Physical-chemical model for the entry of water-insoluble compounds into cells. Studies of fatty acid uptake by the liver.
Physical-chemical model for the entry of water-insoluble compounds into cells. Studies of fatty acid uptake by the liver.
复制标题
水不溶性化合物进入细胞的物理化学模型。
DOI：
10.1021/bi00356a027
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Zakim,D
中科院分区：
文献类型：
--
作者：
Noy,N;Donnelly,TM;Zakim,D
材料和方法棕榈酸酯在脂质双层和白蛋白之间的平衡分布。采用Backer和Dawidowicz（1979）的方法测定了棕榈酸酯在卵磷脂和白蛋白单层囊泡之间的平衡分布。将在10mM三（羟甲基）-氨基甲烷（Tris）、100mM KCl和1mM抗坏血酸盐（pH 7.4）中的牛血清白蛋白（0.2mM）（Sigma，基本上不含脂肪酸）与棕榈酸酯以6：1的摩尔比（棕榈酸酯：白蛋白比）混合。包括痕量的[14 C]棕榈酸（阿默舍姆）。用0.2 mM白蛋白稀释该溶液，以获得各种棕榈酸酯：白蛋白比例。通过超声处理从卵磷脂酰胆碱（Sigma，III-E型，在己烷中）制备囊泡。它们含有10%（w/w）A ′-棕榈酰二氢乳糖苷（Sigma）。然后对脂质进行超声处理以获得澄清悬浮液，将其以10000g离心15分钟以沉淀未分散的物质。将白蛋白-棕榈酸酯的囊泡和复合物在37 ℃下孵育1小时。孵育后，将混合物样品转移到Eppendorf管中，并向其中加入0.3 mg凝集素（RCA 120，Vector Laboratories，Burlingame，CA）。剧烈混合混合物，在37 ℃下保持10分钟，离心10分钟。然后对上清液的等分试样（200 μ L）进行[14C]棕榈酸盐计数。为了校正未沉淀的囊泡，制备含有痕量[14 C]二棕榈酰磷脂酰胆碱（阿默舍姆）和β-D-糖苷的囊泡，用0.2 mM白蛋白溶液孵育，并如所述通过凝集素沉淀，对200 µ上清液中未沉淀的脂质进行计数。在每种棕榈酸酯：白蛋白比例下，计算与白蛋白（上清液中）和脂质囊泡（沉淀中）结合的棕榈酸酯的量，并根据棕榈酸酯（囊泡中）的摩尔数/脂质的摩尔数计算平衡常数。
Materials and Methods Equilibrium Distribution of Palmitate between Lipid Bi-layers and Albumin. The equilibrium distribution of palmitate between unilamellar vesicles of egg phosphatidylcholine and albumin was determined by the method of Backer and Dawidowicz (1979). Bovine serum albumin (0.2 mM)(Sigma, essentially fatty acid free) in 10 mM tris (hydroxymethyl)-aminomethane (Tris), 100 mM KC1, and 1 mM ascorbate, pH 7.4, was mixed with palmitate at a mole ratio of 6: 1 (palmitate: albumin ratio). A trace amount of [14C] palmitic acid (Amersham) was included. This solution was diluted with 0.2 mM albumin to obtain various palmitate: albumin ratios. Vesicles were prepared from egg phosphatidylcholine (Sigma, type III-E in hexane) by sonication. They contained 10%(w/w) A'-palmitoyldihydrolactocerebroside (Sigma). The lipids then were sonicated to obtain a clear suspension that was centrifuged at lOOOOOg for 15 min in order to sediment undispersed material. Vesicles and complexes of albumin-palmitate were incubated at 37 C for 1 h. Following the incubation, a sample of the mixture was transferred to an Eppendorf tube, and 0.3 mg of lectin (RCA 120, Vector Laboratories, Burlingame, CA) was added to it. The mixture was mixed vigorously, keptat 37 C for 10 min, and centri-fuged for 10 min. Aliquots (200 qL) of the supernatant were then counted for [14C] palmitate. To correct for unprecipitated vesicles, vesicles that contained trace amounts of [14C] di-palmitoylphosphatidylcholine (Amersham) and cerebrosides were prepared, incubated with 0.2 mM albumin solution, and precipitated by lectins as described, and 200 µ of supernatant was counted for unprecipitated lipids. Theamounts of pal-mitate boundto albumin (in the supernatant) and to the lipid vesicles (in the pellet) were calculated at each palmitate: albumin ratio, and the equilibrium constant was calculated from the expression mol of palmitate (in vesicles)/mol of lipid