Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.
We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.
Singh
R
R
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada P3E 2C6.
Chénier
D
D
Bériault
R
R
Mailloux
R
R
Hamel
R D
RD
Appanna
V D
VD
D001219:Aspartate Aminotransferases / Q000378:metabolism; D045744:Cell Line, Tumor; D003116:Color; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism*; D005649:Fumarate Hydratase / Q000378:metabolism; D006801:Humans; D008292:Malate Synthase / Q000378:metabolism; D008293:Malates / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D062907:Oxaloacetic Acid / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism
D004798:Enzymes; D008293:Malates; D009097:Multienzyme Complexes; D062907:Oxaloacetic Acid; C030298:malic acid; D008292:Malate Synthase; D001219:Aspartate Aminotransferases; D010730:Phosphoenolpyruvate Carboxylase; D007652:Oxo-Acid-Lyases; C019909:citrate (pro-3S)-lyase; D005649:Fumarate Hydratase