The phospholipid headgroup specificity of an ATP-dependent calcium pump.
The phospholipid headgroup specificity of an ATP-dependent calcium pump.
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ATP 依赖性钙泵的磷脂头基特异性。
DOI：
10.1016/0005-2736(78)90201-8
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发表时间：
1978
期刊：
影响因子：
--
通讯作者：
G. B. Warren
中科院分区：
文献类型：
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作者：
J. P. Bennett;Gerry A. Smith;M. Houslay;T. Hesketh;J. Metcalfe;G. B. Warren
我们用一系列确定的合成二油酰磷脂取代了与纯化的钙转运蛋白相关的脂质，以确定磷脂头基结构对蛋白质ATP酶活性的影响。在37°C下，两性离子磷脂（二油酰磷脂酰胆碱和二油酰磷脂酰乙醇胺）支持最高活性，而具有两个负电荷的磷脂（二油酰磷脂酸）支持至少低二十倍的活性。具有单个净负电荷的二油酰磷脂支持中间ATP酶活性，其不受磷脂头基的精确化学结构的影响。用于测定钙转运蛋白的磷脂头基特异性的方案是新颖的，因为它确定了与蛋白质接触的脂质的组成，而不需要分离确定的脂质-蛋白质复合物。我们还测定了相同磷脂在重构膜中支持钙积累的能力。必须满足两个要求。磷脂必须支持泵蛋白的ATP酶活性，并且必须形成密封的囊泡，如通过电子显微镜测定的。由于许多磷脂符合这些要求，很明显，在体外的脂质特异性的钙积累系统是相当广泛的。
We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37°C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids.We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.