NADP⁺‐isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

Abstract

The activity of NADP+‐dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post‐germinative growth of cucumber (Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP+‐ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43‐kDa subunits. It is specific for NADP+, inhibited by ATP and by 2‐oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP+‐ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.

Purification and characterization of chloroplastic NADP‐isocitrate dehydrogenase from Chlamydomonas reinhardtii

“…This step has also been used for the purification of NADP-IDH 2 from tobacco cell cultures (Ga´lvez et al 1994), although these authors used NADP 1 plus isocitrate to elute the isoenzyme from the column, instead of KCl as we describe in the present study. The same affinity chromatography has also been reported for the purification of NADP-IDH 1 from C. reinhardtii (Martı´nez-Rivas et al 1996), and higher plants (Canino et al 1996). According to the purification method described here, mixotrophic growth of cells is not required to purify the C. reinhardtii NADP-IDH 2 isoenzyme, as was previously reported for the tobacco NADP-IDH 2 , the unique chloroplastic NADP-IDH completely purified so far (Ga´lvez et al 1994).…”

“…Cytosolic isoenzyme (NADP‐IDH 1 ) has been completely purified and characterized from maize (Curry and Ting 1976), pea (Chen et al 1988), tomato (Gallardo et al 1995), cucumber (Canino et al 1996) and Scots pine (Palomo et al 1998); as well as from the eukaryotic alga Chlamydomonas reinhardtii (Martínez‐Rivas et al 1996). In addition, its cDNA has been cloned from several higher plants such as potato (Fieuw et al 1995), tobacco (Gálvez et al 1996), eucalypt (Boiffin et al 1998), soybean (Park and Kahn 1999), and lemon fruit (Sadka et al 2000).…”

The relationship between carbon and nitrogen metabolism in cucumber leaves acclimated to salt stress

“…NADP + -ICHD activity was measured spectophotometrically at 25 °C by monitoring the reduction of NADP (ε = 6.22 mM −1 cm −1 ) at 340 nm according to Canino et al (1996). The reaction medium (2 cm 3 ) contained the following: 0.05 M Tris-HCl, pH 8.0, 0.6 mM NADP + , 1mM MgCl 2 , 9.6 mM isocitrate as a starter of reaction.…”