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.     

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.     

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.     

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).     

NAD+ -dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD⁺-dependent and NADP⁺-dependent malic enzyme activities. The NAD⁺ malic enzyme exhibited more activity with NAD⁺ as cofactor, but also showed some activity with NADP⁺. The NADP⁺ malic enzyme only showed activity when NADP⁺ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NADP⁺ malic enzyme activity (dme) but retained NADP⁺ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N₂. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N₂-fixation would normally begin. These results support the proposal that NAD⁺ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N₂-fixing bacteroids which metabolize C₄-dicarboxylic acids supplied by the plant.     

Isoenzymes of glucose 6-phosphate dehydrogenase from the plant fraction of soybean nodules

Two isoenzymes of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been separated from the plant fraction of soybean (Glycine max L. Merr. cv Williams) nodules by a procedure involving (NH₄)₂SO₄ gradient fractionation, gel chromatography, chromatofocusing, and affinity chromatography. The isoenzymes, which have been termed glucose 6-phosphate dehydrogenases I and II, were specific for NADP⁺ and glucose 6-phosphate and had optimum activity at pH 8.5 and pH 8.1, respectively. Both isoenzymes were labile in the absence of NADP⁺. The apparent molecular weight of glucose 6-phosphate dehydrogenases I and II at pH 8.3 was estimated by gel chromatography to be approximately 110,000 in the absence of NADP⁺ and double this size in the presence of NADP⁺. The apparent molecular weight did not increase when glucose 6-phosphate was added with NADP⁺ at pH 8.3. Both isoenzymes had very similar kinetic properties, displaying positive cooperativity in their interaction with NADP⁺ and negative cooperativity with glucose 6-phosphate. The isoenzymes had half-maximal activity at approximately 10 micromolar NADP⁺ and 70 to 100 micromolar glucose 6-phosphate. NADPH was a potent inhibitor of both of the soybean nodule glucose 6-phosphate dehydrogenases.     

The distribution of the NADPH regenerating mannitol cycle among fungal species

The mannitol cycle is an important NADPH regenerating system in Alternaria alternata. The cycle is built up of the following enzymes: mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, mannitol dehydrogenase and hexokinase. The net reaction of one cycle turn is: NADH+NADP⁺+ATP NAD⁺+NADPH+ADP+P_i. The enzymes needed for an operating cycle were found in Aspergillus, Botrytis, Penicillium, Pyricularia, Trichothecium, Cladosporium and Thermomyces all genera belonging to Fungi Imperfecti. The only genus of this class lacking the cycle was Candida. No genera from the classes Basidiomycetes and Phycomycetes showed any mannitol 1-phosphate dehydrogenase or mannitol 1-phosphatase activities. The genera investigated, belonging to Ascomycetes, Gibberella, Ceratocystis and Neurospora all lacked mannitol 1-phosphate dehydrogenase. It was concluded that the mannitol cycle is an important and widespread pathway for NADH oxidation and NADP⁺ reduction in the organisms belonging to the class Fungi Imperfecti.     

NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182

A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP⁺by an NADP⁺-linked malic enzyme [l-malate: NAD(P)⁺oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP⁺, 67 μmol l-malic acid, 63 μmol MgCl₂ and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP⁺ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ～12 μmol ml^?1of the reaction mixture. No dephosphorylation of NADP⁺to NAD⁺or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)⁺-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].     

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.     

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.     

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.     

Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase

Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate:NADP⁺ 2-oxidoreductase (decar☐ylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of M_r 70 000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2′-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.     

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.     

Assimilation of ammonia inParacoccus denitrificans

Two pathways serve for assimilation of ammonia inParacoccus denitrificans. Glutamate dehydrogenase (NADP⁺) catalyzes the assimilation at a high NH₄ ⁺ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH₄ ⁺ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP⁺) and glutamate-ammonia ligase inP. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP⁺). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP⁺) by the high concentration of glutamine or its metabolic products as in the case when NH₄ ⁺ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP⁺) was observed.     

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.     

Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO₂ into cell material. The product of the initial acetyl-CoA carboxylation with CO₂, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP⁺ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP⁺ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP⁺, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP⁺. Evolution from an NADP⁺ to a bispecific NADP⁺ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP⁺ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.     

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.     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.