Dergunov Alexander D, Shabrova Elena V, Dobretsov Gennady E
National Research Centre for Preventive Medicine, 10, Petroverigsky Street, 101990 Moscow, Russia.
National Research Centre for Preventive Medicine, 10, Petroverigsky Street, 101990 Moscow, Russia.
Arch Biochem Biophys. 2014 Dec 15;564:211-8. doi: 10.1016/j.abb.2014.09.019. Epub 2014 Oct 22.
The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063μMmin(-1)) and catalytic rate constant kcat (0.42s(-1)) together with a similarity of Km (0.9μM CPD) and KI (2.8μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.
研究了重组胆固醇酯转运蛋白（CETP）在重构盘状高密度脂蛋白（rHDL）之间胆固醇酯的转移。供体和受体rHDL中的颗粒分别含有载脂蛋白A-I、不饱和的1-棕榈酰-2-油酰磷脂酰胆碱（POPC）或饱和的二棕榈酰磷脂酰胆碱（DPPC）以及胆固醇酯，胆固醇酯分别为胆固醇1-芘癸酸酯（CPD）或胆固醇月桂酸酯（CL）。探针动力学符合猝灭作用球模型。供体和受体颗粒之间的胆固醇酯交换具有非均相动力学特征；与DPPC复合物相比，在POPC情况下快速交换的CPD池要高得多。CETP可接近的探针分数随温度升高而增加，表明探针分布更均匀。观察到受体颗粒对探针转移的非竞争性抑制。对于含POPC的rHDL，Vmax（0.063μM·min⁻¹）和催化速率常数kcat（0.42s⁻¹）的值以及Km（0.9μM CPD）和KI（2.8μM CL）值的相似性表明，体内新生HDL与不饱和磷脂酰胆碱之间存在有效的胆固醇酯转移。盘状HDL中的磷脂基质可能通过胆固醇酯在双层中的自缔合、扩散性和位置、胆固醇酯对载脂蛋白A-I结构中胆固醇结合位点的可及性以及载脂蛋白A-I可定位的胆固醇酯与CETP的结合来构成CETP活性的基础。
