A high-performance liquid chromatography radio method for determination of L-ascorbic acid and guanosine 5'-diphosphate-l-galactose, key metabolites of the plant vitamin C pathway.
A simple, rapid, and quantitative high-pressure liquid chromatography radio method is described for the determination of in vivo (14)C-labeled l-ascorbate, dehydro-l-ascorbate, and total l-ascorbate of Arabidopsis thaliana cell suspensions upon incubation of cultures with exogenous d-[(14)C]mannose. The same radio-HPLC conditions can be used to follow the products of in vitro enzymatic conversions of GDP-d-mannose by enzyme extracts of A. thaliana, namely GDP-l-galactose, GDP-4"-keto,6"-deoxy-d-mannose, and GDP-l-fucose. In particular, an accurate assay for GDP-d-mannose 3",5"-epimerase, a key enzyme of the plant vitamin C pathway, is presented.
Wolucka
B A
BA
Department of Plant Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, K.L., Ledeganckstraat 35, Gent, B-9000, Belgium. woboe@gengenp.rug.ac.be
Davey
M W
MW
Boerjan
W
W
D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism*; D002250:Carbon Radioisotopes / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D005690:Galactose; D006156:Guanosine Diphosphate Sugars / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D019998:Racemases and Epimerases / Q000378:metabolism*
D002250:Carbon Radioisotopes; D006156:Guanosine Diphosphate Sugars; D019998:Racemases and Epimerases; D001205:Ascorbic Acid; D005690:Galactose