Massey J B, Hickson-Bick D, Via D P, Gotto A M, Pownall H J
Biochim Biophys Acta. 1985 Jun 14;835(1):124-31. doi: 10.1016/0005-2760(85)90038-4.
The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.
利用基于芘基磷脂转移的荧光测定法，研究了人血浆和牛肝磷脂转移蛋白的特异性。该方法先前用于确定磷脂在模型脂蛋白之间自发转移的机制（梅西，J.B.，戈托，A.M.，Jr.和波纳尔，H.J.（1982年）《生物化学》21，3630 - 3636）。芘基磷脂的头部基团部分有所不同；芘基磷脂酰胆碱在sn - 1位含有不同的脂肪酰链。由载脂蛋白A - I和1 - 棕榈酰 - 2 - 油酰磷脂酰胆碱（POPC）组成的模型高密度脂蛋白（R - HDL）用作供体和受体颗粒。如先前所示，牛肝蛋白仅介导磷脂酰胆碱的转移。相比之下，人血浆蛋白转移了所研究的所有种类，包括磷脂酰丝氨酸、磷脂酰胆碱、磷脂酰甘油、磷脂酰乙醇胺、磷脂酸、鞘磷脂、半乳糖脑苷脂和二酰基甘油。这些转移蛋白的活性仅受转移脂质酰链组成变化的轻微影响。芘基和放射性（[³H]POPC）磷脂被人转移蛋白以相同速率转移，这表明该蛋白对芘基和天然磷脂具有相似的结合特性。自发的磷脂转移通过单体脂质的水相扩散发生，其速率高度依赖于脂肪酰链组成。在本研究中，未发现自发转移速率与蛋白介导的转移之间存在相关性。当用作受体时，基于受体颗粒数量，R - HDL和低密度脂蛋白（LDL）的表观Km值相似。牛肝蛋白对R - HDL和LDL的表观Vmax相同，但血浆蛋白对R - HDL的Vmax略高。这些结果表明，与牛肝蛋白一样，血浆蛋白作为一种磷脂结合载体，在膜表面之间交换磷脂。用芘基标记的脂质测定脂质转移蛋白比其他当前方法更快且更易于操作，其他方法需要分离供体和受体颗粒，该方法适用于脂质转移蛋白的功能和作用机制研究。
