Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation.
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.
Ravera
Silvia
S
Department of Biology, University of Genoa, Genova, Italy. silvia.ravera@gmail.com
Calzia
Daniela
D
Panfoli
Isabella
I
Pepe
Isidoro Mario
IM
Morelli
Alessandro
A
D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D000263:Adenylate Kinase / Q000032:analysis*; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis; D008110:Liver Extracts / Q000032:analysis; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D044345:Phosphoric Triester Hydrolases / Q000032:analysis; D012160:Retina / Q000201:enzymology; D012374:Rod Cell Outer Segment / Q000201:enzymology
D007527:Isoenzymes; D008110:Liver Extracts; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D044345:Phosphoric Triester Hydrolases