Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation

The ‘high ammonia pathway’ enzyme glutamate dehydrogenase (NADP⁺) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth in reached. Purified glutamate dehydrogenase (NADP⁺) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP⁺) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP⁺) inactivation is caused or followed by rapid proteolysis.     

Photokinetic microanalysis of NADP+, using bacterial luciferase

Summary Bioluminescence photokinetic assay of NADP⁺ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. The analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP⁺ as measurable product and for nucleotide assay in cell samples of 0.5–10 g dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP⁺.Previously, FMN, NADH, NAD⁺ and NADH have been analysed with the bacterial luciferase system. Its applicability has now been extended by the assay of NADP⁺.     

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

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

Purification and properties of the NAD+-xylitol-dehydrogenase from the yeast Pichia stipitis

Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD⁺ and NADP⁺. During the purification step on DEAE-sephadex A-50 a NAD⁺-dependent xylitol dehydrogenase could be separated from a NADP⁺-dependent. The NAD⁺-xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l-iditol:NAD⁺-5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase).     

Purification and properties of mannitol dehydrogenase from Agaricus bisporus sporocarps

Mannitol dehydrogenase (mannitol: NADP⁺ 2-oxidoreductase: EC 1.1.1.138) was isolated from Agaricus bisporus by fractionation with protamine sulphate and (NH₄)₂SO₄, followed by chromatography on DEAE-Sephadex, then by affinity and gel chromatography. The products of enzyme reaction were identified by GLC and TLC. K_m, optimum pH, MW and pI of the enzyme as well as the influence of temperature, ions and inhibitors on enzymic activity were determined. In the sugar reducing reaction, the enzyme was specific for fructose but, in the reverse direction, some structurally related polyols could substitute for mannitol. The enzyme was very sensitive to alterations in the NADP⁺/NADPH ratio. The results are discussed in relation to the possible role of mannitol dehydrogenase in fungal metabolism.     

Purification and properties of NADP+:Isocitrate dehydrogenase from lactating bovine mammary gland

NADP⁺:isocitrate dehydrogenase has been purified to homogeneity from lactating bovine mammary gland. Purification was achieved through the use of affinity and DEAE-cellulose chromatography. The isolated enzyme gives one band when stained for protein or enzyme activity on discontinuous alkaline gel electrophoresis. The enzyme has a molecular weight of 55,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and a Stokes radius of 4.1 nm as measured by gel chromatography. The enzyme will not use NAD⁺ in place of NADP⁺ and has an absolute requirement for divalent cations. The apparent K_m values for dl-isocitrate, Mn²⁺, and NADP⁺ were found to be 8, 6, and 11 μm, respectively. The Mn²⁺-d_s-isocitrate complex is the most likely substrate for the mammary enzyme with a K_m of 3 μm. The properties of mammary NADP⁺:isocitrate dehydrogenase are compared with those of the homologous enzymes from pig heart and bovine liver, and its characteristics are discussed with respect to the function of the enzyme in lactating mammary gland.     

Activation of 17beta-hydroxysteroid dehydrogenase from human red blood cells

Experiments designed to elucidate the nature of 17β-hydroxysteroid dehydrogenase from human red blood cells have shown that NADP⁺ activates and protects the enzyme, while also serving as substrate for the reaction. Enzyme activity was measured by the conversion of 17β-estradiol to estrone and by the production of NADPH with 17β-estradiol-3-sulfate as substrate. It appears that the reaction sequence is first, binding with NADP⁺ and second, binding with the steroid. The binding with NADP⁺ is essentially irreversible: the activated enzyme is completely protected against loss of activity by dilution. On dilution of the unactivated enzyme, much of the activity is lost. The bireactant rate equation of the sequential type has been restated for the case of activation by one of the reactants. Since it has been found that activation of enzyme is linear with NADP⁺ concentration, it follows that the Michaelis constant for the steroid substrate is independent of the concentration of NADP⁺ activating the enzyme. This is substantiated by the determination of the Michaelis constant for 17β-estradiol-3-sulfate from data on double-reciprocal plots of activated and unactivated enzyme with limiting amounts of steroid. The activating effect increases linearly up to a concentration of 1.2 × 10^?5m of NADP⁺ and then levels off. The activation is highly specific for NADP⁺; neither NAD⁺, ATP, NADPH, nicotinic acid, ncr nicotinamide prevent the loss of activity after storing the enzyme for 1 hr at 37 °C. The steroid substrate appears to interfere with the activation of NADP⁺.     

Alkaloid production during the cultivation with shaking of Claviceps sp: Effects of asparagine

Among the nitrogen sources tested, asparagine stimulated alkaloid production maximally. Ammonium salts supported alkaloid production poorly. During the cultivation with shaking of Claviceps sp. strain SD-58 in asparagine containing medium, the activity of asparagine increased during the exponential growth (up to 8 days) with the intracellular accumulation of ammonium ions. Among the ammonia-assimilating enzymes we studied, NADP⁺-glutamate dehydrogenase (GDH) had a higher activity in the growth phase (up to 6 days), while in the intensive alkaloid producing phase (after 6 days) the activity of glutamine synthetase was higher. The latter was associated with increases in the intracellular level of tryptophan and alkaloid production.The levels of NADP⁺- and NAD⁺-alanine dehydrogenases and glutamate synthase were negligible.     

Isocitrate dehydrogenase from Rhodopseudomonas spheroides: kinetic mechanism and further characterization

Product inhibition studies with Rhodopseudomonas spheriodes NADP⁺ specific isocitrate dehydrogenase indicate that the enzyme mechanism involves the ordered addition of the substrates NADP⁺ and threo-d_s-isocitrate and the ordered release of products CO₂ (HCO_s^?), 2-ketoglutarate, and NADPH. In addition, the presence of a ternary complex consisting of enzyme, NADP⁺, and 2-ketoglutarate is indicated. Binding studies with radioactive substrates support the kinetically derived mechanism. The Rhodopseudomonas enzyme is dimeric and contains but a single active site. Different combinations of substrate were ineffective in causing gross changes in molecular structure as monitored by gel filtration techniques. A comparison of the amino acid composition of this enzyme with the bacterial enzyme from Azotobacter vinelandii indicate very significant differences in the amino acid compositions.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

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

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_m 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.     

Metabolism of glycyrrhetic acid by rat liver microsomes: glycyrrhetinate dehydrogenase

Glycyrrhetic acid, derived from a main component of liquorice, was converted to 3-ketoglycyrrhetic acid reversibly by rat liver homogenates in the presence of NADPH or NADP⁺. Glycyrrhetic acid-oxidizing and 3-ketoglycyrrhetic acid-reducing activities were localized in microsomes among the subcellular fractions of rat liver. Glycyrrhetic acid-oxidizing activity and 3-ketoglycyrrhetic acid-reducing activities showed pH optima at 6.3 and 8.5, respectively, and required NADP⁺ or NAD⁺ and NADPH or NADH, respectively, indicating that these activities were due to glycyrrhetinate dehydrogenase. The dehydrogenase was not solubilized from the membranes by the treatment with 1 M NaCl or sonication, indicating that the enzyme is a membrane component. The dehydrogenase was solubilized with detergents such as Emalgen 913, Triton X-100 and sodium cholate, and then separated from 3β-hydroxysteroid dehydrogenase (5β-androstan-3β-ol-17-one-oxidizing activity) by butyl-Toyopearl 650 M column chromatography. Partially purified enzyme catalyzed the reversible reaction between glycyrrhetic acid and 3-ketoglycyrrhetic acid, but was inactive toward 3-epiglycyrrhetic acid and other steroids having the 3β-hydroxyl group. The enzyme required NADP⁺ and NADPH for the highest activities of oxidation and reduction, respectively, and NAD⁺ and NADH for considerable activities, similar to the results with microsomes. From these results the enzyme is defined as glycyrrhetinate dehydrogenase, being quite different from 3β-hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestine, which is active for both glycyrrhetic acid and steroids having the 3β-hydroxyl group.     

Purification and properties of 6-phosphogluconate dehydrogenase fromMytilus galloprovincialis digestive gland

1. The enzyme 6-phosphogluconate dehydrogenase from digestive gland ofMytilus galloprovincialis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis.2. The enzyme was purified 229-fold with a final specific activity of 2.3 μmol of NADP⁺ reduced/min per mg of protein and overall yield of 10%.3. The molecular weight of the native enzyme is estimated to be 100, 000 from gel-filtration studies.4. The influence of pH and MgCl₂ concentration on enzyme activity have been studied and the results have been compared to those reported by other authors for enzymes from different sources.5. TheK_mvalues for 6-phosphogluconate and NADP⁺ at room temperature (20°C) are approx. 20 and 40 μM respectively.6. NADPH was an inhibitor strictly competitive with respect to NADP⁺ (K_i = 14 μM) and non-competitive with respect to 6-phosphogluconate (K_i = 45 μM).     

Glyceraldehyde-3-phosphate dehydrogenases from Euglena gracilis. Purification and physical and chemical characterization.

NAD⁺-dependent and NADP⁺-dependent glyceraldehyde-3-phosphate (G-3-P) dehydrogenases were isolated from Euglena gracilis and characterized as to their physical and chemical parameters. NAD⁺-G-3-P dehydrogenase was found to have a strong resemblance to similar enzymes from muscle tissue. It has a molecular weight of about 140,000, four subunits of identical size and charge, and a single species of NH₂-terminal amino acid. Two sulfhydryl groups per subunit are present, one of which is directly involved in the catalytic activity and is rapidly titratable. The enzyme also exhibits the “half the sites reactivity” of sulfhydryl groups as defined by O. P. Malhotra and S. A. Bernhard ((1968) J. Biol. Chem. 243, 1243). The pH and temperature optima are also similar to those of the enzymes from muscle tissue, as are the reaction kinetics and the strict specificity for NAD⁺.NADP⁺-dependent G-3-P dehydrogenase is different in many respects. Its molecular weight is slightly lower (~136,000) than that of the NAD⁺ enzyme, though it also consists of four subunits. It has a higher affinity for the reverse reaction substrates, in line with its probable function in vivo in CO₂ fixation. There is only one sulfhydryl group per subunit, and that is not involved in activity, suggesting a difference in reaction mechanisms between the two enzymes. The NADP⁺-dependent enzyme exhibits activation by ATP, whereas the NAD⁺-dependent enzyme is competitively inhibited by this nucleotide.The greatest difference observed is in the physical characteristics of the enzymes. NADP⁺-G-3-P dehydrogenase was highly hydrophobic. Its solubility in a 10% aqueous solution of p-dioxane was approximately four to five times that of the NAD⁺-enzyme. Isolation of the enzyme was accomplished by fractionation in 1,2-dimethoxyethane, which also stabilized the enzymatic activity, as did aqueous p-dioxane. The high axial ratio of the NADP⁺-enzyme (~9) coupled with its very low degree of hydration as well as the high degree of amidation of the dicarboxylic amino acids (>90%) indicates that the exterior of the enzyme molecule is probably hydrophobic in nature. This is in agreement with its in vivo hydrophobic environment in the chloroplast membrane and explains the lability of the enzyme once extracted into an aqueous environment as well as its stabilization in solvents.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.     

Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.     

Partial purification and characterization of geissoschizine dehydrogenase from suspension cultures of Catharanthus roseus

The characterization and partial purification of geissoschizine dehydrogenase from Catharanthus roseus cell suspension cultures are described. The 35-fold purified enzyme removes the 21α-hydrogen of geissoschizine in a NADP⁺-dependent reaction. NAD⁺, FAD or FMN cannot act as cofactors for the dehydrogenation. Structurally related indole alkaloids are not dehydrogenated. In comparison to enzymes of the ajmalicine pathway, geissoschizine dehydrogenase shows an extremely low specific activity.     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.