Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from mixotrophic tobacco cells

Green, 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. Their elution behavior during anion-exchange column chromatography was similar to that described pr...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1994-06, Vol.105 (2), p.593-600
Main Authors: Galvez, S, Bismuth, E, Sarda, C, Gadal, P
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
AHILAMIENTO
CHLOROPLASTE
CLOROPLASTO
CULTIVO DE CELULAS
CULTURE DE CELLULE
ETIOLEMENT
ISOCITRATE DESHYDROGENASE
ISOCITRATO DESHIDROGENASA
ISOENZIMAS
ISOENZYME
NICOTIANA TABACUM
PURIFICACION
PURIFICATION
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Summary:Green, 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. Their 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. ICDH2 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 anion-exchange and affinity chromatography. The 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 ICDH2-specific antibody was used to localize tobacco leaf ICDH2 in situ by an immunogold labeling technique. The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves. ICDH1 and ICDH2 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 Km values for NADP, D(+)isocitrate, and Mg2+ were found for the two enzymes when assayed with Mg2+ as the metal cofactor
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.105.2.593