No abstract

A full-length cDNA (icdb-7) encoding a cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH-1) from potato (Solanum tuberosum L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other eukaryotes such as tobacco, alfalfa, soybean, cattle, pig, and yeast. The gene was transcribed in all tissues tested, with the highest amount of icdb-1 transcript being found in green tissues, i n flowers, and i n roots. In leaves, enzyme activities were dependent on the age, with fully mature leaves showing the highest level of RNA expression and enzyme activity. This observation may indicate that NADP+-dependent ICDH is not only involved in amino acid biosynthesis via the glutamine synthetase/glutamine oxoglutarate aminotransferase cycle but also in cycling, redistribution, and export of amino acids. The latter assumption has been strengthened by our finding of a preferential expression of NADP+-dependent ICDH in leaf veins. Under in vivo conditions, the expression pattern paralleled the enzyine activity, indicating coarse control on the RNA level. Experiments carried out with detached leaves revealed an influence of light, nitrate, and sucrose on icdh-1 transcript levels and i n some cases also on NADP+-dependent ICDH activity. I n darkness, nitrate or sucrose induced icdb-7 mRNA expression.Leaves kept under starvation conditions exhibited a decrease of their protein content, whereas icdh-1 expression and ICDH activity increased significantly.

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.