An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase.
Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.
Yep
Alejandra
A
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Bejar
Clarisa M
CM
Ballicora
Miguel A
MA
Dubay
Jennifer R
JR
Iglesias
Alberto A
AA
Preiss
Jack
J
D000245:Adenosine Diphosphate Glucose / Q000032:analysis / Q000096:biosynthesis*; D002250:Carbon Radioisotopes / Q000032:analysis; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000096:biosynthesis; D006006:Glycogen Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results
D002250:Carbon Radioisotopes; D000245:Adenosine Diphosphate Glucose; D006003:Glycogen; D006006:Glycogen Synthase; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase