Gromov P S, Madsen P, Celis J E
Department of Medical Biochemistry, Aarhus University, Denmark.
Electrophoresis. 1996 Nov;17(11):1728-33. doi: 10.1002/elps.1150171110.
Here we describe a direct approach for two-dimensional (2-D) gel mapping of proteins that are modified by post-translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS-1 cells were metabolically labeled with either [3H]farnesyl-pyrophosphate or [3H]geranylgeranyl-pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2-D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5-diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl-labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2-D autoradiograms. Several [3H]farnesyl-labeled proteins exhibited the same coordinates (M(r) and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.
在此，我们描述了一种直接方法，用于对哺乳动物细胞中经翻译后异戊二烯化修饰的蛋白质进行二维（2-D）凝胶图谱分析。简要地说，在用洛伐他汀处理以阻断甲羟戊酸合成后，将转化的人羊膜细胞（AMA）和转染的COS-1细胞用[3H]法尼基焦磷酸或[3H]香叶基香叶基焦磷酸进行代谢标记。然后通过二维凝胶电泳分离蛋白质，并将其电转移至硝酸纤维素滤膜上。在进行荧光自显影之前，将膜浸入含有10% 2,5-二苯基恶唑的二甲醚中。在二维放射自显影片上鉴定出了40多种[3H]法尼基标记的蛋白质和25多种[3H]香叶基香叶基化的蛋白质。几种[3H]法尼基标记的蛋白质与其[3H]香叶基香叶基化的对应物具有相同的坐标（相对分子质量和等电点），这增加了它们可能是法尼基转移酶和香叶基香叶基转移酶共同底物的可能性。该方法对法尼基化和香叶基香叶基化的蛋白质都具有高分辨率，它可作为一种强大的工具，用于鉴定迄今未知的异戊二烯化蛋白质，以及测定异戊二烯化蛋白质的水平、异戊二烯类修饰的类型和蛋白质异戊二烯基转移酶活性的可能变化。
