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

Abstract

NADP'-isocitrate dehydrogenase (threo-DS-isocitrate: NADP' oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg2" or Mn2, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP', DL-isocitrate, Mn2+, and Mg2+. Their K,. values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP+. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP+ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP+ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP+-IDH in situ is likely to be controlled by intracellular NADPH to NADP+ ratio as well as by the concentration of various substrates and products.

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

“…The product of the reaction, 2-oxoglutarate, is an inhibitor for NADP*-ICDH as indicated by Omran and Dennis (1971) for pea leaves; NAD* at 10 mM, and EDTA at 1 mM slightly inhibit the activity of the purified enzyme, whereas the NADP*-ICDH isoenzymes present in Chlamydomonas reinhardtii are more strongly affected by identical concentration of NAD* and EDTA, resulting in 30-50% and 100% inhibition, respectively (Martinez-Rivas and Vega 1994). ATP seems to inhibit the NADP*-1CDH activity in accordance with data presented by Omran and Dennis (1971), suggesting that the enzyme could be regulated by the concentration of ATP in the cell, but the NADP*-ICDH from chickpea (Gupta and Sing 1988) was not significantly affected by ATP. However, Omran and Dennis (1971) argued that ATP inhibition is the result of the chelation of the metal ion and so it is of poor regulatory meaning.…”

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

“…This value is similar to the native M r of the enzyme from chickpea pod walls (126 kDa; Gupta and Singh 1988) and is in the range of values described for other higher-plant NADP +-IDH enzymes (82-152 kDa; Chen and Gadal 1990b). This value is similar to the native M r of the enzyme from chickpea pod walls (126 kDa; Gupta and Singh 1988) and is in the range of values described for other higher-plant NADP +-IDH enzymes (82-152 kDa; Chen and Gadal 1990b).…”

Isolation and characterization of a cDNA encoding NADP+-specific isocitrate dehydrogenase from soybean (Glycine max)

“…Although NADP-IDH from a number of plants has been isolated and characterized [9,15,23,30,34,35,36,42] and the heritability of IDH has been established in various species, little is known about the molecular genetics of NADP-IDH. On the basis ofisozyme analysis, Kiang and Gorman [27] concluded that there are four active IDH loci in soybeans, two that encode cytosol-associated isozymes and two that encode mitochondrial isozymes.…”