Wetterau J R, Zilversmit D B
Chem Phys Lipids. 1985 Aug 30;38(1-2):205-22. doi: 10.1016/0009-3084(85)90068-4.
A lipid transfer protein was isolated from bovine liver. Following the release of soluble proteins from liver microsomes, the transfer protein was purified 75-fold to near homogeneity by a combination of DEAE-cellulose ion exchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. About 7% of the original activity was recovered. The purified fraction promoted the transfer of triglyceride, cholesteryl ester, phosphatidylcholine, and phosphatidylethanolamine. When the fractional rates of lipid transfer were compared, the transfer of apolar lipids was over 10 times faster than that of phospholipid. The purified transfer complex contained less than 5% lipid. No carbohydrate was detected. Electrophoresis of the purified protein on polyacrylamide gels under non-denaturing conditions showed a single band. Elution of protein from slices of unstained gels showed that lipid transfer activities coincided with the position of the protein band on the stained gel. When the purified protein was electrophoresed in the presence of SDS, two bands, accounting for more than 95% of the staining density, were observed with molecular weights at 58 000 and 88 000. The purified transfer protein eluted from a Sephadex G-200 column at a position corresponding to a protein with a molecular weight of 220 000, which probably represents a complex of two or more polypeptides. The purified transfer protein was activated by increasing NaCl concentrations up to about 100 mM. At higher NaCl concentrations the transfer activity decreased. Maximal transfer activities were observed at pH 7. The protein was inactivated by heating above 50 degrees C. The transfer rates were not greatly increased by changing the assay temperatures between 20 degrees C and 50 degrees C. These activity characteristics of the transfer protein were the same whether triglyceride or cholesteryl ester transfer activities were measured.
从牛肝脏中分离出一种脂质转运蛋白。在从肝脏微粒体中释放出可溶性蛋白质后，通过DEAE - 纤维素离子交换、Sephadex G - 200凝胶渗透和羟基磷灰石色谱法相结合的方法，将转运蛋白纯化了75倍，达到近乎均一的程度。回收了约7%的原始活性。纯化后的组分促进了甘油三酯、胆固醇酯、磷脂酰胆碱和磷脂酰乙醇胺的转运。当比较脂质转运的分数速率时，非极性脂质的转运速度比磷脂快10倍以上。纯化的转运复合物含有的脂质少于5%。未检测到碳水化合物。在非变性条件下，将纯化的蛋白质在聚丙烯酰胺凝胶上进行电泳，显示出一条单一的条带。从未染色凝胶切片上洗脱蛋白质表明，脂质转运活性与染色凝胶上蛋白质条带的位置一致。当在SDS存在下对纯化的蛋白质进行电泳时，观察到两条带，占染色密度的95%以上，分子量分别为58000和88000。从Sephadex G - 200柱上洗脱的纯化转运蛋白在对应于分子量为220000的蛋白质的位置洗脱，这可能代表两种或更多种多肽的复合物。纯化的转运蛋白在NaCl浓度增加至约100 mM时被激活。在更高的NaCl浓度下，转运活性降低。在pH 7时观察到最大转运活性。该蛋白质在50摄氏度以上加热会失活。在20摄氏度至50摄氏度之间改变测定温度，转运速率没有大幅增加。无论测量的是甘油三酯还是胆固醇酯的转运活性，转运蛋白的这些活性特征都是相同的。
