Holmquist L
King Gustaf V Research Institute, Karolinska Insitute, Stockholm, Sweden.
J Biochem Biophys Methods. 1989 Jul;19(1):93-103. doi: 10.1016/0165-022x(89)90054-7.
A high performance gel filtration method for the rapid and reproducible separation of free and apolipoprotein D-associated lecithin: cholesterol acyltransferase (LCAT) originating from human plasma has been developed. Starting from step 3 of a previously invented covalent chromatography procedure, free LCAT was obtained as a well separated fraction in a yield of 55% of that injected into the column. The free LCAT had a specific activity of over 34,000 units/mg and did not contain apolipoprotein D or any other contaminant in the injected sample. Further 28% of LCAT with fully retained activity was recovered in a second fraction, demonstrating a 66,000 u LCAT associated with all apolipoprotein D occurring as a mean 33,000 u and a minor 66,000 u species and with at least two unidentified proteins with apparent molecular masses of 76,000 u and 43,000 u, respectively. Both free and apolipoprotein D-associated LCAT accepted the free cholesterol of heat-inactivated plasma selectively depleted of VLDL and LDL (alpha-LCAT activity) and of HDL (beta-LCAT activity) as substrate.
已开发出一种高效凝胶过滤方法，用于快速且可重复地分离源自人血浆的游离卵磷脂胆固醇酰基转移酶（LCAT）和与载脂蛋白D相关的LCAT。从先前发明的共价色谱法的第3步开始，游离LCAT作为一个分离良好的级分获得，产率为注入柱中量的55%。游离LCAT的比活性超过34,000单位/毫克，且在所注入样品中不含有载脂蛋白D或任何其他污染物。在第二个级分中又回收了28%具有完全保留活性的LCAT，表明与所有载脂蛋白D相关的66,000单位LCAT以平均33,000单位和少量66,000单位的形式存在，并且与至少两种表观分子量分别为76,000单位和43,000单位的未鉴定蛋白质相关。游离的和与载脂蛋白D相关的LCAT均选择性地接受经热灭活的、VLDL和LDL（α-LCAT活性）以及HDL（β-LCAT活性）被选择性去除的血浆中的游离胆固醇作为底物。
