Quantitative determination of isopentenyl diphosphate in cultured mammalian cells

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dc.contributor.authorWasko, Brian
dc.date.accessioned2021-03-08T19:50:16Z
dc.date.available2021-03-08T19:50:16Z
dc.date.issued2012
dc.description.abstractIsopentenyl diphosphate (IPP), an intermediate of the isoprenoid biosynthetic pathway (IBP), has several important biological functions, yet a method to determine its basal level has not been described. Here, we describe a nonradioactive and sensitive analytical method to isolate and specifically quantify IPP from cultured mammalian cells. This method applies an enzymatic coupling reaction to determine intracellular concentrations of IPP. In this reaction, geranylgeranyl diphosphate synthase catalyzes the formation of geranylgeranyl protein transferase I conjugates GGPP with a fluorescently labeled peptide. The geranylgeranylated peptide can be quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, thereby allowing for IPP quantification. The detection lower limit of the fluorescence-labeled geranylgeranyl peptide is approximately 5 pg (~0.017 pmol). This method was used to examine the effects of IBP inhibitors such as lovastatin and zoledronate on intracellular levels of IPP. Inhibition of hydroxymethylglutarly coenzyme A reductase (HMGCR) by lovastatin (50 nM) decreases IPP levels by 78% and 53% in K562 and MCF-7 cells, respectively. Whereas, zoledronic acid (10 uM) increased IPP levels 12.6-fold when compared with untreated cells in the K562 cell line, an astonishing 960-fold increase was observed in the MCF-7 cells.en_US
dc.identifier.citationTong H, Kuder CH, Wasko BM, and Hohl RJ. Quantitative Determination of Isopentenyl Diphosphate in Cultured Mammalian Cells. Anal Biochem. 2012 Sep 18.en_US
dc.identifier.urihttps://hdl.handle.net/10657.1/2561
dc.publisherAnalytical Biochemistryen_US
dc.subjectFarnesyltranstransferase / metabolism Hemiterpenes / analysis* Hemiterpenes / metabolism Humans K562 Cells MCF-7 Cells Organophosphorus Compounds / analysis* Organophosphorus Compounds / metabolism Polyisoprenyl Phosphates / metabolism Reproducibility of Results Terpenes / metabolismen_US
dc.titleQuantitative determination of isopentenyl diphosphate in cultured mammalian cellsen_US
dc.typeArticleen_US

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Brian Wasko