PURIFICATION AND CHARACTERIZATION OF MICROSOMAL TRIGLYCERIDE AND CHOLESTERYL ESTER TRANSFER PROTEIN FROM BOVINE LIVER-MICROSOMES
PURIFICATION AND CHARACTERIZATION OF MICROSOMAL TRIGLYCERIDE AND CHOLESTERYL ESTER TRANSFER PROTEIN FROM BOVINE LIVER-MICROSOMES
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DOI：
10.1016/0009-3084(85)90068-4
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发表时间：
1985-01-01
影响因子：
3.4
通讯作者：
ZILVERSMIT, DB
中科院分区：
文献类型：
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作者：
WETTERAU, JR;ZILVERSMIT, DB
从牛肝中分离到一种脂转移蛋白。从肝微粒体中释放可溶性蛋白后，通过DEAE-纤维素离子交换的组合将转移蛋白纯化75倍至接近均一。Sephadex G-200凝胶渗透和羟基磷灰石色谱。约7%的原始活性被恢复。纯化的馏分促进甘油三酯，胆固醇酯，磷脂酰胆碱和磷脂酰乙醇胺的转移。当比较脂质转移的分数速率时，非极性脂质的转移比磷脂快10倍以上。纯化的转移复合物含有小于5%的脂质。未检测到碳水化合物。纯化蛋白在非变性条件下在聚丙烯酰胺凝胶上电泳显示单一条带。从未染色的凝胶切片中洗脱的蛋白质显示脂质转移活性与染色凝胶上的蛋白质条带的位置一致。纯化后的蛋白质在SDS存在下电泳时，可观察到两条带，占染色密度的95%以上，分子量分别为58000和88000。纯化的转移蛋白从Sephadex G-200柱在对应于分子量为220 000的蛋白质的位置洗脱，其可能代表两种或多种多肽的复合物。纯化的转移蛋白被激活，通过增加NaCl浓度高达约100 mM。在较高的NaCl浓度的转移活性下降。在pH 7下观察到最大转移活性。通过加热至50 ℃以上使蛋白质失活。C.通过在20 ℃和25 ℃之间改变测定温度，转移速率没有显著增加。C和50度。C.这些转移蛋白的活性特征是相同的甘油三酯或胆固醇酯转移活动是否进行了测量。
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 as 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.degree. C. The transfer rates were not greatly increased by changing the assay temperatures between 20.degree. C and 50.degree. C. These activity characteristics of the transfer protein were the same whether triglyceride or cholesteryl ester transfer activities were measured.