-PS located in the outer leaflet of the vesicles was independently verified by resonance energy transfer (between NBD-PS and (lissamine) rhodamine B-labeled phosphatidylethanolamine) and by derivatization with trinitobenzenesulfonic acid (TNBS). These techniques revealed that the exchangeable pool (the fraction of the NBD-PS on the outer leaflet) and the respective fraction of Tnp-(NBD-PS) formed were equivalent to the extent of PS-decarboxylase can be used to generate asymmetric vesicles (i.e., PS inside, PE outside) and determine the intrabilayer distribution of PS in model membranes.