Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Abstract

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves ofPisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K. values of 23 micromolar for DL-iSocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg2+ and Mn2 .NADP-isocitrate dehydrogenase (threo-Ds-isocitrate: NADP oxidoreductase [decarboxylating]; EC 1.1.1.42) has been studied in several different higher plants (3-5, 8, 9). Randall and Givan (9) used protoplasts as a source of organelles and concluded that in pea leaves NADP-IDH2 was localized primarily in the cytosol (90%), to a lesser degree in the chloroplast, but not at significant levels in peroxisomes or mitochondria. The subcellular localization of NADP-IDH suggests that the two forms of the enzyme may have different functions in the metabolism ofthe plant. The chloroplast form of NADP-IDH is generally believed to supply 2-oxoglutarate for glutamate synthesis (3) while the cytosolic enzyme is thought to function in the movement of reducing power between the mitochondria and cytosol (7). However, little is known about the metabolic role of cystosolic NADP-IDH and its relationship with the NADP-IDH present in the chloroplast. In this study, we have purified the cytosolic NADP-IDH to homogeneity and characterized the enzyme. MATERUILS AND METHODSPurification of NADP-Isocitrate Dehydrogenase. Pisum sativum L. cv Little Marvel seedlings were grown on vermiculite in a controlled environment growth chamber (12 h illumination with an intensity of 100 MtE/m2.s, at 25°C) for 2 weeks. Leaves were cut from the seedlings and blended for 5 s with (1:3 w/v) ice-cold isolation medium (25 mm Hepes-NaOH [pH 7.6], 0.35 M sucrose, 2 mm EDTA, and 2 mm sodium isoascorbate). The homogenate was strained through eight layers of cheesecloth and centrifuged at 27,000g for 15 min at 4°C to remove nuclei, ' This work was supported by Grant-841454 from the National Science Foundation.2 Abbreviations: IDH, isocitrate dehydrogenase; ACB, affinity column buffer.chloroplasts, and other organelles. Crystalline (NH4)2SO4 was added to the supernatant with stirring, until a final concentration of 45% was achieved and was left overnight at 4°C. The precipitate was removed by centrifugation at 27,000g for 15 min.Additional crystalline (NH4)2SO4 was added to the supernatant, to achieve a final concentration of 80% and was mixed overnight at 4°C. The precipitate was collected by centrifugation at 27,000g at 4°C and resuspended in affinity column buffer (ACB), pH 7.0 (10 mm K-phosphate, 5 mm citrate, 2 mm MgC12, 10% glycerol, 1 mm benzamidine, and 0.01% NaN3).The protein suspension was desalte...

Partial Purification and Characterization of NADP+-Isocitrate Dehydrogenase from Immature Pod Walls of Chickpea (Cicer arietinum L.)

“…Their K,. values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents.…”

“…It is present in plant cytosol (5,8,15,18), mitochondria (1,5), and chloroplasts (7,11), thus indicating that it may have varying roles in the metabolism ofthe plant. The chloroplastic form of NADP+-IDH is believed to supply 2-oxoglutarate for glutamate synthesis (7), while the cytosolic enzyme probably functions in the transfer of reducing power between the mitochondria and cytosol (12).…”

“…The chloroplastic form of NADP+-IDH is believed to supply 2-oxoglutarate for glutamate synthesis (7), while the cytosolic enzyme probably functions in the transfer of reducing power between the mitochondria and cytosol (12). In addition to the various bacterial and mammalian species, this enzyme has been well studied from a number of plant sources ' including etiolated seedlings (16), leaves (15,18), germinating (21) and maturing seeds (5,20), and root nodules (8).…”

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

“…The enzyme is widely distributed in living organisms. It has been purified to homogeneity, and its properties have been investigated, from bacteria (Barrera and Jurtshuk, 1970;Reeves et al, 1972;Fukunaga et al, 1992), cyanobacteria (Muro-Pastor and Florencio, 1992), fungi (Meixner-Monori et al, 1986), and mammals (Farrell, 1980), as well as from a few plant tissues (Omran and Dennis, 1971;Cuny and Ting, 1976;Henson et al, 1986;Ni et al, 1987;Chen et al, 1988). In higher plants, NADP-ICDH activity has been detected in a11 tissues and organs where it was assayed.…”

Storage protein degradation and proteolytic enzyme activities during germination of water stressed impatiens seeds

“…Isoelecric focussing was performed in a vertical polyacrylamide minigel system (Ni et al, 1987). Enzyme solutions (see enzyme activity) from dry, 96 hr, and PEG germinated (-0.6 MPa) seeds were loaded based on activity (25, 15, and 25 /il, respectively).…”