Huang G, Lee D M, Singh S
Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Biochemistry. 1988 Mar 8;27(5):1395-400. doi: 10.1021/bi00405a001.
Human plasma low-density lipoproteins of 1.032-1.043 g/mL density were totally delipidized. The reduced and carboxymethylated apolipoprotein B was incubated with 50 mM [14C]methylamine at pH 8.5 at 30 degrees C. Covalent incorporation of [14C]methylamine was observed with concomitant generation of new sulfhydryl groups, which could be blocked with [3H]- or [14C]iodoacetic acid. One type of the [14C]methylamine-modified products was separated from the protein and was found to be lipid in nature. Its Rf on thin-layer chromatography (TLC) was similar to that of the synthetic N-methyl fatty acyl amides. After purification with TLC and transesterification in 3 N methanolic HCl, methyl esters of C16 and C18 fatty acids at 1:1 ratio were identified by gas-liquid chromatography. The transesterification method was verified with the known N-methyl fatty acyl amides. These results suggest the presence of labile thiol ester linked palmitate and stearate in apolipoprotein B. Under mild alkaline conditions, the thiol ester bonds are broken by methylamine and form N-methyl fatty acyl amides and release new-SH groups. Intramolecular thiol ester bonds linked between cysteine side chains and acidic amino acid residues were also found present, which will be reported separately.
对密度为1.032 - 1.043 g/mL的人血浆低密度脂蛋白进行了完全脱脂处理。将还原并羧甲基化的载脂蛋白B与50 mM [14C]甲胺在pH 8.5、30℃条件下孵育。观察到[14C]甲胺的共价掺入，并伴随新巯基的产生，这些新巯基可用[3H]-或[14C]碘乙酸阻断。一种[14C]甲胺修饰产物从蛋白质中分离出来，发现其本质为脂质。其在薄层色谱（TLC）上的Rf值与合成的N-甲基脂肪酰酰胺相似。经TLC纯化并在3 N甲醇盐酸中进行酯交换反应后，通过气液色谱法鉴定出比例为1:1的C16和C18脂肪酸甲酯。用已知的N-甲基脂肪酰酰胺对酯交换方法进行了验证。这些结果表明载脂蛋白B中存在不稳定的硫酯连接的棕榈酸酯和硬脂酸酯。在温和碱性条件下，硫酯键被甲胺断裂，形成N-甲基脂肪酰酰胺并释放新的-SH基团。还发现存在连接在半胱氨酸侧链和酸性氨基酸残基之间的分子内硫酯键，这将另行报道。
