Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue.
Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at -80 degrees C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355+/-0.030 and 0.827+/-0.082 units of nmol/g wet tissues in brain, 0.320+/-0.019 and 0.293+/-0.035 units of nmol/g wet tissues in kidney, 0.326+/-0.064 and 0.213+/-0.029 units of nmol/g wet tissues in liver, and 0.364+/-0.015 and 0.349+/-0.023 units of nmol/g wet tissues in heart (means+/-SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.
Tong
Huaxiang
H
Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.
Wiemer
Andrew J
AJ
Neighbors
Jeffrey D
JD
Hohl
Raymond J
RJ
D000818:Animals; D002138:Calibration; D004164:Diphosphonates / Q000494:pharmacology; D004224:Diterpenes / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D020014:K562 Cells; D008297:Male; D051379:Mice; D041681:NIH 3T3 Cells; D009928:Organ Specificity / Q000187:drug effects; D011106:Polyisoprenyl Phosphates / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D015203:Reproducibility of Results; D012717:Sesquiterpenes / Q000032:analysis* / Q000302:isolation & purification; D052616:Solid Phase Extraction; D013729:Terpenes / Q000494:pharmacology
D004164:Diphosphonates; D004224:Diterpenes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D013729:Terpenes; C518651:digeranyl bisphosphonate; C004808:farnesyl pyrophosphate; C002963:geranylgeranyl pyrophosphate