Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Interaction of divalent metal ions with the NADP+-malic enzyme from maize leaves

The effect of several metal ions on NADP⁺-malic enzyme (EC 1.1.1.40) purified from Zea mays L. leaves was studied Mg²⁺, Mn²⁺, Co²⁺ and Cd²⁺ were all active metal cofactors. The malic enzyme from maize has a moderately high intrinsic preference for Mn²⁺ relative to Mg²⁺ at pH 7.0 and 8.0 Negative cooperativity detected in the binding of Mg²⁺ at pH 7.0 and 8.0 and in the binding of Mn²⁺ at pH 7.0 suggests the existence of at least two binding sites with different affinity. All of the activating metal ions have preference for octahedral coordination geometry and have ionic radii of 0.86–1.09 Å. The ions that act as inhibitors are outside this range and/or are incapable of octahedral coordination. Ba²⁺, Sr²⁺, Cd²⁺, Ca²⁺, Be²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Co²⁺, Hg²⁺ showed mixed-type inhibition. The reciprocal of their K₁ values follow the order of their apparence in the Irving-Williams series of stability that derives in part from size effects. It is suggested that the size of the ions may play a partial role in determining the strength of the metal interaction.     

NADP+-malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme

The thioredoxin-dependent light/dark modulation system of the chloroplast is described as a prerequisite enabling the flexible control of fluxes through the various parts of the CO₂-fixation pathway. Both the rapid turnover of the reduced thiol-containing form of the respective target enzyme, and the metabolite effect upon the reductive enzyme modulation, allow rapid adjustment of the amount of active species to the actual requirements. The structural basis of the regulation of chloroplast NADP⁺-malate dehydrogenase (EC 1.1.1.82) is described in more detail. The modulable plastid enzyme is characterized by two sequence extensions not present in any other known NADP⁺- and/or NAD⁺-specific malate dehydrogenase. The NADP⁺-malate dehydrogenase of C₃-plants is part of the "malate valve", which catalyzes the export of reducing equivalents in the form of malate from the chloroplast only when the NADPH to NADP⁺ ratio is high, thus poising the NADPH to ATP ratio required for optimal carbon reduction in the light. The mode of regulation of other light/dark modulated enzymes is discussed.     

Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates

Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m⁻² s⁻¹). The activities of four C₄ enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, P_i dikinase (PPDK; EC 2.7.9.1) and NADP⁺-malic enzyme (NADP⁺-ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP⁺-malate dehydro-genase (NADP⁺-MDH; EC 1.1.1.82) and NADP⁺-ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C.     

Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae

Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO₂ with H₂ or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F₄₂₀-nonreactive hydrogenase, NADP⁺: F₄₂₀ oxidoreductase, NADP⁺-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F₄₂₀ dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F₄₂₀-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F₄₂₀ to provide electrons for biosynthesis, was formulated.     

Differential response of carbon and nitrogen metabolism to fluoride application in fruiting structures of chickpea (Cicer arietinum)

The effect of sodium fluoride (10 and 50 mol·m⁻³) on the activities of sucrose metabolizing enzymes, transaminases and glutamine synthetase in relation to the transformation of free sugars to starch and protein in the fruiting structures (pod wall, seed coat, cotyledons) of chickpea was studied by culturing detached reproductive shoots in a liquid medium. Addition of fluoride to the culture medium drastically reduced starch content of the cotyledons and caused a marked build-up of total free sugars comprised mainly of reducing sugars in the pod wall and seed coat, and sucrose in the cotyledons. Concomitantly, the activity of soluble invertase was stimulated in the pod wall but reduced in the cotyledons. However, soluble protein content of both the pod wall and the cotyledons increased in conjunction with an increase in the activities of glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and glutamine synthetase. Disruption of starch biosynthesis under the influence of fluoride and the resulting accumulation of free sugars possibly resulted in their favoured utilization in nitrogen metabolism. Labelling studies with [U-¹⁴C]-sucrose showed that the ¹⁴C incorporation into total free sugars was enhanced by fluoride in the pod wall but reduced in the seed coat and cotyledons, possibly due to an inhibitory effect on their translocation to the developing seeds.     

Thermal stability and kinetic properties of NADP+-malate dehydrogenase isomorphs in two populations of the C4 weed species Echinohloa crus-galli (Barnyard grass) from sites of contrasting climates

The thermal stability and kinetic properties of purified NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) isomorphs were analyzed from plants of two populations of Barnyard grass from contrasting thermal environments. Plants from Québec (QUE) and Mississippi (MISS) were acclimated under controlled conditions at 26/20°C and 14/8°C (day/night). While the enzyme from QUE showed one isomorph, 3 isomorphs were detected in all plants from MISS, suggesting the presence of gene duplication and fixed heterozygosity for the expression of this dimeric enzyme. This findig raises the possibility that the enhanced acclimatory potential of NADP⁺-MDH from MISS plants, as found from previous studies with the partially purified and unfractioned enzyme, may result from differential kinetic properties of isomorphs which would allow for the proper modulation of catalysis over a wide temperature range. The thermal stability of the QUE isomorph was significantly higher than that of any of the MISS isomorphs. The apparent activation energy of the QUE isomorph was within the range of values found for the 3 MISS isomorphs which were similar to each other. The Michaelis-Menten constants (K_m) for oxalacetic acid were not significantly different among isomorphs or between thermoperiods, but K_m (NADP⁺) values for the QUE isomorph were significantly higher than those of two of the MISS isomorphs over the 15–25°C assay range V_max/K_m ratios for OAA and NADP⁺ were not significantly different among isomorphs or between thermoperiods. Our data indicate that, under highly purified conditions, the single NADP⁺-MDH isomorph of QUE plants possesses good acclimatory potential for maintaining catalytic efficiency under a wide range of temperature conditions. In vitro thermal and kinetic data do not support the hypothesis that the the multiple NADP⁺-MDH isomorphs found in MISS plants may have been selected to optimize the thermal and catalytic efficiency of NADP⁺-MDH under warm temperature conditions.     

Distribution of 14CO2 during ontogeny of chickpea (Cicer arietinum) grown at two moisture levels

The distribution of assimilates of ¹⁴CO₂ in ethanol soluble and insoluble fractions was measured at 20-day intervals from 45–135 days after sowing (DAS) in chickpea (Cicer arietinum) grown at two moisture levels. The contribution of pre-flowering assimilates to pods, although very low, was higher under the stress conditions. At the time of harvest, the recovery of ¹⁴C in pods was 0.4 and 0.9% of the total ¹⁴C fed 45 DAS in soluble and 2.5 and 5.1% in insoluble fractions in control and stressed plants, respectively. The %¹⁴C received by nodules continuously decreased with increasing age of plants. Stressed plants diverted more ¹⁴C to nodules, compared to control, during vegetative and flowering stages. During active seed filling, stressed plants diverted more ¹⁴C to reproductive parts and less to nodules, compared to control. Significant amounts of ¹⁴C were retgined by the stem and leaves during the seed-filling period and it appears that there is scope for the remobilisation of pre-flowering, as well as post-flowering assimilates for seed-filling of chickpea.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

Field evaluation of eco-friendly biocontrol package for the management of pod borer,Helicoverpa armigera Hubner,and fusarium wilt in chickpea,Cicer arietinum

Helicoverpa armigera is a serious pest of chickpea and causes great damage to crop. Extensive and indiscriminate use of insecticides has led to the development of resistance in H. armigera. Among the several alternative methods for management of H. armigera, the nuclear polyhedrosis virus (NPV) is promising, whereas Trichoderma sp. have shown promising results against chickpea wilt. The experiments to evaluate biocontrol package against H. armigera and wilt disease under field conditions were conducted. Lowest H. armigera larval population (0.71 larvae/plant) was recorded in chemical control, which was at a par with biocontrol package (0.91 larvae/plant), and both the treatments were significantly better than control. Lowest per cent pod damage (3.85%) was recorded in chemical control followed by biocontrol treatment (5.08%) and unsprayed control (8.61%). The yields from biocontrol package (13.45 q/ha) and chemical control (15.37 q/ha) were significantly higher than unsprayed control (10.7 q/ha). There was no disease incidence in all treatments in both 2008 and 2009.     

Control of K+ (Rb+) influx and transport with changed internal K+ concentration and age in two varieties of barley

The influx of Rb⁺ into the roots of two barley varieties (Hordeum vulgare L. cv. Salve and cv. Ingrid) from a K⁺-free ⁸⁶Rb-labelled nutrient solution with 2.0 mM Rb⁺, was checked at intervals from day 6 to day 18. The control plants were continuously grown in complete nutrient solution containing 5.0 mM K⁺, while two other groups of plants were grown in K⁺-free nutrient solution starting on day 6 and between day 6 and day 9, respectively. The pattern of Rb⁺ influx was similar for both varieties, although their efficiencies in absorbing Rb⁺ were different. The relationship between Rb⁺ influx and K⁺ concentration of the root could be interpreted in terms of negative feedback through allosteric control of uptake across the plasmalemma of the root cells. Hill plots were bimodal, but in the opposite direction. The Hill coefficients, reflecting the minimum number of interacting allosteric binding sites for K⁺ (Rb⁺), were low (≤–3.0). It is discussed whether the threshold value, that is the breaking point in the Hill plot, is indicative of a changed efficiency of transporting units for K⁺ (Rb⁺) transport to the xylem. Moreover, feedback regulation might be involved in transport of K⁺ between root and shoot. The variation in K⁺ concentrations in the roots and shoots of control plants were cyclic but in phase opposition despite an exponential growth. The average K⁺ concentration varied only slightly with age.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Constitutive and elicitation induced metabolism of isoflavones and pterocarpans in chickpea (Cicer arietinum) cell suspension cultures

Constitutive phenolics of chickpea cell suspension cultures are the isoflavones formononetin and biochanin A, the isoflavanones homoferreirin and cicerin and the pterocarpans medicarpin and maackiain. They accumulate as vacuolar malonylglucosides. The biosynthetic pathways to isoflavones, pterocarpans and malonylglucoside conjugates together with their enzymes are explained. Elicitation of cell cultures leads to pronounced increases in the activities of biosynthetic enzymes with differential effects on the enzymes involved in conjugate metabolism. Low elicitor doses favour pterocarpan conjugate formation whereas high doses lead to pterocarpan aglycone accumulation accompanied by vacuolar efflux of formononetin and pterocarpan malonylglucosides. Elicitor-induced changes in enzyme activities and vacuolar efflux of conjugates are prevented by application of 10^-3M concentrations of cinnamic acid. Cinnamate is alternatively metabolized to a glucose ester, a S-glutathionyl conjugate and to cell wall bounds forms; these reactions are intensified by elicitation. Isoflavone and pterocarpan biosynthesis and conjugate metabolism as regulated by elicitation and cinnamate is depicted in a metabolic grid to explain the complex regulatory pattern of phenolic accumulation in chickpea cell cultures.Abbreviations AOPP L--aminooxy--phenylpropionic acid - BGM biochanin A 7-0-glucoside-6-0-malonate - FGM formononetin 7-0-glucoside-6-0-malonate - HPLC high performance liquid chromatography - MaGM maackianin 3-0-glucoside-6-0-malonate - MeGM medicarpin 3-0-glucoside-6-0-malonate     

NADP+-dependent glutamate dehydrogenase from the obligate methylotroph methylobacillus flagellatum

Abstract NADP-dependent glutamate dehydrogenase (GDH; E.C.1.4.1.4) was purified from an obligate methylotroph Methylobacillus flagellatum using ammonium sulphate precipitation, DEAE-Sepharose and dye-ligand Procion red HE3B column chromatography and Sephacryl S-200 gel-filtration. The M_r of the native enzyme was estimated to be 300 000 (±5000). The enzyme consists of six identical subunits with an M_{r of} 47 000 (±3000) (SDS-PAGE). The enzyme has a pH optimum of 8.0 when participating in amination and 9.5 in deamination. Michaelis-Menten kinetics were observed for both reactions. The apparent K_m values were 1.33 mM, 0.032 mM, 11.5 mM, 7.0 mM and 0.014 mM for α-ketoglutarate, NADPH, NH₄⁺, glutamate and NADP⁺, respectively. The enzyme was highly specific for all the substrates and was insensitive to inhibitors. It plays an exclusively anabolic role in the cells.     

Growth, contents of K+ and kinetics of K+(86Rb) uptake in barley cultured at different low supply rates of potassium

Plants of barley ( Hordeum vulgare L. cv. Salve) were grown with 6.5–35% relative increase of K⁺ supply per day (RKR) using a special computer-controlled culture unit. After a few days on the culture solution the plants adapted their relative growth rate (RGR) to the rate of nutrient supply. The roots of the plants remained in a low salt status irrespective of the rate of nutrient supply, whereas the concentration of K⁺ in shoots increased with RKR. Both V_max and K_m for K⁺(⁸⁶Rb) influx increased with RKR. It is concluded that with a continuous and stable K⁺ stress, the K⁺ uptake system is adjusted to provide an effective K⁺ uptake at each given RKR. Allosteric regulation of K⁺ influx does not occur and efflux of K⁺ is very small.     

Growth and accumulation of N, K+, Ca2+ and Mg2+ in barley exposed to various nutrient regimes and root/shoot temperatures

Six cultivars of spring barley ( Hordeum vulgare L. cvs Salve, Nümberg II, Bomi, Risø 1508, Mona and Sv 73 608) were grown in water culture for three weeks with various combinations of mineral supply and differential roots/shoot temperatures during the growth period. Most important for growth and accumulation of N, K⁺, Ca²⁺ and Mg²⁺ was the mineral supply, followed by the root temperature and the choice of cultivar. Treatments with low mineral supply or low root temperature induced a uniform reduction in growth and accumulation of the ions studied. The effects of low mineral supply and low root temperature on growth and N accumulation was additive, which indicates that these factors exert their influence independently of each other.
Roots grown at 10°C were smaller and Rb⁺(⁸⁶Rb) influx was higher than in roots grown at 20°C. It is suggested that the control of Rb⁺(⁸⁶Rb) influx is affected by the root temperature and the age of the plants. The higher ⁸⁶Rb⁺ (⁸⁶Rb) influx into the low temperature roots could not compensate for the smaller root size. However, the lower total mineral accumulation made up for the needs of the smaller plants and cannot explain the reduction in growth.     

Effect of thiourea on the ionic content and dark fixation of CO2 in embryonic axes of Cicer arietinum seeds

During the first 12 h of germination of Cicer arietinum L. (cv. Castellans) seeds, K⁺ is first lost into the surrounding medium and is later reabsorbed. Thiourea accelerates this reabsorption. Since there is an increase in the mobilization of K⁺ in response to thiourea, a greater accumulation of malate due to the carboxylation of phosphoenolpyruvic acid takes place as compared with that occurring in water. The subapical zone of the radicle accumulates the greatest amounts of water, K⁺ and malate. The variation in the "in vitro" activity of phosphoenolpyruvate carboxylase does not explain the difference in malate in response to the different treatments, consequently there must be chemical changes in the cytoplasm which favour this carboxylation "in vivo". These results show that thiourea accelerates the mobilization of K⁺ and stimulates the dark fixation of CO₂ in embryonic axes of Cicer arietinum.     

Isoflavones from black chickpea (Cicer arietinum L) sprouts with antioxidant and antiproliferative activity

Black chickpea is a good source of bioactive compounds, particularly isoflavones. Sprouting improves nutraceutical value in chickpea seeds. This study aimed to explore the role of sprouting of black chickpea seeds on the synthesis of isoflavones and evaluate the impact of the soluble isoflavone on cellular antioxidant activity (CAA) and antiproliferative activity in breast cancer cells. Isoflavones were identified and quantified by HPLC-UV-MS. The CAA and antiproliferative activity were determined in HepG2 cells and MDA-MB-231 cancer cells, correspondingly. In sprouted black chickpea, six isoflavones (formononetin, biochanin-A, and its glycosides) were identified and the total isoflavones content increased (0.31 to 35.72 µgBA/mg of extract). The CAA was increased five times from 137.2 to 788.2 µMEQ/100 g of sample. The bioactive compounds in sprouted chickpea decreased the proliferation of MDA-MB-231 cell line. Also caused morphological changes such as cell shrinkage, rounding and nuclear fragmentation. The results herein suggest that bioactive compounds, as isoflavones, in sprouted black chickpea showed a potential antioxidant and antiproliferative activity. Therefore, it may be considered as a value-added product or ingredient for produce functional foods.     

Ferredoxin–NADP+ Reductase from Tsuga canadensis. A Procedure for Isolation and Properties

Activity of ferredoxin-NADP⁺ reductase in leaf extracts of eastern hemlock [Tsuga canadensis (L.) Carr.] was relatively low, but could be markedly increased by use of protective agents. The best method employed polyvinylpolypyrrolidone (PVP) in the extraction medium plus removal of phenolic compounds by filtering the extracts through an insoluble PVP (Polyclar AT) column. Further purification of the enzyme was achieved by means of DEAE cellulose chromatography and DEAE Sephadex chromatography. A 94-fold purification of the enzyme with a total recovery of 43% was obtained. The eastern hemlock ferredoxin-NADP⁺ reductase was characterized by its diaphorase activity, i.e. the transfer of electrons from NADPH to an electron acceptor. 2,6-dichlorophenol indophenol. The pH optimum for the oxidation of NADPH is between 8.5 and 9.0. The enzyme is highly specific for its electron donor. NADPH, but shows low specificity for electron acceptors. The apparent Michaelis constant values of the enzyme for NADPH. NADH, and 2,6-dichlorophenol indophenol are 2.4 × 10^?5, 5.4 × 10^?3, and 4.7 × 10^?5M respectively. The molecular weight of the enzyme, as estimated by gel filtration, is about 45,000. The enzyme is inhibited by both organic and inorganic mercurials and certain cations. Comparison of properties of eastern hemlock ferredoxin-NADP⁺ reductase and spinach ferredoxin-NADP⁺ reductase shows that both enzymes are similar.