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     

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M _r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M _r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K _m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K _m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]_0.5) value was 2.5 mM.Abbreviations DEAE diethylaminoethyl - M _r molecular weight     

Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

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.     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH     

Genetics of glucose phosphate isomerase and phosphoglucomutase in Aedes albopictus (diptera: culicidae)

Summary Glucose phosphate isomerase (E.C. 5.3.1.9) and phosphoglucomutase (E.C. 2.7.5.1) were found to be polymorphic in a laboratory colony of Aedes albopictus. The glucose phosphate isomerase locus is represented by two alleles resulting in three genotypes, while the phosphoglucomutase locus is represented by at least five alleles giving rise to a total of 15 genotypes. The inheritance of these two enzymes is of the Mendelian type with codominant alleles. Present data indicate that these genes are not linked.Of 105 mosquitoes analysed for these two gene-enzyme systems, the frequencies for glucose phosphate isomerase alleles are Gpi ^S=0.68 and Gpi ^F=0.32, while the frequencies for phosphoglucomutase alleles are Pgm ^A=0.16, Pgm ^B=0.11, Pgm ^C=0.19, Pgm ^D=0.30 and Pgm ^F= 0.24. The frequencies of the three glucose phosphate isomerase genotypes are in accord with Hardy-Weinberg expectations (X ₁ ² =2.74). Similarly, the frequencies of the 15 phosphoglucomutase genotypes probably do not differ significantly from Hardy-Weinberg expectations (X ₁₀ ² = 18.45).     

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

Electrophoretic Analysis of Isoenzymes in the Identification of Trypanosomatids of the Genus Phytomonas1

Five strains of trypanosomatids of the genus Phytomonas, isolated from different species of Euphorbia {Euphorbia heterophylla, E. characias, E. pinea, E. hyssopifolia) and from Manihot escutenta, were cultured and compared through the electrophoretic mobility of isoenzymes of six enzymes: aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucosephosphate isomerase (EC 5.3.1.9), and malate dehydrogenase (EC 1.1.1.40). The strains could be distinguished from one another by their respective isoenzyme profiles.     

PARTIAL CHARACTERIZATION OF SOLUBLE ACETYLCHOLINESTERASE ISOENZYMES OF THE RAT BRAIN

(1) The 105,000 g supernatant fluid obtained from rat brain was separated by agar-gel electrophoresis. (2) Three isoenzymes, capable of hydrolysing acetylthiocholine, one of them also hydrolysing butyrylthiocholine, were detected. (3) The pH optima and K_m for hydrolysis of acetyl- and butyrylthiocholine by the supernatant fluid were determined. (4) After extraction of acetylcholinesterase isoenzymes from the gel, individual isoenzymes were characterized by pH optima and K_m values. (5) Two of the enzymes were characterized as acetylcholinesterase (EC 3.1.1.7) and one as butyrylcholinesterase (EC 3.1.1.8).     

Fertile progeny of a hybridization between soybean [Glycine max (L.) Merr.] and G. tomentella Hayata

Summary A colchicine-doubled F₁ hybrid (2n=118) of a cross between PI 360841 (Glycine max) (2n=40) x PI 378708 (G. tomentella) (2n=78), propagated by shoot cuttings since January 1984, produced approximately 100 F₂ seed during October 1988. One-fourth of the F₂ plants or their F₃ progeny have been analyzed for chromosome number, pollen viability, pubescence tip morphology, seed coat color, and isoenzyme variation. Without exception, all plants evaluated possessed the chromosome number of the G. max parent (2n=40). Most F₂ plants demonstrated a high level of fertility, although 2 of 24 plants had low pollen viability and had large numbers of fleshy pods. One F₂ plant possessed sharp pubescence tip morphology, whereas all others were blunt-tipped. All evaluated F₂ and F₃ plants expressed the malate dehydrogenase and diaphorase isoenzyme patterns of the G. max parent and the endopeptidase isoenzyme pattern of the G. tomentella parent. Mobility variants were observed among progeny for the isoenzymes phosphoglucomutase, aconitase, and phosphoglucoisomerase. This study suggests that the G. Tomentella chromosome complement has been eliminated after genetic exchange and/or modification has taken place between the genomes.Journal Paper No. J-13776 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, USA, Project 2763     

Purification and Characterization of Class Alpha and Mu Glutathione S-Transferases From Porcine Liver

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M_r = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M_r values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M_r = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.     

Effects of various metabolites on two phosphoglucomutase allozyme activities from Drosophila melanogaster

The effects of various metabolites on the two most common phosphoglucomutase allozymes (PGM^A and PGM^B) in Drosophila melanogaster have been investigated in vitro. 2,3-Diphosphoglycerate (2,3DPG) inhibited PGM^A and PGM^B to the same degree in the presence of 25 µM glucose-1,6-diphosphate (G1, 6P₂). However a higher concentration of G1,6P₂ partially reversed the inhibition of PGM^A exerted by 2,3DPG, so that in the presence of 150 µM G1,6P₂ the inhibition of PGM^A was half that of PGM^B at pH 6.0. Glycerol-3-phosphate (G3P) had no significant effect at pH 7.4 but exerted an activating effect at pH 6.0 which was more pronounced in the case of PGM^B. ATP, citrate, and fructose-1, 6-diphosphate (F1,6P₂) inhibited both PGM^A and PGM^B. The differences found in vitro between these two allozymes can have a significant impact on in vivo function and, therefore, on the maintenance of PGM polymorphism in experimental populations of D. melanogaster studied in the laboratory.     

Prostaglandins, not cyclic GMP, inhibit oxytocinase isoenzymes from human amniotic fluid in vitro

Two isoenzymes of oxytocinase (EC 3.4.11.3) activity were fractionated from human amniotic fluid samples between the 14th and 22nd weeks of gestation by Ultrogel acrylamide-agarose gel filtration and partially characterized. The isoenzymes were competitively inhibited by PGE₁, PGE₂ and PGF_2α more at pH 6.2 than at pH 6.8, whereas cyclic GMP (cGMP) and its 8-bromo derivative had no effect at either pH. The implications of these findings are discussed and it is suggested that since the activity of amniotic fluid oxytocinases is very low or minimal at or near term, inhibition of these by prostaglandins may not have physiological significance in the initiation of human parturition.     

Purification and characterization of chorismate mutase isoenzymes from ruta graveolens l.

Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K₁ = 4 · 10^?6 M) and tyrosine (K₁ = 8. 10^?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The K_m-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens.     

On the role of chloroplastic phosphoglucomutase in the regulation of starch turnover

When spinach (Spinacia oleracea L.) leaf disks were incubated in 10% polyethylene glycol to induce water stress, the ratio of glucose-1-phosphate to glucose-6-phosphate increased. This increase indicated an imbalance in the phosphoglucomutase (EC 2.7.5.1) reaction, which was earlier observed to be close to equilibrium, and was accompanied by higher fructose-1,6-bisphosphate and ribulose-1,5-bisphosphate concentrations. Because starch degradation was assumed to be the source of the glucose-1-phosphate accumulation, the kinetic properties of plastidic phosphoglucomutase were analysed. It was found that physiological concentrations of both sugar bisphosphates inhibited phosphoglucomutase by about 50%. From this observation it was concluded that under conditions in which fructose-1,6-bisphosphate and ribulose-1,5-bisphosphate accumulated, an inhibition of phosphoglucomutase activity restricted the carbon exchange between the Calvin cycle and starch turnover. Received: 23 March 1998 / Accepted: 26 August 1998     

Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize

Electrophoretic characterization of adenosine diphosphate glucose pyrophosphorylase from the developing endosperms of nine shrunken-2 and four brittle-2 mutants revealed that (1) all mutants had low but detectable levels of activity, (2) mutation at either locus decreased activity of pyrophosphorylases A and B, and (3) differences in mobility were not found. However, pyrophosphorylase B extracted from several shrunken-2 and brittle-2 mutants differed from normal in extent of urea denaturation, K _m (glucose-1-phosphate) or type of glucose-1-phosphate saturation kinetics. Pyrophosphorylase B from sh2-m (association of Dissociation with the sh2 locus) appears to differ from normal in K _m (glucose-1-phosphate).This research was supported by the College of Agricultural and Life Sciences and by National Institutes of Health Grant No. 15422.The investigations reported were included in the thesis submitted by L. C. Hannah to the Graduate School, University of Wisconsin, Madison, Wisconsin, in partial fulfillment of requirement for the Ph.D. degree. Laboratory of Genetics Paper No. 1922.     

Glucose and glutamate metabolism ofLegionella pneumophila

Two serotype 1 strains ofLegionella pneumophila, Phildelphia 2 and Bellingham, were tested for their ability to metabolize five common substrates by measuring¹⁴CO₂ released and¹⁴C-carbon incorporated into macromolecules. No major differences were noted between the two strains or preparations grown in the yolk sac of chick embryos or agar-broth diphasic medium, following 2 or 14 pasaages on agar. Glutamate was the most actively metabolized substrate, followed by glutamine. Acetate, glucose, and succinate were utilized at much more moderate rates. Changes in cell density and substrate concentration altered the channeling of glutamate and glucose into CO₂ and macromolecules. Specific CO₂ felease from glutamate was greatest at low cell density and high substrate concentration, while carbon incorporation was increased at high substrate concentration. A reciprocal relationship was noted with glucose: the proportion of carbon incorporation was enhanced at low substrate concentration, but CO₂ release paralleled increases in substrate concentration. The pH optimum for glutamate carbon incorporation and CO₂ release was 5.5 and 6.1, respectively, but 25% of both activities were retained at pH 3.1. CO₂ release from glucose was maximal at pH 7.5 with negligible activity at pH 3.1. Pathways of glucose metabolism were explored by employing glucose, glucose-1-phosphate, and glucose-6-phosphate labeled in various carbon positions. The glycolytic pathway appeared to play a lesser role than the pentose phosphate and/or Entner-Doudoroff pathways. Glucose-1-phosphate was metabolized at a much higher rate than glucose or glucose-6-phosphate. We conclude that glutamate is utilized primarily as an energy source while glucose may serve as an important metabolite for the nutrition ofL. pneumophila.     

Molecular characterization reveals that YMR278w encoded protein is environmental stress response homologue of Saccharomyces cerevisiae PGM2

The uncharacterized ORF YMR278w of Saccharomyces cerevisiae is a member of D-phosphohexomutase super family, annotated as phosphoribomutase. In order to evaluate its functional role, we cloned, over-expressed and purified YMR278w protein. The protein product of YMR278w exhibits phosphoglucomutase activity. S158T mutant derivative of YMR278w protein lost phosphoglucomutase activity. Purified YMR278w protein has higher K_m for glucose-1-phosphate compared to other known phosphoglucomutases. Trehalose content was reduced in YMR278w disruptant as compared to the wild type strain. Based on the above results we suggest that YMR278w encodes phosphoglucomutase and not phosphoribomutase.     

Alterations in carbohydrate metabolism of γ-irradiated cavendish banana

γ-Irradiation of preclimacteric banana resulted in a gradual increase in fructose content, which reached a maximum in 6 days. Although the catabolism of glucose-U-¹⁴C was less in irradiated banana, incorporation of label into fructose was high. Initial fructose accumulation in irradiated banana may be due to a shift in glucose utilization from the glycolytic to the pentose phosphate pathway. The ratio of resporatory CO₂ from glucose-6-¹⁴C and glucose-1-¹⁴C was halved in irradiated bananas indicating predominance of the pentose phosphate pathway. The radioactivity of fructose derived from glucose-6-¹⁴C was almost twice that from glucose-1-¹⁴C in irradiated bananas, whilst in control both fruit the labelled precursors yielded equal amounts. Studies on individual enzymes in these two pathways showed an increase in phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and fructose-6-phosphatase and a decrease in hexokinase in irradiated banana.