Document Detail


Quantitative determination of geranyl diphosphate levels in cultured human cells.
MedLine Citation:
PMID:  19856009     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.
Authors:
Sarah A Holstein; Huaxiang Tong; Craig H Kuder; Raymond J Hohl
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2009-10-24
Journal Detail:
Title:  Lipids     Volume:  44     ISSN:  1558-9307     ISO Abbreviation:  Lipids     Publication Date:  2009 Nov 
Date Detail:
Created Date:  2010-01-21     Completed Date:  2010-03-23     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0060450     Medline TA:  Lipids     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  1055-62     Citation Subset:  IM    
Affiliation:
Department of Internal Medicine, SE 313 GH, University of Iowa, Iowa City, IA, 52242, USA.
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MeSH Terms
Descriptor/Qualifier:
Cells, Cultured
Diphosphates / analysis*
Diterpenes / analysis*
Humans
K562 Cells
Limit of Detection
Polyisoprenyl Phosphates / analysis
Sesquiterpenes / analysis
Chemical
Reg. No./Substance:
0/Diphosphates; 0/Diterpenes; 0/Polyisoprenyl Phosphates; 0/Sesquiterpenes; 0/geranyl diphosphate; 13058-04-3/farnesyl pyrophosphate; 6699-20-3/geranylgeranyl pyrophosphate

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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