A high-performance liquid chromatography-based radiometric assay for sucrose-phosphate synthase and other UDP-glucose requiring enzymes.
A method for product analysis that eliminates a problematic step in the radiometric sucrose-phosphate synthase assay is described. The method uses chromatography on a boronate-derivatized high-performance liquid chromatography column to separate the labeled product, [14C]sucrose phosphate, from unreacted uridine 5'-diphosphate-[14C]glucose (UDP-Glc). Direct separation of these compounds eliminates the need for treatment of the reaction mixtures with alkaline phosphatase, thereby avoiding the problem of high background caused by contaminating phosphodiesterase activity in alkaline phosphatase preparations. The method presented in this paper can be applied to many UDP-Glc requiring enzymes; here we show its use for determining the activities of sucrose-phosphate synthase, sucrose synthase, and uridine diphosphate-glucose pyrophosphorylase in plant extracts.
Salvucci
M E
ME
U.S. Department of Agriculture, University of Kentucky, Lexington 40546-0076.
Crafts-Brandner
S J
SJ
D001897:Boronic Acids; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D010944:Plants / Q000201:enzymology; D011874:Radiometry / Q000379:methods; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000378:metabolism
D001897:Boronic Acids; D005964:Glucosyltransferases; C022699:sucrose synthase; C022698:sucrose-phosphate synthase; C059055:N-acetylglucosaminyldiphosphoundecaprenol glucosyltransferase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase