Quantitative determination of isopentenyl diphosphate in cultured mammalian cells

Huaxiang Tong, Craig H. Kuder, Brian M. Wasko, Raymond Hohl

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Isopentenyl 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 diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, 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 hydroxymethylglutaryl 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 μM) 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.

Original languageEnglish (US)
Pages (from-to)36-42
Number of pages7
JournalAnalytical Biochemistry
Volume433
Issue number1
DOIs
StatePublished - Feb 1 2013

Fingerprint

Cultured Cells
Cells
zoledronic acid
Lovastatin
Biosynthetic Pathways
Terpenes
MCF-7 Cells
Peptides
Fluorescence
Farnesyltranstransferase
K562 Cells
isopentenyl pyrophosphate
High performance liquid chromatography
Coenzyme A
Transferases
Limit of Detection
Oxidoreductases
High Pressure Liquid Chromatography
Detectors
Cell Line

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tong, Huaxiang ; Kuder, Craig H. ; Wasko, Brian M. ; Hohl, Raymond. / Quantitative determination of isopentenyl diphosphate in cultured mammalian cells. In: Analytical Biochemistry. 2013 ; Vol. 433, No. 1. pp. 36-42.
@article{708343723d6a407099fa245ec3a6ea4e,
title = "Quantitative determination of isopentenyl diphosphate in cultured mammalian cells",
abstract = "Isopentenyl 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 diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, 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 hydroxymethylglutaryl 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 μM) 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.",
author = "Huaxiang Tong and Kuder, {Craig H.} and Wasko, {Brian M.} and Raymond Hohl",
year = "2013",
month = "2",
day = "1",
doi = "10.1016/j.ab.2012.09.001",
language = "English (US)",
volume = "433",
pages = "36--42",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

Quantitative determination of isopentenyl diphosphate in cultured mammalian cells. / Tong, Huaxiang; Kuder, Craig H.; Wasko, Brian M.; Hohl, Raymond.

In: Analytical Biochemistry, Vol. 433, No. 1, 01.02.2013, p. 36-42.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Quantitative determination of isopentenyl diphosphate in cultured mammalian cells

AU - Tong, Huaxiang

AU - Kuder, Craig H.

AU - Wasko, Brian M.

AU - Hohl, Raymond

PY - 2013/2/1

Y1 - 2013/2/1

N2 - Isopentenyl 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 diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, 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 hydroxymethylglutaryl 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 μM) 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.

AB - Isopentenyl 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 diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, 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 hydroxymethylglutaryl 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 μM) 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.

UR - http://www.scopus.com/inward/record.url?scp=84869880620&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84869880620&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2012.09.001

DO - 10.1016/j.ab.2012.09.001

M3 - Article

VL - 433

SP - 36

EP - 42

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -