The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis

Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada
Electrophoresis (Impact Factor: 3.03). 04/2008; 29(7):1484 - 1489. DOI: 10.1002/elps.200700697
Source: PubMed


Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of γ-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival.

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    • "Ketoacids can detoxify ROS with the concomitant formation of the corresponding carboxylic acid and CO 2 . For instance, when ␣KG participates as an ROS scavenger, succinate is produced (Lemire and Appanna 2011; Mailloux et al. 2008). The involvement of pyruvate and oxaloacetate as antioxidants helps liberate acetate and malonate respectively (Campos et al. 2012; Ramsey et al. 2014). "
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    • "2.2. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) and in-gel activity assays BN-PAGE was performed as described in Mailloux et al. (2008) and Schagger and von Jagow (1991). A gradient gel (4–16%) was used for these assays. "
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    • "Blue Native polyacrylamide gel electrophoresis (BN PAGE) and in-gel activity stains were performed as described previously [16]. Membrane proteins were prepared in blue native (BN) buffer (50 mM Bis-Tris, 500 mM 6-aminohexanoic acid, pH 7.0, 4°C) and 1% β-dodecyl-D-maltoside. "
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