Jonas A, Wald J H, Toohill K L, Krul E S, Kézdy K E
Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801.
J Biol Chem. 1990 Dec 25;265(36):22123-9.
We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.
我们制备了纯形式的球形重组高密度脂蛋白（rHDL）颗粒，并将其与均一的盘状rHDL前体在载脂蛋白A-I（apoA-I）组分的结构和稳定性、表面脂质的动力学以及与卵磷脂胆固醇酰基转移酶的相对反应性方面进行了比较。通过圆二色性和荧光光谱法以及针对apoA-I表位的单克隆抗体的结合来检测rHDL颗粒中的apoA-I结构。通过盐酸胍对载脂蛋白最大固有荧光波长的影响来评估apoA-I在rHDL颗粒上的稳定性。利用荧光探针研究了酰基链区域的脂质动力学和脂质-水界面的极性。发现球形93-A rHDL颗粒中apoA-I的构象与96-A rHDL盘状颗粒中的非常相似，但与78-A rHDL盘状颗粒中的apoA-I结构不同。apoA-I对盐酸胍变性的稳定性在93-A rHDL球体中最高。脂质实验表明，相对于盘状颗粒，球体中的酰基链更有序且运动受限，但表面极性相似。这些结果表明，apoA-I在这三种颗粒上的折叠和组织包括由每个apoA-I分子羧基末端区域中相互作用的α-螺旋片段和氨基末端区域中的球状结构域组成的蛋白质结构域。与卵磷脂胆固醇酰基转移酶的反应性在96-A rHDL盘状颗粒中最高，在78-A rHDL盘状颗粒和93-A rHDL球体中分别低16倍和34倍，这可能是由于这些颗粒中apoA-I结构的差异和产物抑制所致。
