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.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

NADP+-isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons

NADP⁺-isocitrate dehydrogenase (IDH, EC 1.1.1.42) was studied during the post-germinative growth of pumpkin ( Cucurbita pepo L. cv. Alberello di Sarzana) seedlings. In cotyledons. IDH activity increased in the dark and declined after illumination. Native PAGE showed that at least two isozymes of low electrophoretic mobility are present in cotyledons and absent in other pumpkin tissues. Anion exchange chromatography performed on extracts both from 4-day-old etiolated cotyledons and from illuminated cotyledons confirmed the trend of the IDH isoforms. In senescing cotyledons an additional IDH isoform with higher electrophoretic mobility appears. Overall the data indicate the presence of specific NADP⁺-IDH isoforms in etiolated cotyledons and senescing cotyledons, when glyoxylate cycle enzymes are active. A possible role for these IDH isoforms is proposed.     

Characterization of NADP+-isocitrate dehydrogenase from the host plant cytosol of lucerne (Medicago sativa) root nodules

NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) was purified more than 1500-fold from the host-plant cytosol of Medicago sativa L. cv. Saranac root nodules by ion exchange and affinity chromatography. The forward reaction was characterized. The enzyme exhibited an absolute requirement for a divalent cation (preferably Mn²⁺), had a broad activity optimum from pH 7.5 to 9.0, and was most stable at pH 7.5. The apparent Arrhenius energy of activation was 70.7 kJ mol⁻¹ (20 to 30°C) indicating that the reaction rate of the enzyme was relatively sensitive to temperature. The K_m for isocitrate was 20 μ M and for NADP⁺ 10.7 μ M . Initial velocity and end product inhibition studies of the forward reaction indicate a random bi ter mechanism. End product studies indicated that NADPH was a competitive inhibitor and α-ketoglutarate was a non-competitive inhibitor with respect to isocitrate and NADP⁺. Citrate was a competitive inhibitor with respect to isocitrate. Glutamine was identified as a positive effector when assays were conducted at non-saturating isocitrate concentrations. The potential significance of glutamine regulation of α-ketoglutarate production in a dinitrogen-fixing tissue is discussed.     

Citrate synthases from germinating castor bean seeds – II. Time course of synthesis and immunochemical comparison

The time course of total citrate synthase activity in castor bean ( Ricinus communis L., type Sanzibariensis) endosperm showed a 7-fold increase during the initial 5 days of germination and a decrease thereafter. All citrate synthase activity in the ungerminated seeds was due to the mitochondrial isoenzyme. After two days of germination the glyoxysomal isoenzyme began to appear. After 5 days the glyoxysomal citrate synthase represented 50 to 55% of the total activity and the mitochondrial enzyme the remainder. This was estimated from (a) inactivation of the glyoxysomal citrate synthase by 5,5'-dithiobis(2-nitrobenzoic acid); (b) solid phase adsorption of the glyoxysomal synthase by a specific antiserum; (c) separation of isoenzymes by (NH₄)₂SO₄ gradient solubilization.
The increase of both citrate synthases during the initial 4 days of germination could be prevented by 10 μg cycloheximide ml⁻¹, but not by 40 or 400 μg chloramphenicol ml⁻¹, indicating a synthesis on 80 S ribosomes. Actinomycin D completely inhibited the appearance of the glyoxysomal enzyme while the mitochondrial enzyme was not affected. Antisera against the two isoenzymes revealed major structural differences between two citrate synthases, however, also some common determinants. No cross-reaction was observed with the citrate synthase from pig heart or E. coli.     

Citrate synthases from germinating castor bean seeds – I. Purification and properties

The mitochondrial and glyoxysomal citrate synthase (EC 4.1.3.7) from the endosperm of germinating castor beans ( Ricinus communis L., type Sanzibaricnsis) were purified to a final specific activity of 76 and 78 U (mg protein)⁻¹, respectively. Both citrate synthases could be bound to ATP-Sepharose. However, only the mitochondrial enzyme could be eluted by either 100 μ M oxaloacetate or 100 μ M coenzyme A (indicative of affinity chromatography), while the glyoxysomal enzyme was only eluted by 0.5 M KCI (indicative of ion-exchange chromatography). Many properties of the two isoenzymes were similar including the pH dependence and temperature dependence of activity, the pH stability, and the inactivation of the enzyme at elevated temperatures. The most pronounced differences between the two citrate synthases were the isolelectric points of pH 5.9 for the mitochondrial and of pH 9.1 for the glyoxysomal enzyme. Both citrate synthases are dimers in the native form with a molecular weight of 95000 each, as determined by gel filtration on Sepharose CL-6B and by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. However, the glyoxysomal citrate synthase existed also as a tetramer with a molecular weight of 200000 in the presence of 10 m M MgCl₂.     

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

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.     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

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.     

Mg2+- or Ca2+-Activated ATPase in Squid Giant Fiber Axoplasm

A divalent cation-activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of enzyme with those of myosin and Na+-K+-ATPase suggests that differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.     

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.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Regulation of 109Cd2+ Uptake into Isolated Neurohypophysial Peptidergic Nerve Terminals

Cadmium (109Cd2+) uptake was studied in a preparation of isolated neurohypophysial nerve terminals. By use of a filter separation method, together with a permeabilizing agent (Triton X-100), two cellular Cd2+ pools have been distinguished. The uptake into the intraterminal pool was governed mainly by a process that displayed saturable kinetics, with a Vmax of 0.15 nmol of Cd2+/mg of protein/min and a Km of 0.18 mM, consistent with a transport system. The superficially bound Cd2+ pool (Triton insensitive), which represented 30-50% of the total Cd2+ bound to the cellular system, was very sensitive to the ionic composition of the incubation medium. Reducing the extracellular Ca2+ or Na+ concentration caused a significant increase in the size of the Triton-insensitive Cd2+ pool. Whereas Na+ did not affect Cd2+ uptake, Ca2+ induced a small, but significant, increase of Cd2+ uptake into the terminals. It is concluded that there is a significant intraterminal uptake of Cd2+, which could explain several physiological effects of this ion.     

Incubation of Tissue Slices in the Absence of Ca2+ and Mg2+ Can Cause Nonspecific Damage

Superfusion of striatal slices with a medium deficient in Ca2+ and Mg2+ caused a large and sustained increase in release of lactate dehydrogenase, a finding indicative of the disruption of plasma membranes. This was associated with an efflux of dopamine (DA) and the depletion of DA from the tissue. In addition, whereas DA efflux was stimulated by either D-amphetamine (10 microM) or L-glutamate (10 mM) in the absence of Ca2+, these effects were greatly reduced when Mg2+ also was withdrawn from the buffer. These results suggest that (a) incubation in a Ca2+/Mg2(+)-free buffer disrupts plasma membranes, (b) this disruption affects dopaminergic neurons as well as those of other striatal elements, and (c) the failure of a treatment to stimulate DA release in a Ca2+/Mg2(+)-free buffer cannot be used as a test of Ca2+ dependence.     

NADP+-dependent glutamate dehydrogenase from the facultative methylotroph Hyphomicrobium X

Abstract NADP⁺-dependent glutamate dehydrogenase (GDH; EC 1.4.1.4) was purified using acetone precipitation, heat, DEAE-cellulose and dye-ligand Ramazol Red column chromatography. The M _r of the native enzyme was estimated to be 380 000 (± 10 000) by polyacrylamide gel electrophoresis. The same technique in the presence of sodium dodecyl sulphate (SDS) gave one subunit band with an M _r of 63 400 (±4000). Thus the enzyme has a hexameric structure. The enzyme has a pH optimum of 8.5 and has K _m apparent values of 1.6 mM, 0.015 mM and 10.2 mM for α-ketoglutarate, N NADPH and L -glutamate, respectively. Michaelis-Menten kinetics were not observed when the ammonium concentration was increased. A progressive increase in the ammonium concentration resulted in a progressively increasing K _m value. The enzyme was highly specific for all substrates and markedly insensitive to inhibitors.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Purification and Characterization of Posterior Pituitary Calmodulin and Its Activation of Neurosecretosome Ca2++ Mg2+-ATPase Activities

Abstract: Calmodulin was isolated as an electrophoretically homogeneous protein from bovine posterior pituitary glands. The yield indicated that this gland is a particularly rich source. Purified bovine posterior pituitary calmodulin and bovine brain calmodulin had identical electrophoretic mobilities on 10% and 12% polyacrylamide gels. The protein was further identified by molecular weight determination and by amino acid analysis which showed that it contained trimethyllysine, one residue per molecule. Bovine posterior pituitary calmodulin was found to activate a preparation of calmodulin-deficient phosphodiesterase from bovine heart. In addition, pituitary calmodulin stimulated Ca2⁺+ Mg²⁺-ATPase activity associated with a purified nerve ending plasma membrane fraction. This dependence could only be demonstrated after successive washing of the membranes with EGTA buffers, a procedure designed to remove endogenous calmodulin.     

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.