Farnsworth C C, Wolda S L, Gelb M H, Glomset J A
Howard Hughes Medical Institute Laboratory, University of Washington, Seattle 98195.
J Biol Chem. 1989 Dec 5;264(34):20422-9.
We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative's structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B.
我们最近发现，HeLa细胞的核纤层蛋白B被一种甲羟戊酸衍生物修饰。在此，我们鉴定了被修饰的氨基酸，确定了其与甲羟戊酸衍生物的连接方式，并确定了该衍生物的结构。一个半胱氨酸残基被修饰，因为用[3H]甲羟戊酸或[35S]半胱氨酸进行生物合成标记，然后用蛋白酶进行广泛消化的核纤层蛋白B实验产生了在五个连续系统中共同色谱分离的3H或35S标记产物。涉及的是硫醚键而不是硫酯键，因为甲羟戊酸衍生物可以通过用雷尼镍处理而不是用甲醇氢氧化钾处理以戊烷可萃取的形式从3H标记产物中释放出来。该衍生物是一个法尼基部分，因为通过气相色谱和质谱联用，雷尼镍释放的物质被鉴定为2,6,10-三甲基-2,6,10-十二碳三烯。硫醚修饰的半胱氨酸残基似乎位于核纤层蛋白B的羧基末端附近，因为用溴化氰处理3H标记的核纤层蛋白B产生了一个单一的标记多肽，该多肽定位于人核纤层蛋白B的cDNA推断序列的该末端。
