Lee D M, Singh S
Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
SAAS Bull Biochem Biotechnol. 1990 Jan;3:74-9.
Intramolecular thiolester bonds in apolipoprotein B (ApoB) were studied using [14C]methylamine (MA) to cleave the thiolester and [3H]- or [14C]iodoacetate (IA) to titrate the newly generated sulfhydryls. Covalent incorporation of [14C]MA and [3H]carboxylmethyl group into the previously carboxymethylated LDL or the reduced and carboxymethylated ApoB was observed and both radioactivities coincided with ApoB-100 band on SDS-polyacrylamide gel electrophoresis. The [14C]MA-labeled ApoB was completely trypsinized and cross-linked to the activated thiol Sepharose 4B beads. The peptides were eluted with DTT and the free -SH groups blocked with IA then separated on FPLC. Two fractions contained [14C]MA. Sequence analyses showed that these labeled peptides contained Cys-51 and Cys-3734, respectively. Evidence suggests that the thiolester is formed between Cys-51 and gamma-Glu-54 for one, and Cys-3734 and beta-Asp-3737 for the other, with Lys and a hydrophobic amino acid, Val/Leu, in between. This is the first evidence for the presence of intramolecular thiolester linkages in ApoB. The presence of high energy, labile thiolester bonds may explain many of the unusual properties of ApoB and LDL.
利用[14C]甲胺（MA）裂解硫酯键以及[3H]-或[14C]碘乙酸盐（IA）滴定新生成的巯基，对载脂蛋白B（ApoB）中的分子内硫酯键进行了研究。观察到[14C]MA和[3H]羧甲基基团共价掺入先前羧甲基化的低密度脂蛋白（LDL）或还原且羧甲基化的ApoB中，并且两种放射性在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上均与ApoB-100条带重合。[14C]MA标记的ApoB完全被胰蛋白酶消化，并与活化的巯基琼脂糖4B珠交联。用二硫苏糖醇（DTT）洗脱肽段，并用IA封闭游离的-SH基团，然后在快速蛋白质液相色谱（FPLC）上进行分离。两个组分含有[14C]MA。序列分析表明，这些标记的肽段分别含有半胱氨酸（Cys）-51和Cys-3734。有证据表明，一个硫酯键在Cys-51与γ-谷氨酸（Glu）-54之间形成，另一个在Cys-3734与β-天冬氨酸（Asp）-3737之间形成，中间夹着赖氨酸以及一个疏水氨基酸缬氨酸（Val）/亮氨酸（Leu）。这是首次证明ApoB中存在分子内硫酯键连接。高能、不稳定硫酯键的存在可能解释了ApoB和LDL的许多异常特性。
