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G Kakefuda

Publications and source records attributed to G Kakefuda.

12 recordsLinked to original sources
PubMed1996

Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase.

Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo. The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.

Acetolactate Synthase↗
PubMed1992

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120.

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.

Amino Acid Sequence↗
PubMed1992

Involvement of arginine residues in the allosteric activation and inhibition of Synechocystis PCC 6803 ADPglucose pyrophosphorylase.

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacterium Synechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl2 enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form. Vmax at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.

Amino Acid Sequence↗
PubMed1991

Mutants of Arabidopsis with altered regulation of starch degradation.

Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes alpha-amylase, beta-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.

Journal Article↗
PubMed1991

Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases.

ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A(0.5) = 0.81 millimolar; n(H) = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A(0.5) = 0.12 millimolar; n(H) = 1.0), having a maximal stimulation of 17-fold. I(0.5) values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A(0.5) = 10 micromolar; n(H) = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.

Journal Article↗
PubMed1989

Characterization of Pea Chloroplast D-Enzyme (4-alpha-d-Glucanotransferase).

Pea (Pisum sativum L.) chloroplast D-enzyme (4-alpha-d-glucanotransferase, EC 2.4. 1.25) was purified greater than 750-fold and partially characterized. It is a dimer with a subunit M(r) of ca. 50,000. Optimal activity is between pH 7.5 and 8.0 with maltotriose as substrate and the enzyme's K(m) for maltotriose is 3.3 millimolar. Chloroplast D-enzyme converts maltotriose to maltopentaose and glucose via the exchange of alpha-1,4-glycosidic linkages. Maltotriose acts either as a donor or acceptor of a maltosyl group. The enzyme has highest activity with maltotriose as substrate. As initial substrate degree of polymerization is increased to maltoheptaose, D-enzyme activity drops to zero at 10 millimolar substrate concentrations and by 70% at 1 millimolar concentrations. The enzyme cannot use maltose as a substrate. Glucose was found to be a suitable acceptor substrate for this D-enzyme. Addition of glucose to incubation mixtures, or production of glucose by D-enzyme, prevents the synthesis of maltodextrins larger than maltopentaose. Removal of glucose produced by D-enzyme activity with maltotriose as substrate resulted in the synthesis of maltopentaose and maltodextrins with sufficient degrees of polymerization to be suitable substrates for pea chloroplast starch phosphorylase. The possible role of D-enzyme in pea chloroplast starch metabolism is discussed.

Journal Article↗
PubMed1984

Electrophoretic transfer as a technique for the detection and identification of plant amylolytic enzymes in polyacrylamide gels.

An electrophoretic transfer technique was developed for the specific identification of isozymes of starch debranching enzyme, alpha-amylase, and beta-amylase. Amylolytic enzymes are separated by native polyacrylamide slab gel electrophoresis and proteins in gels are electrophoretically transferred through starch-containing polyacrylamide gels. Each amylolytic enzyme degrades starch in the transfer gel to its characteristic limit dextrin as it moves through the gel. Various limit dextrins in the starch gel are identified by their characteristic color development in KI/I solution. Isozymes of starch debranching enzyme, alpha-amylase, and beta-amylase can be easily identified in the same gel.

Journal Article↗
PubMed1983

Differential leakage of intracellular substances from imbibing soybean seeds.

Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max [L.] Merr.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25 degrees C) of 3.0 and 15.8 kilocalories per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intracellular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells.

Journal Article↗
PubMed1983

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings.

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO(3) (-), tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO(3) (-) and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO(3) (-) and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO(3) (-). The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO(3) (-), further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO(3) (-) requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO(3) (-) requirement for induction were quite different. K(m) values for NO(3) (-) were identical for both nitrate reductases.

Journal Article↗
PubMed1981

Role of the testa in preventing cellular rupture during imbibition of legume seeds.

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.

Journal Article↗