In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis.
Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.
Mailloux
Ryan J
RJ
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada P3E 2C6.
Singh
Ranji
R
Appanna
Vasu D
VD
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007202:Indicators and Reagents; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013194:Staining and Labeling; D013379:Substrate Specificity
D007202:Indicators and Reagents; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D005954:Glucosephosphate Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase