Weinberg R B, Jordan M K, Steinmetz A
Department of Medicine, University of Texas Health Science Center, Houston 77225.
J Biol Chem. 1990 Oct 25;265(30):18372-8.
We have investigated the molecular structure, phospholipid binding, and lecithin-cholesterol acyltransferase catalytic activity of pure apoA-IV-2, a basic variant isoform of apoA-IV which is inherited as a classical Mendelian allele with a gene frequency of 0.09. Circular dichroism spectroscopy established that the alpha-helical content of apoA-IV-2 was 75% in the native state (versus 56% for apoA-IV-1), and increased to 88% in the presence of phospholipid. Fluorescence titration established that apoA-IV-2 bound to egg phospholipid vesicles with a Ka of 3.3 x 10(6) liter/mol, 2.4-fold greater than the affinity of apoA-IV-1. Fluorescence quenching studies revealed that, unlike apoA-IV-1, binding of apoA-IV-2 to phospholipid vesicles induced strong shielding of the amino-terminal tryptophan against iodide quenching. Enzyme kinetic studies using both saturated and unsaturated phospholipid substrates demonstrated that apoA-IV-2 was 36-71% more efficient in activating lecithin-cholesterol acyltransferase than apoA-IV-1. We conclude that apoA-IV-2 has more alpha-helical structure, is more stable in solution, and is more hydrophobic than apoA-IV-1, and that these distinctive structural features are associated with a higher affinity for phospholipid surfaces and an increased catalytic efficiency of lecithin:cholesterol acyltransferase activation. The biophysical basis for this latter characteristic may be the ability of apoA-IV-2 to penetrate phospholipid surfaces to a greater depth than apoA-IV-1. These molecular properties may be responsible for the increased levels of high density lipoproteins which have been observed in apoA-IV-2 heterozygotes.
我们研究了纯载脂蛋白A-IV-2的分子结构、磷脂结合及卵磷脂胆固醇酰基转移酶催化活性。载脂蛋白A-IV-2是载脂蛋白A-IV的一种碱性变异亚型，作为经典孟德尔等位基因遗传，基因频率为0.09。圆二色光谱法确定，载脂蛋白A-IV-2在天然状态下的α-螺旋含量为75%（载脂蛋白A-IV-1为56%），在磷脂存在时增加到88%。荧光滴定确定，载脂蛋白A-IV-2与鸡蛋磷脂囊泡结合的解离常数Ka为3.3×10⁶升/摩尔，比载脂蛋白A-IV-1的亲和力高2.4倍。荧光猝灭研究表明，与载脂蛋白A-IV-1不同，载脂蛋白A-IV-2与磷脂囊泡的结合诱导氨基末端色氨酸对碘化物猝灭产生强烈屏蔽。使用饱和和不饱和磷脂底物的酶动力学研究表明，载脂蛋白A-IV-2在激活卵磷脂胆固醇酰基转移酶方面比载脂蛋白A-IV-1效率高36%-71%。我们得出结论，载脂蛋白A-IV-2比载脂蛋白A-IV-1具有更多的α-螺旋结构，在溶液中更稳定，疏水性更强，并且这些独特的结构特征与对磷脂表面的更高亲和力以及卵磷脂：胆固醇酰基转移酶激活的催化效率提高相关。后一特征的生物物理基础可能是载脂蛋白A-IV-2比载脂蛋白A-IV-1能够更深入地穿透磷脂表面。这些分子特性可能是在载脂蛋白A-IV-2杂合子中观察到的高密度脂蛋白水平升高的原因。
