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

Abstract

NADP؉ -isocitrate dehydrogenase (NADP ؉ -IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP ؉ -IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP ؉ -IDH from angiosperm, with a higher catalytic efficiency (10 5 M ؊1 s ؊1 ) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP ؉ -IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP ؉ -IDH were coordinated with GS during seed germination and were associated with GS/ GOGAT enzymes during chloroplast biogenesis, suggesting that NADP ؉ -IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP ؉ -IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP ؉ -IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP ؉ -IDH may have other, as-yet-unknown, biological functions.

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

“…4d). NADP + -IDH has been reported to be linked with hypocotyl diVerentiation in pine (Palomo et al 1998). Accordingly, these data suggest that GAD, like NADP + -IDH, is connected with the diVerentiation of the hypocotyl in pine.…”

“…4e), suggesting that GAD activity is the major regulator of hypocotyl GABA levels during seedling development. To further characterize GAD in pine, we compared GAD expression with the expression of other genes encoding enzymes associated with seedling diVerentiation, including cytosolic NADP + -IDH (Palomo et al 1998;Pascual et al 2008), ascorbate peroxidase (APX), and oxidase (AOX) (Pignocchi and Foyer 2003;de Pinto and De Gara 2004), enzymes linked with ligniWcation and signaling, including laccase (LAC, Gavnholt and Larsen 2002), and 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and oxidase (ACO), enzymes involved in ethylene synthesis that has been described to be connected to ligniWcation of plant stem (Klintborg et al 2002). Northern blots hybridized with speciWc probes indicated that these genes were diVerentially expressed in organs of pine seedlings (Fig.…”

“…Antibodies against pine GAD were raised in rabbits by immunization with the recombinant protein as described by Palomo et al (1998). Several dilutions of the serum were tested against pine GAD in crude extracts, and a 1:20,000 dilution was selected to detect GAD polypeptide in Western blots.…”

“…Protein proWles were analyzed by fractionation of samples by SDS-PAGE followed by Coomassie blue staining. SpeciWc detection of proteins was conducted by Western blot analysis using antisera raised against pine NADP + -IDH (Palomo et al 1998) or recombinant GAD.…”

Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence

“…NADP-ICDH is widely distributed in living organisms, and there are numerous reports describing its characterization and subcellular distribution (Barroso, 1993;Gálvez and Gadal, 1995). In higher plant cells, NADP-ICDH activity has been detected and characterized in the cytosol (Chen et al, 1988(Chen et al, , 1989Fieuw et al, 1995;Canino et al, 1996;Palomo et al, 1998), mitochondria (Rasmusson and Møller, 1990;Attucci et al, 1994;Møller and Rasmusson, 1998), and chloroplasts (Randall and Givan, 1981;Gálvez et al, 1994). Breidenbach and Beevers (1967) and Donaldson (1982) reported the association of NADP-ICDH activity with glyoxysomes, and Yamazaki and Tolbert (1970) localized traces of this activity in leaf peroxisomes.…”