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The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis
Author(s) -
Mailloux Ryan J.,
Darwich Rami,
Lemire Joseph,
Appanna Vasu
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700697
Subject(s) - nucleoside diphosphate kinase , chemistry , biochemistry , polyacrylamide gel electrophoresis , nucleotide , gel electrophoresis , enzyme , microbiology and biotechnology , chromatography , biology , gene
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.