Bick R J, Youker K A, Pownall H J, Van Winkle W B, Entman M L
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Arch Biochem Biophys. 1991 May 1;286(2):346-52. doi: 10.1016/0003-9861(91)90050-s.
When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.
当用非离子去污剂C12E8（八乙二醇十二烷基醚）剥离快肌骨骼肌囊泡（SR）和纯化的钙泵蛋白时，并非所有膜磷脂都从钙泵蛋白上被去除。最大程度的提取会产生每摩尔钙ATP酶（CaATPase）残留6 - 8摩尔磷脂。与天然SR和预剥离的纯化CaATPase相比，在这两种制剂中，剩余的磷脂显著富含磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）；剩余的脂质也富含主要为不饱和的磷脂。此外，几乎所有相关的PE都是缩醛磷脂（96%，而天然SR中为63%）。利用氨基特异性交联试剂DFDNB（1,5 - 二氟 - 2,4 - 二硝基苯磺酸）和氨基结合试剂TNBS（2,4,6 - 三硝基苯磺酸）来确定这些磷脂所在的天然制剂的单层，并确定去污剂处理后哪些磷脂与钙泵蛋白紧密相关。这些研究表明，PE和PS与泵蛋白紧密相关，PE几乎只存在于SR的外层单分子层，而PS存在于内层单分子层。非特异性磷脂交换蛋白能够将供体囊泡中的磷脂交换到与CaATPase相关的那些磷脂中；用C12E8剥离脂质交换囊泡在头部基团种类方面表现出相同的特异性（即，PE在提取的蛋白质相关部分中显著富集）。结果表明存在特定的蛋白质 - 脂质相互作用，有利于缩醛磷脂氨基磷脂与钙泵蛋白的结合。
