Abstract
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 (∼0.01 pmol). A linear response has been established over a range of 5-1000 pg (∼0.01-2 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 51-59 |
| Number of pages | 9 |
| Journal | Analytical Biochemistry |
| Volume | 336 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2005 |
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Keywords
- Farnesyl pyrophosphate
- Fluorescence detection
- Geranylgeranyl pyrophosphate
- High-performance liquid chromatography
- Isoprenoid pyrophosphate
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells. / Tong, Huaxiang; Holstein, Sarah A.; Hohl, Raymond J.
In: Analytical Biochemistry, Vol. 336, No. 1, 01.01.2005, p. 51-59.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells
AU - Tong, Huaxiang
AU - Holstein, Sarah A.
AU - Hohl, Raymond J.
PY - 2005/1/1
Y1 - 2005/1/1
N2 - 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 (∼0.01 pmol). A linear response has been established over a range of 5-1000 pg (∼0.01-2 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.
AB - 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 (∼0.01 pmol). A linear response has been established over a range of 5-1000 pg (∼0.01-2 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.
KW - Farnesyl pyrophosphate
KW - Fluorescence detection
KW - Geranylgeranyl pyrophosphate
KW - High-performance liquid chromatography
KW - Isoprenoid pyrophosphate
UR - http://www.scopus.com/inward/record.url?scp=9944233982&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=9944233982&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2004.09.024
DO - 10.1016/j.ab.2004.09.024
M3 - Article
C2 - 15582558
AN - SCOPUS:9944233982
VL - 336
SP - 51
EP - 59
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -