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Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation
Author(s) -
Ravera Silvia,
Calzia Daniela,
Panfoli Isabella,
Pepe Isidoro Mario,
Morelli Alessandro
Publication year - 2007
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.200600353
Subject(s) - adenylate kinase , chemistry , polyacrylamide gel electrophoresis , biochemistry , electrophoresis , enzyme , isozyme , kinase , chromatography , gel electrophoresis , gene isoform , fluorescence , phosphotransferases , gene , physics , quantum mechanics
Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark‐blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long‐wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.