title: Nonradioactive assay for cellular dimethylallyl diphosphate.
abstract: A sensitive, nonradioactive method was developed to measure cellular levels of dimethylallyl diphosphate (DMAPP), a central intermediate of isoprenoid metabolism in nature. The assay is based on the hydrolysis of DMAPP in acid to the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene), with subsequent analysis of isoprene by headspace gas chromatography with reduction gas detection. In the assay, cell samples are directly acidified with 4 M H(2)SO(4) in sealed reaction vials. Therefore, there is no need to extract metabolites, purify them, and keep them stable prior to analysis, and degradative enzymatic activities are destroyed. DMAPP levels of 23 +/- 4 nmol (g fresh weight)(-1) [ca. 85 nmol (g dry weight)(-1)] and 80 +/- 14 nmol (g fresh weight)(-1) [ca. 296 nmol (g dry weight)(-1)] were measured in dark- and light-adapted leaves of Populus deltoides (Eastern cottonwood), respectively. Evidence is presented to show that DMAPP is the major leaf metabolite giving rise to isoprene following acid hydrolysis. DMAPP levels in Bacillus subtilis and Saccharomyces cerevisiae were determined to be 40.8 +/- 16.7 pmol (OD(600))(-1) [ca. 638 pmol (mg dry weight)(-1)] and 6.3 +/- 3.7 pmol (OD(600))(-1) [ca. 139 pmol (mg dry weight)(-1)], respectively. The method should be suitable for any cell or tissue type and isolated cellular organelles.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Fisher
authors.forename: T N
authors.initials: TN
authors.lastname: Rosenstiel
authors.forename: M C
authors.initials: MC
authors.lastname: Shirk
authors.forename: R
authors.initials: R
authors.lastname: Fall
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry* / Q000378:metabolism; D002070:Butadienes / Q000032:analysis / Q000378:metabolism; D002849:Chromatography, Gas; D002852:Chromatography, Ion Exchange; D045782:Hemiterpenes; D006868:Hydrolysis; D007700:Kinetics; D019684:Magnoliopsida / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000378:metabolism; D010420:Pentanes; D018515:Plant Leaves / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000032:analysis / Q000378:metabolism; D011849:Radioactive Tracers; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D002070:Butadienes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010420:Pentanes; D011106:Polyisoprenyl Phosphates; D011849:Radioactive Tracers; C005059:isoprene; C043060:3,3-dimethylallyl pyrophosphate