Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis.
We describe a blue native polyacrylamide gel electrophoretic technique that allows the facile detection, quantitation and purification of three NADPH-producing enzymes. Glucose 6-phosphate dehydrogenase, malic enzyme and NADP-dependent isocitrate dehydrogenase were detected simultaneously. Activity staining based on the formation of NADPH from the respective substrates and the subsequent precipitation of formazan enabled the relative quantitation of enzymatic activities, while Coomassie staining on one-dimensional or two-dimensional gels helped monitor the amount of protein associated with these enzymatic activities. This technique provides a simple and effective route to obtain homogeneous protein for further analyses and also enables the screening of these NADPH-producing enzymes in various cellular systems.
Bériault
Robin
R
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
Chénier
Daniel
D
Singh
Ranji
R
Middaugh
Jeff
J
Mailloux
Ryan
R
Appanna
Vasu
V
D004591:Electrophoresis, Polyacrylamide Gel; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification*; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification*; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D005954:Glucosephosphate Dehydrogenase