Two different cDNAs that encode NADP-specific isocitrate dehydrogenase (NADP-IDH) isozymes of soybean (Glycine max) were characterized. The nucleotide sequences of the coding regions of these cDNAs have 74% identity to each other and give predicted amino acid sequences that have 83% identity to each other. Using PCR techniques, their coding regions were subcloned into a protein overexpression vector, pQE32, to yield pIDH4 and pIDH1, respectively. Both IDH4 and IDH1 enzymes were expressed in Escherichia coli as catalytically active His6 tagged proteins, purified to homogeneity by affinity chromatography on nickel chelate resin and rabbit polyclonal antibodies to each were generated. Surprisingly, antiserum to IDH4 did not react with IDH1 protein and IDH1 antiserum reacted only very weakly with IDH4 protein. IDH4 antibody reacts with a protein of expected molecular weight in cotyledon, young leaf, young root, mature root and nodules but the reaction with mature leaf tissue was low compared to other tissues. Western blot results show that IDH1 was not expressed in young roots but a protein that reacts with the IDH1 antibody was highly expressed in leaves, showing that there was tissue-specific accumulation of NADP-IDH isozymes in soybean.
Plant Molecular Biology – Springer Journals
Published: Sep 29, 2004
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