Determination of the starch-phosphorylating enzyme activity in plant extracts.
For quantification of alpha-glucan, water dikinase (GWD) activity in crude extracts of plant tissues a radio-labeling assay was established that uses soluble starch and (33)P-labeled ATP as phosphate acceptor and donor, respectively. A constant rate of starch labeling was observed only if the ATP applied was labeled at the beta position. In wild-type extracts from leaves of Arabidopsis thaliana (L.) Heynh. the maximum rate of starch phosphorylation was approximately 27 pmol min(-1) (mg protein)(-1). Leaf extracts from the GWD-deficient sex1 mutants of Arabidopsis showed no significant incorporation of phosphate whereas extracts from potato (Solanum tuberosum L.) tuber expressing a GWD antisense construct exhibited less activity than the wild-type control. To our knowledge this is the first time that a quantification of the starch-phosphorylating activity has been achieved in plant crude extracts.
Ritte
Gerhard
G
Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25 Haus 20, 14476 Golm, Germany.
Steup
Martin
M
Kossmann
Jens
J
Lloyd
James R
JR
D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism; D005936:Glucans / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010936:Plant Extracts / Q000302:isolation & purification / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D011198:Solanum tuberosum / Q000201:enzymology*; D013213:Starch / Q000378:metabolism*; D024941:Starch Phosphorylase / Q000378:metabolism*
D000255:Adenosine Triphosphate; D029681:Arabidopsis Proteins; D005936:Glucans; D009004:Monosaccharide Transport Proteins; D010761:Phosphorus Radioisotopes; D010936:Plant Extracts; D013213:Starch; D024941:Starch Phosphorylase; C435922:At1g10760 protein, Arabidopsis