Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells
Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells
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DOI：
10.1016/j.ab.2004.09.024
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发表时间：
2005-01-01
影响因子：
2.9
通讯作者：
Hohl, RJ
中科院分区：
文献类型：
--
作者：
Tong, HX;Holstein, SA;Hohl, RJ
建立了一种灵敏的非放射性分析方法，可同时测定培养细胞中焦磷酸法呢酯（FPP）和焦磷酸香叶基香叶酯（GGPP）的浓度。提取后，进行涉及重组法呢基蛋白转移酶或香叶基香叶基蛋白转移酶I的酶测定，以将FPP或GGPP缀合至丹磺酰化肽。然后通过与荧光检测器偶联的高效液相色谱法在激发波长335 nm和发射波长528 nm下分离和定量反应产物。法呢基肽和香叶基香叶基肽的保留时间分别为8.4和16.9 min。检测下限为5 pg FPP或GGPP（近似于0.01 pmol）。线性范围为5-1000 pg（与0.01 -2 pmol相似），重现性好。该方法已用于测定NIH 3 T3细胞中FPP（0.125 +/- 0.010 pmol/10（6）细胞）和GGPP（0.145 +/- 0.008 pmol/10（6）细胞）的水平。此外，测量了用类异戊二烯生物合成途径抑制剂处理细胞后FPP和GGPP水平的变化。该方法适用于测定任何细胞类型或组织中FPP和GGPP的浓度。(C)2004年爱思唯尔公司All rights reserved.
A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl-peptide and geranylgeranyl-peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP (similar to0.01 pmol). A linear response has been established over a range of 5-1000 pg (similar to0.01-2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125 +/- 0.010 pmol/10(6) cells) and GGPP (0.145 +/- 0.008 pmol/10(6) cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue. (C) 2004 Elsevier Inc. All rights reserved.