Studies concerning the identity of the so-called 72 kDa NADP-malic enzyme

MARÍA VALERIA LARA; GABRIELA LETICIA MÜLLER; VERÓNICA MAURINO; MARIANA SAIGO; FEDERICO BOLOGNA; PAULA CASATI; CARLOS ANDREO; MARÍA FABIANA DRINCOVICH

Montreal, Canadá

Congreso; XIII International Congress of Photosynthesis; 2004

Different isoforms of NADP-malic enzyme (NADP-ME) are involved in a wide range of pathways, being the most abundant the C4-enzyme. Different batches of antibodies against purified maize leaves 62 kDa NADP-malic enzyme (NADP-ME) cross-react with a 72 kDa protein from different tissues of several plants. A 72 kDa protein with low NADP-ME activity was also purified from several species. Thus, this 72 kDa-protein was pointed out as a non-photosynthetic NADP-ME. Here, we present evidence suggesting that the 72 kDa protein is not a NADP-ME. i) A cDNA supposed to encode for this NADP-ME in maize roots, codifies in fact for a novel highly active 66 kDa NADP-ME. ii) After a NADP-ME purification procedure from maize roots followed by activity, a 72 kDa protein is obtained which cross-reacts with antibodies against purified maize leaves NADP-ME. Nevertheless, 72 kDa purified protein MS-MS assay shows best match of the peptides obtained with those of Heat Shock Protein (Hsp70). iii) By screening a cDNA tobacco library with antibodies raised against 62 kDa purified maize NADP-ME; the insets of the clones obtained present 82-92% identity to Hsp70 mRNAs (AY253326) and codify for a 70,876 Da protein. iv) When the cDNA for this Hsp70 was expressed in E. coli , the recombinant protein cross-reacts with antibodies against purified 62 kDa NADP-ME. The explanation that could account for the results previously obtained is that NADP-ME/Hsp70 in vivo association can lead to a co-purification of both proteins, explaining the cross-reaction of the antibodies. In this way, the NADP-ME activity of the purified enzyme from maize roots could be due to a SDS-PAGE undetectable 66 kDa protein, being the 72 kDa protein the only one detected. In addition, preliminary data suggest a possible association between recombinant Hsp70 and NADP-ME.