Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells

The soluble protein fraction of tobacco bright yellow 2 cells contained ade nosine 3',5'-cyclic monophosphate (cAMP)-binding activity, detected with bo th a conventional binding assay and a surface plasmon resonance biosensor. A cAMP-agarose-based affinity purification procedure yielded three proteins which were identified by mass spectrometry as glyceraldehyde 3-phosphate d ehydrogenase (GAPDH) and two nucleoside diphosphate kinases (NDPKs). This i s the first report describing an interaction between cAMP and these protein s in higher plants. Our findings are discussed in view of the reported role of the interaction of cAMP with GAPDH and NDPK in animals and yeast. In ad dition, we provide a rapid method to isolate both proteins from higher plan ts. (C) 2001 Federation of European Biochemical Societies. Published by Els evier Science B.V. All rights reserved.