In Vitro Inhibition of the Plastid and Cytosolic Isozymess of 6-Phosphogluconate Dehydrogenase from Developing Endosperm of Ricinus communis by Fructose 2,6-bisphosphate

Activities of the cytosolic and plastid isozymes of 6-phosphogluconate dehydrogenase from developing endosperm of Ricinus communis L. seeds were inhibited in vitro by hexosebisphosphates. Inhibition constants for glucose 1,6-bisphosphate were 221 and 209 micromolar for the cytosolic and plastid isozymes, respectively, and corresponding values for fructose 2,6-bisphosphate were 10.5 and 8.6 micromolar. In each case inhibition was of a mixed noncompetitive nature relative to 6-phosphogluconate. While the levels and distribution of fructose 2,6-bisphosphate in castor oil seed endosperm cells are not yet known, the levels reported to occur in leaf cytosol would be high enough to significantly inhibit carbon flux through the pentosephosphate pathway due to inhibition of 6-phosphogluconate dehydrogenase activity.     

Comparison of tryptic peptide maps of two isoenzymes of 6-phosphogluconate dehydrogenase from tobacco tissue cultures

In a previous study by the authors, two isoenzymes of 6-phosphogluconate dehydrogenase were isolated from cultures of tobacco tissue Nicotiana tabacum W-38 and shown to be similar in their pH optima and MWs and in their affinities toward 6-phosphogluconate or NADP⁺. In an attempt to clarify the structural relationships between these two isoenzymes, peptide mapping of trypsin digests of the purified isoenzymes was performed. The maps were found to be similar, with at least 29 peptide groups from the trypsin digestion of each isoenzyme being alike. There were, however, definite minor differences in the peptide maps of the two isoenzymes.     

Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves

Isoenzymes of glucose-6-phosphate dehydrogenase and 6-P-gluconate dehydrogenase from a 70% ammonium sulfate precipitate of spinach leaf homogenate were separated by differential solubilization in a gradient of 70-0% ammonium sulfate and analyzed by disc gel electrophoresis. Isolated whole chloroplasts contained isoenzyme 1 of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase 1, whereas isoenzyme 2 of each was found in the soluble cytosol fraction. Both isoenzymes of each dehydrogenase were present in about equal amounts. Glucose-6-phosphate dehydrogenase isoenzymes 1 and 2 had pH optima of 9.2 and 9.0 and K_m values of 400 and 330 μm, respectively. Molecular weights for both isoenzyme of glucose-6-phosphate dehydrogenase were very similar at about 105,000 ± 10% as estimated by sedimentation velocity measurements. For 6-phosphogluconate dehydrogenase isoenzymes 1 and 2 the pH optima were 9.0 and 9.3, respectively, the K_m values were 100 and 80 μm, and the apparent molecular weights were also nearly identical at about 110,000 ± 10%. The data support the hypothesis that leaf cells have two oxidative pentose phosphate pathways, one in the chloroplast and the other in the cytosol.     

Purification and characterization of cytosolic 6-phosphogluconate dehydrogenase isozymes from maize

Cytosolic isozymes of 6-phosphogluconate dehydrogenase were purified from roots of maize (Zea mays L.). The final preparation contained two 55-kD proteins. Affinity-purified dehydrogenases from a maize line that is null for both cytosolic 6-phosphogluconate dehydrogenase isozymes (Pgd1-null, Pgd2-null) lacked the 55-kD proteins. The substrate kinetics of the purified enzyme were determined.     

Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L

The metabolism of sucrose to long chain fatty acids in the endosperm of developing castor bean (Ricinus communis L.) seeds requires a combination of cytosolic and proplastid enzymes. The total activity and the subcellular distribution of the intermediate enzymic steps responsible for the conversion of sucrose to pyruvate have been determined. Hexose phosphate synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase. The proplastids contain the necessary complement of glycolytic enzymes to account for the in vivo rates of acetate synthesis from glucose 6-phosphate. These organelles also contain the majority of the cellular 6-phosphogluconate dehydrogenase, transketolase, and transaldolase activities.     

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.     

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.     

Isoenzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinis communis L

Two isoenzymes each of hexose-P isomerase, aldolase and 6-P-gluconate dehydrogenase have been found in the endosperm of developing castor beans (Ricinus communis L.). One isoenzyme for each activity is present in the proplastid fraction. Only one form of glucose-6-P dehydrogenase was found. It is suggested that the partition of an enzyme activity between cytosol and plastid is regulated by the synthesis of isoenzymes which are subcellular site specific. In addition, this report describes the use of diethylaminoethyl-Sephadex A-25 sievorptive chromatography for the preparation of plant enzymes.     

Some properties of a ficin-papain inhibitor from avian egg white

A procedure has been established for the isolation, from sheep liver, of 6-phosphogluconate dehydrogenase which is homogeneous according to the criteria of the analytical ultracentrifuge, and isoelectric focusing. A systematic determination of the effects of pH, ionic strength, metal ions, and temperature, on the kinetic parameters of the isolated 6-phosphogluconate dehydrogenase has been carried out. Double-reciprocal plots of enzyme rate measurements as a function of substrate concentration indicate K_m values of 15 μm for 6-phosphogluconate, and 7 μm for NADP⁺, under optimum assay conditions, and show no effect of the concentration of one substrate on the K_m of the other substrate under the assay conditions employed. Ionic strength, monovalent and divalent metals, are apparently interchangeable in their ability to activate the enzyme, and act by decreasing the K_m values of the enzyme, not by increasing the reaction rate. Concentrations of metals above the optimum are strongly inhibitory. Plots of ?log K_m vs pH show inflection points at 8.3 for 6-phosphogluconate, and 6.5 for NADP⁺. At low substrate concentrations the pH optimum of the enzyme is at pH 7.7, but plots of V vs pH increase up to pH 9.1 (the enzyme is unstable at higher pH values). An Arrhenius plot shows a straight line between temperatures of 8.6 and 39.4 °C, and an energy of activation of 15,450 cal mole^?1.     

Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway

The aim of this work was to examine the extent to which the oxidative steps of the pentose phosphate pathway in the cytosol contribute to the provision of reductant for biosynthetic reactions. Maize (Zea mays L.) contains at least two loci (pgd1 and pgd2) that encode 6-phosphogluconate dehydrogenase. Ten genotypic combinations of wild-type (Pgd1+3.8;Pgd2+5) and null alleles of pgd1 and pgd2 were constructed in the B73 background. The maximum catalytic activity of 6-phosphogluconate dehydrogenase in the roots of seedlings of these lines correlated with the number of functional pgd1 and pgd2 alleles. Enzyme activity in the double-null homozygote (pgd1-null;pgd2-null) was 32% of that in B73 wild-type suggesting the presence of at least one other isozyme of 6-phosphogluconate dehydrogenase in maize. Subcellular fractionation studies and latency measurements confirmed that the products of pgd1 and pgd2 are responsible for the vast majority, if not all, of the cytosolic 6-phosphogluconate dehydrogenase activity in maize roots. Essentially, all of the residual activity in the double-null homozygote was confined to the plastids. Low concentrations (0.1–0.5 mM) of sodium nitrite stimulated ¹⁴CO₂ production by detached root tips of both wild-type and 6-phosphogluconate dehydrogenase-deficient maize seedlings fed [U-¹⁴C]glucose. Analysis of the ratio of ¹⁴CO₂ released from [1–¹⁴C]glucose relative to [6–¹⁴C]glucose (C1/C6 ratio) showed that stimulation of the oxidative pentose phosphate pathway by nitrite correlated with the dosage of wild-type alleles of pgd1 and pgd2. The failure of 6-phosphogluconate dehydrogenase-deficient lines to respond to nitrite indicates that perturbation of the cytosolic oxidative pentose phosphate pathway can influence the provision of reductant in the plastid. We conclude that the plastidic and cytosolic oxidative pentose phosphate pathways are able to co-operate in the provision of NADPH for biosynthesis.     

Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans

Two isoenzymes each of phosphoglucomutase, hexose phosphate isomerase, aldolase, fructose diphosphatase, phosphofructokinase, and 6-phosphogluconate dehydrogenase have been separated by DEAE-cellulose column chromatography of extracts from endosperm of germinating castor beans (Ricinus communis cv. Hale). One of each of the enzymes is localized in the cytosol and the other is confined to plastids. Developmental studies of these isoenzymes were carried out to clarify their roles in the endosperm. In extracts from ungerminated seeds the activities of marker enzymes of mitochondria (fumarase), plastids (ribulose bisphosphate carboxylase), and glyoxysomes (catalase) were low, but phosphoglucomutase, hexose phosphate isomerase, aldolase, and 6-phosphogluconate dehydrogenase were present in relatively high activity. The total amounts of these enzymes increased 3- to 4-fold during the first 5 days of growth. The activities of isoenzymes in the plastids rose in parallel with that of ribulose bisphosphate carboxylase to reach a maximum at day 4, and like the carboxylase they declined sharply thereafter. The activities of the cytosolic isoenzymes peaked at day 5. These changes are consistent with the roles previously proposed for the sequences present in plastid and cytosol.     

Repression of the Plastidic Isoenzymes of Aldolase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase in the Barley Mutant "albostrians"

White leaves of the mutant line albostrians and green leaves of the wild-type cultivar Salome of barley (Hordeum vulgare L.) were screened for the presence of plastidic and cytosolic isoenzymes of sugar-phosphate metabolism. Isoenzyme separation was achieved by anion-exchange chromatography on Fractogel TSK DEAE-650(S). The mutant tissue had a markedly reduced level of plastidic 3-phosphoglycerate kinase, triosephosphate isomerase, and aldolase activity. In contrast, the activity of plastidic glucosephosphate isomerase, fructose 1,6-bisphosphatase, 6-phosphogluconate dehydrogenase, starch phosphorylase, and ADP-glucose pyrophosphorylase was in the same range as in wild-type leaf tissue. The activity of the corresponding cytosolic isoenzymes (including UDP-glucose pyrophosphorylase) showed essentially no differences in mutant and wild type. The same trend was observed in dark-grown mutant and wild-type leaves. Interestingly, the total activity levels of all isoenzymes were about the same when comparing dark-grown and light-grown mutant or wild-type plants. From these data, it is concluded that mutant leaves exhibit a selective decrease of a subgroup of plastidic isoenzymes associated with the Calvin cycle.     

Capacity for NADPH/NADP turnover in the cytosol of barley seed endosperm: The role of NADPH-dependent hydroxypyruvate reductase

Barley (Hordeum vulgare L.) endosperm from developing seeds was found to contain relatively high activities of cytosolic NAD(P)H-dependent hydroxypyruvate reductase (HPR-2) and isocitrate dehydrogenase (ICDH). In contrast, activities of peroxisomal NADH-dependent hydroxypyruvate reductase (HPR-1) and glycolate oxidase as well as cytosolic NAD(P)H-dependent glyoxylate reductase were very low or absent in the endosperm both during maturation and seed germination, indicating the lack of a complete glycolate cycle in this tissue. In addition, activities of cytosolic glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were low or absent in the endosperm. The endosperm HPR-2 exhibited similar properties to those of an earlier described HPR-2 from green leaves, e.g. activities with both hydroxypyruvate and glyoxylate, utilization of both NADPH and NADH as cofactors, and a strong uncompetitive inhibition by oxalate (K_i in the order of micromolar). In etiolated leaves, both HPR-1 and HPR-2 were present with the same activity as in green leaves, indicating that the lack of HPR-1 in the endosperm is not a general feature of non-photosynthetic tissues. We conclude that the endosperm has considerable capacity for cytosolic NADP/NADPH cycling via HPR-2 and ICDH, the former being possibly involved in the utilization of a serine-derived carbon.     

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.     

Characterization of Plasmodium falciparum 6-Phosphogluconate Dehydrogenase as an Antimalarial Drug Target

The enzyme 6-phosphogluconate dehydrogenase (6PGD) of the malaria parasite Plasmodium falciparum catalyzes the third step of the pentose phosphate pathway converting 6-phosphogluconate (6PG) to ribulose 5-phosphate. The NADPH produced by 6PGD is crucial for antioxidant defense and redox regulation, and ribose 5-phosphate is essential for DNA and RNA synthesis in the rapidly growing parasite. Thus, 6PGD represents an attractive antimalarial drug target. In this study, we present the X-ray structures of Pf6PGD in native form as well as in complex with 6PG or nicotinamide adenine dinucleotide phosphate (NADP⁺) at resolutions of 2.8, 1.9, and 2.9?Å, respectively. The overall structure of the protein is similar to structures of 6PGDs from other species; however, a flexible loop close to the active site rearranges upon binding of 6PG and likely regulates the conformation of the cofactor NADP⁺. Upon binding of 6PG, the active site loop adopts a closed conformation. In the absence of 6PG, the loop opens and NADP⁺ is bound in a waiting position, indicating that the cofactor and 6PG bind independently from each other. This sequential binding mechanism was supported by kinetic studies on the homodimeric wild-type Pf6PGD. Furthermore, the function of the Plasmodium-specific residue W104L mutant was characterized by site-directed mutagenesis. Notably, the activity of Pf6PGD was found to be post-translationally redox regulated via S-nitrosylation, and screening the Medicines for Malaria Venture Malaria Box identified several compounds with IC₅₀s in the low micromolar range. Together with the three-dimensional structure of the protein, this is a promising starting point for further drug discovery approaches.     

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K_m values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg²⁺ and Mn²⁺.     

6-Phosphogluconate dehydrogenase from Drosophila melanogaster. I. Purification and properties of the a isozyme

6-Phosphogluconate dehyrogenase is evident at all developmental stages of Drosophila melanogaster. The activity level is highest in early third instar larvae and declines to a lower, but relatively constant, level at all later stages of development. The enzyme is localized in the cytosolic portion of the cell. The A-isozymic form of 6-phosphogluconate dehydrogenase was purified to homogeneity and has a molecular weight of 105,000. The enzyme is a dimer consisting of subunits with molecular weights of 55,000 and 53,000. For the oxidative decarboxylation of 6-phosphogluconate the K_m for substrate is 81 µm while that for NADP⁺ is 22.3 µm. The optimum pH for activity is 7.8 while the optimum temperature is 37 C.This work was supported by National Research Council of Canada Grant A5860 and by the University of Calgary Research Policy and Grants Committee.     

A survey for isoenzymes of glucosephosphate isomerase,phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C4-and crassulacean-acid-metabolism plants,and green algae

Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH₄)₂SO₄ gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C₃-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C₄-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C₄-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C₄ plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells. New address: Institut für Pflanzenphyiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a, D-1000 Berlin 33     

Rat liver 6-phosphogluconolactonase: A low Km enzyme

By specific enzymic synthesis of the substrate 6-phosphogluconolactone in situ, the K_m for rat liver 6-phosphogluconolactonase was found to be 80 μM. This value is approximately 100 fold lower than the previously determined value, and is compatible with the kinetic paramaters of both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and hence with the flux through the oxidative segment of the pentose phosphate pathway.     

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg²⁺ and exhibited hyperbolic kinetics with all substrates including Mg²⁺. K_m values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. Kinetic constants were determined in the presence of fructose-2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K_i values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.