A capillary zone electrophoresis assay for the nucleoside transfer enzyme adenosine diphosphate-glucose pyrophosphorylase.
Conditions are described for a capillary zone electrophoresis-based assay of ADP-glucose pyrophosphorylase, the first enzyme in the de novo synthesis for alpha-glucan synthesis in plants and bacteria. ATP, ADP-glucose, and other adenine derivatives are effectively resolved and quantified in 12 min of electrophoresis. The method requires minimal sample manipulations and no postincubation hydrolyses, is effective over a wide range of enzyme and substrate concentrations, has a sensitivity comparable to radioisotope assays, and yields reproducible results in either the forward and reverse directions. Moreover, the assay is applicable to other enzymes that utilize nucleosides or their bases as substrates or products.
Roberts
M W
MW
Institute of Biological Chemistry, Washington State University, Pullman 99163-6340, USA.
Preiss
J
J
Okita
T W
TW
D000227:Adenine Nucleotides / Q000032:analysis; D000245:Adenosine Diphosphate Glucose / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D001419:Bacteria / Q000201:enzymology; D002197:Capillary Action; D004586:Electrophoresis / Q000379:methods; D051599:Glucose-1-Phosphate Adenylyltransferase; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D011865:Radioisotope Dilution Technique; D012680:Sensitivity and Specificity; D013997:Time Factors
D000227:Adenine Nucleotides; D000245:Adenosine Diphosphate Glucose; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase