6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L

The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl₂ stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6-phosphogluconate were 12 and 9.6 micromolar and for NADP⁺ were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development.     

The effect of some phenolic compounds on the activity of 6-phosphogluconate dehydrogenase from tobacco tissue cultures

Anodic polyacrylamide gel electrophoresis of extracts of cultures of tobacco tissue Nicotiana tabacum W-38 revealed the presence of two 6-phosphogluconate dehydrogenases (6PGD). The slow and the fast anodic migrating zones were designated I and II, respectively. After purification, enzymes from both zones exhibited no major differences in their affinity towards 6-phosphogluconate (6PG) or NADP⁺, and were found to have approximately the same pH optima and MWs (69 000–72 000). The coumarins scopoletin and esculetin showed some inhibitory effect on each isozyme at 0.4 mM. Below 0.3 mM, however, esculetin stimulated the activity of zone I when lower amounts of 6PG (S_0.25) were used. The glucosylated compounds, scopolin and esculin, were much more inhibitory towards the 6PGDs than their respective aglycones. Ferulic, p-coumaric and caffeic acids seemed to have an inhibitory effect dependent on 6PG concentration. A larger inhibition was observed in each case at the lower 6PG levels used. Zone I activity appeared to be inhibited to a greater degree than zone II activity by 0.4 mM p-coumaric acid with low 6PG. Of the phenolic compounds tested, chlorogenic acid was most effective, completely inhibiting the enzyme activity at 0.4 mM. Of the non-phenolic compounds investigated, glucose 1,6-diphosphate inhibited both isoenzymes of 6PGD at lower 6PG concentrations. On the other hand, 2,3-diphosphoglycerate activated both isoenzymes up to 200% of their original activity.     

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.     

Characterization of human alcohol dehydrogenase isoenzymes by high-performance liquid chromatographic peptide mapping

Human liver alcohol dehydrogenase (ADH, EC 1.1.1.1) is a large and heterogeneous family of isoenzymes and the high-performance liquid chromatographic peptide mapping technique which was developed here recognizes differences and similarities between them. Isoenzymes were S-carboxymethylated, digested with trypsin, and the mixtures of tryptic peptides fractionated by reverse-phase gradient chromatography on octadecylsilane columns, using perchlorate-phosphate buffer and acetonitrile as eluants. The resultant peptide maps were reproducible, showing great similarities between the αβγ-ADH isoenzymes (now called Class I) on the one hand and remarkable differences between these and both the π- and χ-ADH isoenzymes (now called Class II and III, respectively) on the other. This implies that these three isoenzyme groups have characteristic primary structures which correspond to their typical substrate specificities and kinetics.     

Studies on muscular dystrophy: a comparison by physical and chemical means of the normal human ATP-creatine transphosphorylases (creatine kinases) with those from tissues of Duchenne muscular dystrophy.

The four human Duchenne dystrophic isoenzymes (M-M, M-B, B-B, from the muscle and B-B from the brain) of ATP-creatine transphosphorylase (S. A. Kuby, H. J. Keutel, K. Okabe, H. K. Jacobs, F. Ziter, D. Gerber, and F. H. Tyler, 1977, J. Biol. Chem.252, 8382–8390) have now been compared physically and chemically with their normal human counterparts (viz., with the three isoenzymes, M-M, M-B, B-B, 2). All isoenzymes proved to be composed of two noncovalently linked polypeptide chains, by sedimentation equilibrium analyses in the presence and absence of disruptive agents. In the presence of 2-mercaptoethanol at 0.16(Γ/2), pH 7.8, the two native muscle types yielded identical values for s_20,w, concentration dependencies, and molecular weight, and similarly for the brain types (from the brain). But the human brain type proved to be slightly heavier than the muscle type (viz. 88,400 vs 85,900). All of the isoenzymes showed similar electrophoretic behavior between their several counterparts between pH 5–8, except perhaps between pH 8–10, where small differences appeared. The three native normal human isoenzymes, as well as the dystrophic human isoenzymes (M-M from the muscle and B-B from the brain) all contain 2 reactive sulfhydryl groups per mole or 1 per polypeptide chain of these two-chain proteins, which may be titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of 10 -SH groups per mole of each brain type and 8 -SH groups per mole of muscle type, in the case of man, dystrophic man, calf, and rabbit. The kinetics of reactions between Nbs₂ and the sulfhydryl groups of all three normal human isoenzymes and two dystrophic human isoenzymes have been measured under several sets of denaturing conditions. A comparison of their reactive calculated second-order velocity constants reveal significant differences between these three normal human isoenzymes, but the k_{second order} values for the reactions of the sulfhydryl groups of the dystrophic M-M and B-B with Nbs₂, when compared with their normal counterparts, gave identical values in the presence of 7.3 m urea or 1.8% laurylsulfate, from which it may be inferred that very similar, if not identical, environments surround these two sets of sulfhydryl groups. A comparison of the amino acid compositions of the normal human muscle type and brain type with the human dystrophic M-M and B-B (from the brain) reveal essentially identical values for the muscle types but nearly identical values for the brain types, with a few differences. Their respective tryptic peptide maps have been compared of the S-carboxy-methylated proteins (alkylated with iodo[2-¹⁴C]acetic acid at the two exposed -SH groups per mole). Thus, the muscle types, normal and dystrophic, yield identical maps, but the brain types nearly identical maps, with a few significant differences. Isolation of the tryptic tridecapeptide from the S-carboxymethylated normal human and dystrophic human dimeric muscle-type ATP-creatine transphosphorylases, labeled at the single exposed SH group per polypeptide chain with iodo[2-¹⁴C]acetate, yielded the following sequence for both proteins: ValLeuThrCys(CH₂COOH)ProSerAsnLeuGlyThr GlyLeuArg [where Cys(CH₂COOH) is S-carboxymethyl cysteine]. This sequence showed remarkable homology with a few other equivalent peptides reported to be derived from the exposed SH group of other ATP-creatine transphosphorylases. In conclusion, there does not appear to be a mutation in the structural genes for the muscle-type creatine kinases detectable by the analyses presented here. However, the brain types warrant further investigation.     

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.     

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     

Incorporation of carbohydrate residues into peroxidase isoenzymes in horseradish roots

Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-¹⁴C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-³H and mannose-U-¹⁴C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of ¹⁴C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems.     

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.     

Purification and structural comparisons of the cytosolic and mitochondrial fumarases from baker's yeast

1. The cytosolic and mitochondrial fumarases (EC 4.2.1.2) from baker's yeast (Saccharomyces cerevisiae) have been purified to homogeneity. 2. Subunit molecular weights for the cytosolic and mitochondrial isoenzymes were 53,000 and 48,000 respectively. 3. Peptide maps obtained after digestion of the two isoenzymes with trypsin were almost identical but showed significant small differences. The same was true of peptide maps obtained after digestion with the glutamic acid-specific proteinase from S. aureus.     

Decrease of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenase Activities in the Xylem of Populus gelrica on Budding

The activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases, transketolase, phosphoglucose isomerase, and fructose 6-phosphate kinase were studied in extracts of wintering poplar (Populus gelrica) xylem. The xylem of wintering poplar showed high levels of transketolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases. On recommencement of growth, the two dehydrogenase activities decreased. The three remaining enzymes appeared to be unchanged. In spring and early summer, glucose 6-phosphate dehydrogenase of the xylem was extremely low. On the other hand, 6-phosphogluconate dehydrogenase, which also became lower during the metabolic shift from winter to spring, was readily detected, and was several times higher than glucose 6-phosphate dehydrogenase throughout the year. The low dehydrogenase activities lasted into late October and then appeared to resume their original activity. A shift of metabolism at the beginning of growth was also observed by measuring the amount of sugar phosphates, soluble amino acids and amides, and proteins in the xylem. In contrast to the decrease of the two dehydrogenases and soluble proteins at the time of budding, incorporation of lysine-U-¹⁴C into the xylem protein ramained constant. A method to transfuse radioactive compounds into a section of stem was described.     

Subunit composition and substrate binding region of potato l-lactate dehydrogenase

Four of the six electrophoretically distinguishable isoenzymes of the l-lactate dehydrogenase (EC 1.1.1.27) from potato tubers were purified from crude extracts. The isoenzymes are tetrameric and exhibit MWs around 145000. They are composed of mixtures of different subunits. Two of the isoenzymes together contain at least three, the other two together contain six different subunits indicating that the actual number of isoenzymes may be even greater than the number of electrophoretically detectable isoenzymes. Since the isoenzymes agree largely with respect to their enzymatic properties and to their primary structure as suggested from fingerprinting and amino acid analysis, it is suggested that the variation of the subunits is caused by proteolytic processing in vivo rather than by different genetic coding. The amino acid sequence of the substrate-binding region (Arg₆ peptide) shows a high homology to that of the l-lactate dehydrogenases of animals and bacteria indicating a common origin of plant, animal and bacterial enzymes.     

Purified desmin from adult mammalian skeletal muscle: a peptide mapping comparison with desmins from adult mammalian and avian smooth muscle.

Comparative one-dimensional peptide maps were prepared by the electrophoresis of digests derived from treatment of desmins with Ca²⁺-activated muscle protease, trypsin, $\text{Staphylococcus}\text{aureus}$ V8 protease, and cyanogen bromide. Desmins from adult mammalian skeletal and smooth muscles were very similar. Avian smooth muscle desmin, although homologous with respect to many peptides, was different from the mammalian smooth and skeletal desmins. The amino acid compositions of the three desmins were quite similar.     

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

Electrophoretic studies of several dehydrogenases in relation to the cold tolerance of alfalfa.

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening conditions to determine the solubility characteristics and relationships of several dehydrogenases to cold tolerance.Soluble enzymatic proteins, extracted with three extractants, from lyophilized crown and root tissues, were separated by polyacrylamide disc gel electrophoresis.Gels assayed for glutamate, NAD-malate, NADP-malate, isocitrate, lactate, 6-phosphogluconate, and glucose-6-phosphate dehydrogenases showed quantitative differences in isoenzymes that were influenced by cultivar, extractant, and environmental differences.For both cultivars, enzyme activity was lowest during summer, increased in winter, and decreased during dehardening. Dehydrogenase activity, therefore, was closely associated with the fluctuations in soluble protein concentration, which were related to environmental changes and cold tolerance.Additional isoenzymes of isocitrate, lactate, and glucose-6-phosphate dehydrogenases were detected in the winter samples of both cultivars; however, most of the qualitative differences observed were generally due to the differential solubilities of isoenzymes in the three extractants.Comparison of data obtained from the use of frozen and unfrozen extracts indicated differential stabilities of the dehydrogenases to freezing in the different extractants. Glutamate, NAD-malate, and NADP-malate dehydrogenases were fairly stable to freezing whereas isocitrate, lactate, 6-phosphogluconate, and glucose-6-phosphate dehydrogenases were labile. Detectable levels of the latter dehydrogenases in frozen extracts were evident only in certain extracts of winter samples, indicating the importance of the nature of the extraction medium in protecting against enzyme denaturation.Since both cultivars showed similar changes in dehydrogenase activities at most times, the increased enzyme levels during winter coincided with increased levels of soluble protein and soluble sugars, which are indicative of the broad spectrum of metabolic changes involved in the attainment of the cold-tolerant state.     

Amino Acid Sequence of Mung Bean Trypsin Inhibitor and Its Modified Forms Appearing during Germination

The amino acid sequence of the major trypsin inhibitor, F, of ungerminated mung beans (Vigna radiata [L.] Wilczek) was determined by a combination of automatic solid phase and manual sequencing techniques. F is a typical Bowman-Birk-type proteinase inhibitor with 80 amino acid residues and exhibits a high degree of identity with the other sequenced members of the Bowman-Birk family of inhibitors. Thin layer peptide maps of mung bean inhibitors E and C (which appear during germination) indicate that both are derived from inhibitor F by limited specific proteolysis. Loss of the carboxyl-terminal residues 77 to 80 from F produces inhibitor E, while the loss of an additional two carboxyl-terminal residues, the loss of the amino-terminal residues 1 to 8, and an internal cleavage at Ala³⁵-Asp³⁶ produces inhibitor C from E. Another inhibitor species, E′, was isolated from ungerminated seeds. It differs from F in the loss of residues 1 to 6. The majority of the proteolytic cleavages noted in the F-E-C-E′ system are at peptide bonds involving aspartyl residues.     

Identification of two binding sites of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach for D-ribulose 1,5-bisphosphate and effectors of the carboxylation reaction.

Fluorimetric studies of the binding of d-ribulose 1,5-bisphosphate (RuP₂) and the effectors 6-phosphogluconate and fructose 1,6-bisphosphate to the d-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach were correlated with the functions of these sugar phosphates in the carboxylation reaction. These agents compete for two binding sites of the enzyme. At relatively low concentrations they bind to an allosteric site, where 6-phosphogluconate and fructose 1,6-bisphosphate display their stimulating effect on the fixation of CO₂. At higher concentrations these compounds inhibit the carboxylation reaction and compete with RuP₂ for the reaction center of the carboxylase. Preincubation of the enzyme with low concentrations of RuP₂ (0.1–5 μm) inhibits the activity of these effectors as well as the effector-induced fluorescence changes of the enzyme-2-p-toluidinonapthalene-6-sulfonate (TNS) complex by competition for the regulatory center which could be identified as the high affinity binding site of the enzyme for RuP₂ with a K_D = 0.6 μm. The deactivation of the carboxylase which is observed on preincubation of the enzyme with RuP₂ in the absence of bicarbonate and Mg²⁺ cannot be correlated to the binding of RuP₂ to the effector site. The deactivation process occurs in an RuP₂ concentration range similar to that for CO₂ fixation.     

Activation of Clostridium botulinum type E toxin purified by two different procedures

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.     

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.     

Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli

The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories. Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C. elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C. elegans 6-phosphogluconate dehydrogenase gene. The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing. The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence. The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences. Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences. The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid–E. coli system (pQE30). The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity. Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer. Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity. The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The C. elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree. Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters.