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During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum ‘Xanthi’) and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [35S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplast's translational machinery to salt stress by scavenging hydrogen peroxide.

A reporter gene assay revealed that promoters derived from Synechococcus PCC7942 (S.7942) psbAI and Synechocystis PCC6803 (S.6803) psbAII were suitable for the expression of foreign ribulose-bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) in S.7942 cells. Transformational vectors with a promoter and a foreign RuBisCO gene, cvrbc originated from Allochromatium vinosum, were constructed on a binary vector, pUC303, and introduced to S.7942 cells. When the cvrbc was expressed with the S.7942 psbAI promoter, the total RuBisCO activity increased 2.5- to 4-fold than that of the wild type cell. The S.6803 psbAII promoter increased the activity of the transformant 1.5-2 times of that of wild type cell. There was a significant increase in the rate of photosynthesis depending on the increase of RuBisCO activity. The maximum rate of photosynthesis of the transformant cell was 1.63 times higher than that of the wild type under the illumination of 400 micromol m(-2) s(-1), at 20 mM bicarbonate and at 30 degrees C. Although the photosynthesis of the higher plant is limited by the ability of photosystems under high irradiance and the high CO(2 )concentration, that of the S.7942 cell is limited by the RuBisCO activity, even at high CO(2) concentrations and under high irradiance.

The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato ( Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens -mediated transformation. Catalase activity in progeny from transgenic plants was approximately three-fold higher than that in wild-type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µ M paraquat under moderate light intensity, whereas leaf discs from wild-type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild-type leaf discs at 24 h after treatment with 1 µ M paraquat and 10 m M H 2 O 2 , respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wildtype plants were sprayed with 100 µ µ µ M paraquat and exposed to high light illumination (800 µ mol m − 2 s − 1 ). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild-type plants. The catalase activity and the photosynthesis activity (indicated by the F v / F m ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO 2 fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 ° C) under high light intensity (1000 µ mol m − 2 s − − − 1 ). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photooxidative stress invoked by paraquat treatment, drought stress and chilling stress.

When cyanobacterium cells are grown under extremely low CO2 concentration, the number of carboxysomes, structures containing ribulose-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), is known to increase. This suggests that Rubisco helps to regulate photosynthesis in cyanobacteria. However, no studies have been done on the changes of Rubisco content and activity in response to the extracellular CO2 concentration, and no information is available on its effect on photosynthesis. To elucidate the relationship between the expression responses of Rubisco and extracellular CO2, wild-type cells (Synechococcus PCC7942) and carboxysome-lacking cells were grown under various CO2 concentrations, and Rubisco activity was determined. In both strains, Rubisco activity increased when the cells were grown under a CO2 concentration around, or less than, K1/2(CO2) of photosynthesis. In carboxysome-lacking cells, Rubisco activity increased five to six times at most, and a simultaneous increase in the rate of photosynthesis was observed. These results suggest that stimulation of expression of Rubisco occurs to compensate for the decrease in the rate of photosynthesis under CO2-limited conditions.

A novel photosynthetic bioelectrochemical cell that utilizes biocatalysts in both anode and cathode compartments was constructed for the first time. in the anodic half-cell, some parts of the electrons produced by the oxidation of water in the photosystem of cyanobacteria are transferred to the carbon felt anode through quinonoid electron transfer mediators. The electron is passed to dioxygen to regenerate water in the cathodic half-cell reaction with an aid of bilirubin oxidase reaction via a mediator. The maximum electric power was about 0.3–0.4 W m-2 for the projective electrode surface area at an apparent efficiency of the light energy conversion of 2–2.5%. The factors governing the cell output are discussed on the basis of the potential-current curves of each half-cell.

The electron transfer from the photosynthetic system in cyanobacteria, Synechococcus sp. PPC7942 to exogenous electron acceptors was examined using several electrochemical techniques. 1,4-Benzoquinone (BQ) and 2,6-dimethyl-1,4-benzoquinone (DMBQ) were found to function as good exogenous electron acceptors for the photosystem. Kinetic analysis with rotating disk amperometry revealed that the photoreduction of these quinones proceeds in Michaelis–Menten type kinetics for the concentration of the quinones and the light intensity. The electron transfer rate of the BQ reduction was as high as 68% compared with that of the photosynthetic oxygen evolution. Synechococcus sp. cell-entrapped and DMBQ-embedded carbon paste electrodes provided steady-state current ascribed to the photoelectrochemical oxidation of water. Although several inhibitors against the photosynthetic system suppressed the photoelectrochemical response, phenylmercury acetate, which inhibits ferredoxin and ferredoxin–NADP oxidoreductase, was found to enhance the photocurrent. Some electrochemical aspects of this system are discussed.

Grafting is an easy way to produce a new seedling, which can tolerate against various stresses. During the acclimation after grafting, however, the seedlings still suffer a severe water stress. It is well known that water stress produces active oxygen to oxidize ascorbic acid. The concentration of ascorbic acid in the leaves was analyzed by HPLC equipped with an electrochemical detector. The column used was SP-120-5-ODS-BP (DAISO, JAPAN) and elution was performed with 0.1 ᴍ phosphate buffer, pH 3.0. After grafting the seedlings were acclimated under a 6-hr light/dark regimen. The content of ascorbic acid increased gradually during 2 days compared with control. The ascorbate peroxidase showed about constant activity, so the increase of ascorbic acid may be due to its requirement to cure the grafting

The process of the reaction of carbon molecules in the dark reaction of photosynthesis was chosen as a BDA (Biosystem-Derived Algorithm). In this paper, the BDA was referred to as the 'Photosynthetic Learning Algorithm (PLA)'. The PLA utilizes the rules governing the conversion of carbon molecules from one substance to another in the Benson-Calvin cycle and Photorespiration reactions. In this paper, the principles of the PLA are presented in detail. Neural network training was selected as a typical optimization or parameter estimation problem to demonstrate the performance of the PLA. The neural network model for the logical functions . was successfully trained TSP was chosen to demonstrate the capability of photosynthetic Algorithm because it is a representative problem for a wide variety of combinatorial optimization problems where the solution space is all permutations of n objects. We see again that the photosynthetic algorithm has been able to find a good solution.

The process of the reaction of carbon molecules in the dark reaction of photosynthesis was chosen as a BDA (Biosystem-Derived Algorithm). In this paper, the BDA was referred to as the 'Photosynthetic Learning Algorithm (PLA)'. The PLA utilizes the rules governing the conversion of carbon molecules from one substance to another in the Benson-Calvin cycle and Photorespiration reactions. In this paper, the principles of the PLA are presented in detail. Neural network training was selected as a typical optimization or parameter estimation problem to demonstrate the performance of the PLA. The neural network model for the logical functions was successfully trained

The dependence of the activity of phosphoribulokinase isolated from a cyanobacterium, Synechococcus PCC7942, on Mg2+ showed that its real substrates were Mg-ATP and free D-ribulose 5-phosphate. On the basis of results of kinetic inhibition studies and previously reported result of affinity chromatography, an ordered bi bi mechanism in which Mg-ATP binds before ribulose 5-phosphate is proposed. The Km values for ATP and D-ribulose 5-phosphate were 0.09 and 0.27 mM, respectively. Ki values of ADP and D-ribulose 1,5-bisphosphate were 0.32 and 10.0 mM, respectively. Inhibition constants Ki1 and Ki2 for 6-phosphogluconate were 9.3 and 0.49 mM. Kia was 0.13 mM. New kinetics on PRK gave higher control coefficient than the kinetics on Spinach PRK did in the model with PRK activity from 175 to 1000 µmol min−1 mg−1 chl.

It is important to understand the characteristics of grafted seedlings during the acclimation process, especially the moisture content inside the scion, which depends on the balance between transpiration and supply from the stock. The joining rate between the scion and the stock can be done effectively by measuring the wilting rate of a grafted seedling. The wilting behavior of the seedlings was non linear. An attempt was made to model this behavĩor after the balance equation of a spring-mass system, using a synthetic spring coefficient as the variable. The wilting behaviors of different seedlings were successfully identified.

Two preparations of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO; EC 4.1.2.39) were obtained from the cyanobacterium Synechococcus PCC7942. In one preparation, the majority of RuBisCO was insoluble to be localized in carboxysomes ("Carboxysomal RuBisCO"), whereas in the other, the enzyme was solubilized ("Solubilized RuBisCO"). The kinetic properties of both RuBisCO preparations were determined to elucidate the changes of the activity based on packing the enzyme in carboxysomes. The activity of Carboxysomal RuBisCO decreased with increasing reaction time. The apparent affinity of the Carboxysomal RuBisCO for RuBP was lower than that of the Solubilized RuBisCO. The 3-phosphoglycerate produced in carboxysomes was detected with a lag of three minutes. These results suggest that RuBisCO activity changes dependent on packing in carboxysomes and have a diffusion resistance to RuBP and PGA.

The production system for grafted seedlings mainly consists of three processes; 1) growth of seedlings, 2) grafting of seedlings, and 3) acclimation of grafted seedlings. Of the three processes, the duration of acclimation is highly influenced by the acclimation conditions. The acclimation environment after grafting was controlled to be satisfied the demands of grafted seedlings in the point of the physiological reaction such as photosynthesis, respiration, transpiration, and translocation nutrients. In the present study, a preliminary experiment was conducted to understand the relationship between the factors concerned with the acclimation of grafted seedlings, using a new acclimation apparatus. The factors of interest were air temperature, relative humidity, light, and carbon dioxide concentration. In the presence of light, the air temperature and relative humidity were interfered each other, so that both factors were difficult to keep at a constant value. Furthermore, the concentration of carbon dioxide was remarkably fluctuated by the relative humidity regulated by the humidifier and dehumidification which was controlled by the temperature differences between water and ambient air. A new device of acclimation system which is automatically controlled would be expected to construct in near future. Such a device will make it possible to shorten the duration of acclimation and produce high quality of grafted seedlings.

A rapid and sensitive method to determine the relative specificity (r) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) using anion-exchange chromatography is described. We employed the open gas system for the reaction of RuBisCO to get the most reliable CO2 and O2 concentrations in the reaction mixture. 3-Phosphoglycerate and 2-phosphoglycolate which were formed in the RuBisCO reaction were completely separated and directly measured with anion-exchange chromatography without using radioisotopes. The determination of the r value was accomplished in 3 h. The r values of RuBisCO enzymes from higher land plants were between 90 and 96, and those from bacteria including cyanobacterium were close to 45. These values were in agreement with previously reported values. The enzyme of the red macroalga Porphyra yezoensis exhibited a r value of over 140, as expected from the reported value of the enzyme from the red microalga Porphyridium cruenteum. RuBisCO from the green macroalga Ulvapertusa had a rvalue close to 70 and similar to that of the enzyme from the green microalga Chlamydomonas reinhardtii.

This paper aims at clarifying the cause of the time-dependent, partial loss of the activity during reaction (so-called fallover) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from plant sources. This was done by comparing the reaction courses calculated using the reaction models constructed here based on the present conflicting two ideas on fallover with directly measured courses obtained with RuBisCO purified from spinach leaves. Since the ordinary methods with 14CO2 and indicator enzymes were not adequate for analyzing the progress of fallover, we followed the reaction by measuring the change of the light absorbance of ribulose 1,5-bisphosphate (RuBP) at 280 nm. Direct measurements of the reaction course showed that RuBisCO lost its activity with a rate constant of 6.1 to 6.5 x 10(-3) s-1 at both 0.5 and 2 mM RuBP. The rate constant of the recovery of the enzyme to show the original fallover was determined as 1.2 to 1.3 x 10(-3) s-1 with RuBisCO that had just experienced fallover. These constants were used in the models. Calculation with a model assuming the binding of xylulose 1,5-bisphosphate (XuBP) to the catalytic sites of the enzyme as the cause of fallover and using the reported dissociation constant of XuBP in the binding and the reported rate of the formation of XuBP from RuBP gave a rather linear reaction course. The minimum requirements for the model to be valid were that the rate of XuBP formation was more than once for every 600 turnovers, the dissociation constant of XuBP for the catalytic sites was less than 0.1 nM, and the binding of XuBP to the sites showed a strong negative cooperativity. Inclusion of non-catalytic RuBP-binding sites in the model was essential to elucidate the course at higher RuBP concentrations. The model constructed assuming that hysteresis was the cause of fallover could calculate the measured reaction courses for the initial 20 min of reaction of both 0.5 and 2mM RuBP. The rate constants of the hysteretic conformational changes of the predicted enzyme forms to others were given. The direct measurement of the long-term reaction course revealed the two phases in the decay of the activity; fast decay for the initial several minutes and subsequent slow decrease. Although the fast decay could be predicted by the hysteresis model, the slower one required the participation of inhibition by XuBP. We reasoned from these results and the reported characteristics of the binding of other sugar phosphates to the catalytic sites that the initial fast decay of the activity in fallover was due to the hysteretic property of the enzyme and the slower phase of fallover was due to the inhibition of XuBP.

This study aims at clarifying the cause of the time-dependent, partial loss of the activity during reaction, fallover, of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of plant sources. This was done by comparing the reaction courses calculated using the reaction models constructed here based on the present conflicting two ideas on fallover with directly measured courses with RuBisCO purified from spinach leaves. Calculation with a model assuming the binding of xylulose 1,5-bisphosphate (XuBP) to the catalytic sites of the enzyme as the cause of fallover and using the reported dissociation constant of XuBP in the binding and the reported rate of the formation of XuBP from ribulose 1,5-bisphosphate (RuBP) gave a rather linear reaction course. The model constructed assuming that hysteresis was the cause of fallover could calculate the measured reaction courses for the initial 20 minutes of reaction at both 0.5 and 2 milimolar RuBP. The rate constants of the hysteretic conformational changes of the predicted enzyme forms to others were given.

SummaryThe reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.

3-Hydroxykynurenine (3-HOK) accumulated in may tissues of A. grahami, especially in the Malpighian tubules. The compound was present in the insect during its whole life cycle. Its content in the insect seemed to be more than that needed for the synthesis of ommochrome, and was also 5–10-fold of the concentration of other reductants. It can protect the fat body cells of A. grahami from peroxidation and also suppress the decrease of respiration of the cells due to injury by t-butylhydroperoxide (t-BuOOH). The effects of the compound are superior to those of reduced glutathione and uric acid, and approach those of ascorbic acid. The protection of the respiration against injury from t-BuOOH means that 3-HOK effectively scavenged radicals and the more 3-HOK the tissue contained, the less the fat body was injured by t-BuOOH.

Two δ-endotoxins, CryIA(a) and CryIA(b), from Bacillus thuringiensis subsp.. aizawai were used to investigate the specificity in insecticidal activity. CryIA(a) was 17-fold more toxic to Bomhyx mori than CryIA(b). After in vitro solubilization and digestion of these δ-endotoxins, the specificity of toxicity was retained. Trypsin-activated CryIA(a) and CryIA(b) showed specific, high affinity and saturable binding to brush border membrane vesicles (BBMV) from B. mori midguts. These two toxins competed for the same binding site. Dissociation constant for CryIA(a) and CryIA(b) binding to B. mori BBMV was O.89nM and 1.46 nM, respectively. In both toxins, dissociation reaction followed a biphasic process with a fast and a very slow component, suggesting that binding of the toxins proceeds through a reversible component and an apparently irreversible component. In the CryIA(a) dissociation reaction, the irreversible component comprised a large portion of total binding. On the other hand in that of CryIA(b), the reversible component was major. These results suggest that the specific toxicity of the toxins to B. mori may depend mainly on irreversibility.

This study was attempted to elucidate the mechanism of the regulation of the turnover number on the catalytic sites by the regulatory sites of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO]. To this end, we analyzed the effects of the binding of 6-phosphogluconate (6-P-Gln) to the regulatory sites of the enzyme on the progress in the subsequent catalysis assayed with 0.5 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. This concentration of Rbu(1,5)P2 hardly binds to the regulatory sites but competes with 6-P-Gln for the catalytic sites. An equilibrium binding assay showed that Rbu(1,5)P2CO bound 8 mol 6-P-Gln/mol enzyme in addition to the catalytic sites. The binding to the eight regulatory sites was positively cooperative. The biphasic reaction course, inherent in the carboxylase reaction of plant Rbu(1,5)P2CO and composed of a burst for an initial few minutes and a subsequent linear phase, became faint with increasing binding of 6-P-Gln to the sites. The change of the reaction progress from the biphasic to linear course was ascribed to the suppression of the functioning form of the enzyme from the high-activity to low-activity form and to the increased turnover number on the catalytic sites though the binding of 6-P-Gln to the regulatory sites.

Bacillus thuringiensis δ-endotoxin, CryIA(a), increased ion permeability of brush border membrane vesicles isolated from midgut epithelia of Bombyx mori larvae. This ion permeability change was measured with a potential-sensitive fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. This effect was observed at concentrations of the toxin that correspond to normal effective doses in vivo. CryIA(b) and heat-treat CryIA(a) did not show this effect. CryIA(a) did not show this effect on rat renal brush border membranes. The effects depended on the toxin dose in saturable manner. These suggest that this assay system reflects the mode of action of δ-endotoxin in vivo. The toxin increased various ion permeabilities, suggesting that δ-endotoxin forms non-selective cation pores on brush border membranes.

Both microperoxidase and cytochrome c could be immobilized on a gel for HPLC without losing their activity, the specificity of each immobilized catalyst being similar to that of the free catalyst. With up to 40% of methanol, the chemiluminescence increased as the alcohol concentration increased, while the count decreased with more than 10% methanol for the free cytochrome c. When used as the reactant for flow injection analysis, they do not need to occupy the cell for the emission of chemiluminescence. The immobilized reactant can measure from 400 pmol to 10 nmol of tert-butylhydroperoxide (t-BuOOH) without staining the emission cell.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of a thermophilic cyanobacterium, Thermosynechococcus vulcanus, was cloned and expressed in Escherichia coli. The purified enzyme had higher thermostability than RuBisCOs isolated from mesophilic cyanobacteria. Prediction of the tertiary structure was performed using the software Molecular Operating Environment (MOE). The predicted structure did not give any clue about the basis of thermostability. Then, the molecular docking of substrates and inhibitors in the catalytic site were carried out to test analogs for consistency of ribulose 1,5-bisphosphate, a RuBisCO substrate. The analogs were searched in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and 99 compounds were selected for the docking. The mol files from LIGAND Database in KEGG were changed to a three dimensional (3D) structure for use in docking simulation. The docking simulation was performed on ASEDock of MOE, and the SiteFinder command suggested about 20 candidates for the docking site of the compounds. Based on the homology of these candidate sites with the xylulose 1,5-bisphosphate (XBP)-binding site of RuBisCO isolated from Synechococcus PCC 6301, one site was selected for the docking simulation. The 40 compounds with the highest docking energies included synthetic organic substances that had never been demonstrated to be inhibitors of RuBisCO. The total docking energies were -102 kcal/mol, -104 kcal/mol, -94.0 kcal/mol, and -57.7 kcal/mol for ribulose 1,5-bisphosphate (RuBP), etidronate, risedronate, and citrate respectively. Kinetic analysis of RuBisCO revealed a K(m) value of 315 microM for RuBP, and K(i) values of 1.70, 0.93, and 2.04 mM for etidronate, risedronate, and citrate respectively. From these values, the binding energies were estimated to be -4.85, -3.84, -4.20, and -3.73 kcal/mol for RuBP, etidronate, risedronate, and citrate respectively. The differences between the values estimated from experimental data and by simulation may mainly depend on the dissimilarity of the environment for the protein and ligands between the experiments and the simulation. The results obtained here suggested a few new inhibitors, which might be useful as tools for studying the relationship between the structure and the function of RuBisCO.

When cyanobacterium cells are grown under extremely low CO(2) concentration, the number of carboxysomes, structures containing ribulose-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), is known to increase. This suggests that Rubisco helps to regulate photosynthesis in cyanobacteria. However, no studies have been done on the changes of Rubisco content and activity in response to the extracellular CO(2) concentration, and no information is available on its effect on photosynthesis. To elucidate the relationship between the expression responses of Rubisco and extracellular CO(2), wild-type cells (Synechococcus PCC7942) and carboxysome-lacking cells were grown under various CO(2) concentrations, and Rubisco activity was determined. In both strains, Rubisco activity increased when the cells were grown under a CO(2) concentration around, or less than, K (1/2)(CO(2)) of photosynthesis. In carboxysome-lacking cells, Rubisco activity increased five to six times at most, and a simultaneous increase in the rate of photosynthesis was observed. These results suggest that stimulation of expression of Rubisco occurs to compensate for the decrease in the rate of photosynthesis under CO(2)-limited conditions.

Resemblances and differences on photosynthesis of C3 plant and cyanobacterium Synechococcus PCC7942 were discussed with a simplified model of photosynthesis and photorespiration. Then the transformation of the cyanobacterium was shown with Chromatium vinosum (Cv) RuBisCO. Total RuBisCO activity and photosynthesis were compared with wild type S. 7942 and RuBisCO high expression mutant. Results were explained with RuBisCO environment of S. 7942 mutant. For excluding the influence of inorganic carbon concentrating mechanism (CCM) on the effect of RuBisCO, carboxysome less mutant was created with homologous recombination of ccm operon to be replaced with kanamycine resistant gene. And the deletion of the operon was confirmed by genomic PCR on the transformant. The confirmation was performed with three primers. Electron micrograms of S. 7942 show the existence of carboxysome in wild type and its absence in the mutant. Growth rate of CL mutant on different CO2 concentration shows that there is a possibility to increase a photosynthetic ability of a cyanobacterium with increases the RuBisCO activity.

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.

The binding protein for Bacillus thuringiensis δ-endotoxin, CryIAa, from the brush border membrane of the midgut of Bombyx mori was purified by the dot blot method and δ-endotoxin affinity chromatography. The binding protein was purified to 235-fold enrichment from cholic acid extracts of brush border membranes from B. mori midgut by activated CryIAa-affinity chromatography and DEAE ion-exchange chromatography. The purified binding protein showed a single band of 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and this band specifically reacted to 125I-labeled CryIAa on Immobilon membrane. The affinity of the binding protein for CryIAa was equivalent to that of the brush border membrane vesicles and solubilized membrane proteins. Partial amino acid sequences of the binding protein showed sequence similarity to the cadherin-like binding protein for CryIAb from Manduca sexta, but not for CryIAc binding protein from M. sexta and Heliothis virescens.

The control coefficient is an important index to show how an enzyme regulates a metabolic pathway. On higher plants, antisence technique decrease RuBisCO protein for the elucidation of a regulatory role of the enzyme in photosynthetic inorganic carbon anabolism (1). The technique can not be applicable to cyanobacteria because of their high possibility of homologous recombination. On the other hand, the control coefficient could be estimated dependent on the increase of the enzyme concerned. As a trial for the estimation, RuBisCO protein of purple sulfur photosynthetic bacterium Chromatium vinosum was expressed in the cyanobacterium Synechococcus PCC7942 by the transformation with bidirectional expression vector. In this transformant MT1, we confirmed that introduced expression vector replicated stably And C. vinosum RuBisCO was expressed in the cytosol in active L8S8 form. But the expression level of C. vinosum RuBisCO was around 30% of total RuBisCO activity. The low expression level makes it difficult to analyze the effect of the foreign enzyme on the photosynthetic physiology of the transformant. Thus, in this paper, we try to increase its expression level after checking promoters derived from cyanobacterium and E. coli with luciferase as a reporter.

Phosphoribulokinase (PRK) was purified to electrophoretic homogeneity from Synechococcus PCC7942 with high specific activity. Molecular masses of the native enzyme and its subunit were 178 and 42 kDa, respectively. Cys-17 and Cys-38 were conserved in the cyanobacterial PRK, but 18 amino acid residues between them were missing among the 40 residues found in higher plant PRKs.

Ribulose bisphosphate carboxylase (RuBisCO) binds carboxyarabinitol bisphosphate (CABP) on its regulatory sites [Yokota, A. (1991) J. Biochem. 110, 246-252]. The characteristics of the equilibrium binding of CABP to the sites were examined by the gel-filtration method. Since RuBisCO binds CABP on the substrate sites with a dissociation constant of less than 10 pM, CABP bound exclusively to the substrate sites at less than 5 microM. Plotting the number of CABP bound to the sites other than the substrate sites against the concentration of CABP gave a typical "bumpy" curve; the binding number in the intermediate plateau at 20 to 40 microM CABP was 3.7 to 4.4 mol per mol of RuBisCO and that at the saturating concentration of CABP was 7.6 to 7.8 mol per mol of RuBisCO. The Hill plot of their relationship gave a line which bent strongly at 20 to 40 microM CABP. The best fitting of the data to the equations derived from the binding model constructed according to the reported model [Teipel, J. & Koshland, D.E., Jr. (1969) Biochemistry 8, 4656-4663] showed that the binding of CABP to the regulatory sites proceeded with positive cooperativity both before and after the plateau. The dissociation constant decreased from 31 to 14 microM by the factor of 1/1.3 in the former group and 490 to 0.7 microM by the factor of 1/8.9 in the latter with increasing binding number of CABP.

1. 1. Glutamyl kinase in a homogenate of blowfly abdomens was not inhibited by proline and its analogues, and was found only in the mitochondrial fraction. 2. 2. Proline was biosynthesized from glutamate only in a cell-free system prepared from blowfly abdomen.

In a blowfly, Aldrichina grahami, the enzyme which oxidizes hypoxanthine to uric acid, was not an oxidase but a dehydrogenase and the enzyme activity was found throughout all stages. The xanthine dehydrogenase activity in the adult stage was enhanced by a high protein diet. Adenine and inosine also caused an increase of this enzyme activity, but guanine had little effect. The enzyme was purified by the methods of charcoal treatment, ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 column chromatography. The enzyme was purified ca. 100 fold. Michaelis constants of the enzyme for hypoxanthine, xanthine, and NAD were 3.50 × 10−2 mM, 1.25 × 10−2 mM, and 6.25 × 10−2 mM, respectively. The enzyme was inhibited by allopurinol and adenine but not by inosine.

Non-enzymatic cytochrome c reducing activities were found in homogenates of a blowfly. The activity was maximum 5 days after the insect hatched, and rapidly decreased thereafter. Cytochrome c reductant in the pupae was purified as a yellow needle-like crystalline product. We investigated some chemical and electrochemical properties, and determined the structure, using NMR, MS, i.r. and u.v. spectroscopic analyses.

1. 1. A peak of the activity during growth was observed within 5 days after hatching. The relative specific activity of OA (ornithine aminotransferase E.C. 2.6.1.13) in different tissues was found to be: fat body > gut > malpighian tube > muscle. 2. 2. Hypo-osmotical shock experiment suggests that OA is absolutely located in the intracellular particles of the fat body. 3. 3. In the cell fractionation, one-half of OA in the fat body cell was distributed in the crude mitochondrial fraction and the activity was apparently increased after treatment with Triton X-100 or sonication. 4. 4. In the sucrose density gradient, the peak fraction of OA was almost overlapped with mitochondrial marker enzymes. 5. 5. These findings show that most of the OA activity in blowfly is localized in intramitochondrial sites.

In adults of A. grahami , xanthine dehydrogenase (EC 1.2.1.37; XDH) activity increased significantly during the first few days after emergence. The activity was high in the fat body, low in the gut, and no activity was detected in the Malipighian tubules. XDH activity increased in relation to the protein content of the diet, and the increase was localized mainly in the fat body. XDH activity increased by 1.5–3 times when the diet contained either amino acids related to purine biosynthesis, or adenine or hypoxanthine. Guanine and xanthine, however, had no effect on XDH activity whilst uric acid lowered the activity. The amino acids and purines which increased XDH activity also augmented the levels of AMP, inosine and hypoxanthine. The increase in XDH activity was due to a de novo synthesis of the enzyme protein.

We purified a proteolytic enzyme from pupae of the blowfly Aldrichina grahami. The purification procedure consisted of fractionation with ammonium sulfate, ethanol treatment, affinity chromatography on Con A-Sepharose, and ion exchange chromatography on CM-Sepharose CL-6B. The purified enzyme was very labile. The lability was reduced by the use of MES buffer containing 10% ethanol, which enabled us to purify the enzyme to homogeneity. The lower the polarity of the alcohol added, the more stable the enzyme became. The enzyme had a molecular weight of 41,000, an optimum pH of 3.5, high susceptibility to pepstatin and two pI forms of 5.4 and 6.2. This enzyme resembled cathepsin D, a lysosomal proteinase.

1. (1) In the larval stage of Aldrichina grahami the high proline diet (semi-synthetic diet consisting mainly of milk casein) resulted in a much higher concentration of free proline in the haemolymph than did the low proline diet (semi-synthetic diet consisting mainly of egg albumin). Therefore, proline accumulation in 4-day-old larvae reared on the high proline diet, is mainly due to the proline derived from the diet, and its accumulation seems to have no physiological significance. 2. (2) The synthesis of proline from glutamic acid in A. grahami was first observed in the adult stage. Proline was synthesized much more from glutamic acid than from arginine in the adult stage. 3. (3) Unnecessary interconversion between Δ 1 -pyrroline-5-carboxylic acid (P5A) and proline seems to be prevented by separate compartmentation of P5A reductase and proline oxidase in the cell.

Pyrroline-5-carboxylate (P5A) reductase was purified about 200 fold by ammonium sulphate fractionation, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-50 gel filtration chromatography. The partially purified enzyme reduced 1.69 μmole of P5A per min per mg protein. The enzyme was unstable without the addition of glycerol or ethylene glycol. The optimum pH of the enzyme was found to be 6.8. The enzyme required NADH specifically as the hydrogen donor. It was inhibited by Hg2+, PCMB and KCN. Its Km values for P5A and NADH were 0.27 mM and 0.28 mM, respectively. Proline exhibited competitive inhibition on the reductase against P5A and its Ki value was 16.8 mM.

1. 1. The occurrence of acid protease activity is demonstrated in the pupal stage of the blowfly A. grahami. 2. 2. Two proteases involved in larval tissue histolysis are found; one is a carboxy protease like cathepsin D, and the other is a thiol protease. 3. 3. The thiol protease was purified to homogeneity and characterized. It is a cathepsin-L-like protease with an apparent mol. wt of 23,000 and an optimum pH of 5.0. This enzyme can be detected by using a synthetic substrate of N-carbobenzyloxy-lysine-p-nitrophenyl (Z-Lys-ONP).