Connolly D T, McIntyre J, Heuvelman D, Remsen E E, McKinnie R E, Vu L, Melton M, Monsell R, Krul E S, Glenn K
Cardiovascular Diseases Research Department, Searie, St. Louis, Missouri 63167, USA.
Biochem J. 1996 Nov 15;320 ( Pt 1)(Pt 1):39-47. doi: 10.1042/bj3200039.
Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.
胆固醇酯转运蛋白（CETP）介导血浆中脂蛋白之间甘油三酯（TGs）、胆固醇酯（CEs）和磷脂（PLs）的交换。为了更好地理解脂质转运过程，我们使用了在培养的哺乳动物细胞中表达的重组人CETP，通过免疫亲和色谱法纯化至同质。通过沉降平衡测定，纯化的重组CETP的重均相对分子质量（MW）为69561，比吸收系数为0.83升·克⁻¹·厘米⁻¹。通过动态光散射测定，该蛋白相应的流体动力学直径（Dh）为14纳米，几乎是这种分子量的球状蛋白预期值的两倍。这些数据表明CETP在溶液中具有非球状形状。通过圆二色性估计CETP的二级结构包含32%的α-螺旋、35%的β-折叠、17%的转角和16%的无规卷曲。与血浆中的天然蛋白一样，重组蛋白由几种糖型组成，使用N-糖苷酶F只能部分去糖基化。对CETP进行有机萃取后进行薄层层析显示，CE、未酯化胆固醇（UC）、PL、TG和脂肪酸（FA）与纯蛋白相关。定量分析证实，每摩尔CETP含有1.0摩尔胆固醇、0.5摩尔TG和1.3摩尔PL。CETP介导了CE、TG、PL和UC在脂蛋白之间或无蛋白脂质体之间的转运。在双标记转运实验中，发现CE或TG从高密度脂蛋白（HDL）向低密度脂蛋白（LDL）的转运速率与供体HDL颗粒中各自配体的初始浓度成正比。CE转运的动力学分析与载体机制一致，对LDL颗粒的Km为700 nM，对HDL颗粒的Km为2000 nM，kcat为2秒⁻¹。因此，每个底物的Km值处于正常生理浓度的低范围。在“半反应”实验中，独立验证了CE、TG、PL和UC的载体机制。
