Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

Abstract

Creen, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the exponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes. lheir elution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplastic (ICDH2) enzymes from pea (Pisum sativum) leaves. lCDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process. Both isoforms were purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation and anionexchange and affinity chromatography. l h e isoenzymes were separated on a DEAE-Sephacel column, but the most effective step was a Matrex Red-A column, which enabled an overall purification of 833-and 1328-fold for ICDH1 and ICDH2, respectively. Polyclonal antibodies were raised against each isoform. The ICDHZspecific antibody was used to localize tobacco leaf lCDH2 in situ by an immunogold labeling technique. The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves. ICDHl and lCDH2 were shown to have apparent native molecular weights of 117,000 and 136,000, respectively, and to consist of identical, 48.5-kD subunits. Similar apparent K,,, values for NADP, D(+)isocitrate, and Mgz+ were found for the two enzymes when assayed with Mgz+ as the metal cofactor.

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

“…The first is IDH, a tricarboxylic acid cycle enzyme localized in the mitochondria of eukaryotic cells that use NAD. The second is ICDH, consisting of several isoenzymes, localized in the cytosol (Gá lvez et al, 1996), the chloroplasts (Gá lvez et al, 1994), the mitochondria (Gá lvez et al, 1998), and the peroxisomes (Corpas et al, 1999). Both enzymes are potential candidates for supplying 2OG for the amination reactions of GOGAT and GDH.…”

“…It was of interest to test whether or not the increase in the transcript of Nt gdh-NAD;A1 and immunoreactive GDH protein brought about by NaCl ( Figures 5A and 5B) was accompanied by similar trends in the transcript(s) and protein of I(C)DH. Thus, the abundance of Nt idh;A and Nt cyt icdh transcripts (Gá lvez et al, 1994) and the levels of immunoreactive mt ICDH and cyt ICDH proteins were examined in the shoots of tobacco seedlings 36 and 48 h after salt treatment (250 mM NaCl) and with and without MSX. RNA gel blot analysis revealed that the increase in the Nt gdh-NAD;A1 transcript in the shoots of salt-treated plants (with and without MSX) at 36 h (Figure 11) was accompanied by a concomitant increase in the transcript of Nt idh;A (2.5-and 2-fold at 36 h and 6-and 2.2-fold at 48 h; Figure 11A) but not in the plants treated only with MSX ( Figure 11A).…”

Purification and Characterization of NADP+-Linked Isocitrate Dehydrogenase from Scots Pine

“…It occurs as several isozymes located in different subcellular compartments, including the cytosol, chloroplast, mitochondria, and peroxisome (Chen and Gadal, 1990b, and refs. therein;Rasmusson and Moller, 1990;Gálvez et al, 1994;Gallardo et al, 1995;Cornu et al, 1996).…”

“…This hypothesis was based on the findings that (a) plant organs exhibit very low NAD ϩ -IDH activity, (b) NADP ϩ -IDH has a higher affinity for substrates, and (c) cytosolic aconitase is present in plants and may produce isocitrate outside of the mitochondria. In angiosperms NADP ϩ -IDH has been studied in a limited number of species, including pea (Omran and Dennis, 1971;Randall and Givan, 1981;Chen et al, 1989a), several members of the Solanaceae (Gálvez et al, 1994;Fieuw et al, 1995;Gallardo et al, 1995), and cucumber (Canino et al, 1996). It occurs as several isozymes located in different subcellular compartments, including the cytosol, chloroplast, mitochondria, and peroxisome (Chen and Gadal, 1990b, and refs.…”

Mild Reductions in Mitochondrial Citrate Synthase Activity Result in a Compromised Nitrate Assimilation and Reduced Leaf Pigmentation But Have No Effect on Photosynthetic Performance or Growth

“…In recent years much research effort has been expended on elucidating the metabolic and physiological functions of the various isoforms of isocitrate dehydrogenase (Galvez et al, 1994;Hodges et al, 2003). In contrast, directed studies, such as those described above, but concerning the metabolic role of citrate synthase are rare (Pracharoenwattana et al, 2005) and do not directly concern the mitochondrial isoform.…”