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Nonradioactive Assay for Cellular Dimethylallyl Diphosphate by Alison J. Fisher; Todd N. Rosenstiel; Megan C. Shirk; Ray Fall is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Nonradioactive Assay for Cellular Dimethylallyl Diphosphate about?

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 [

Who reads Nonradioactive Assay for Cellular Dimethylallyl Diphosphate?

Author

Alison J. Fisher; Todd N. Rosenstiel; Megan C. Shirk; Ray Fall

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2001

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)