PURIFICATION AND CHARACTERIZATION OF CHLOROPLASTIC NADP-ISOCITRATE DEHYDROGENASE FROM MIXOTROPHIC TOBACCO CELLS - COMPARISON WITH THE CYTOSOLIC ISOENZYME

Green, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the ex ponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes. Their el ution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplas tic (ICDH2) enzymes from pea (Pisum sativum) leaves. ICDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process. Both isoforms were purified to apparent electrophoretic homo geneity by ammonium sulfate fractionation and anion-exchange and affin ity chromatography. The isoenzymes were separated on a DEAE-Sephacel c olumn, but the most effective step was a Matrex Red-A column, which en abled an overall purification of 833- and 1328-fold for ICDH1 and ICDH 2, respectively. Polyclonal antibodies were raised against each isofor m. The ICDH2-specific antibody was used to localize tobacco leaf ICDH2 in situ by an immunogold labeling technique. The enzyme was found lar gely, if not exclusively, in the chloroplasts of green leaves. ICDH1 a nd ICDH2 were shown to have apparent native molecular weights of 117,0 00 and 136,000, respectively, and to consist of identical, 48.5-kD sub units. Similar apparent K-m values for NADP, D(+)isocitrate, and Mg2were found for the two enzymes when assayed with Mg2+ as the metal cof actor.