Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus PCC7942 cells.

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.

Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa delta-endotoxin of Bacillus thuringiensis.

The binding protein for Bacillus thuringiensis delta-endotoxin, CryIAa, from the brush border membrane of the midgut of Bombyx mori was purified by the dot blot method and delta-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.

Design of an acclimation system capable of controlling carbon dioxide concentration.

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.

Comparison of the primary structure of waxy proteins (granule-bound starch synthase) between polyploid wheats and related diploid species.

The waxy proteins encoded by the genomes A, B, and D in polyploid wheats and related diploid species were isolated by SDS-PAGE. The N-terminal amino acid sequences of mature proteins and V8 protease-induced fragments were determined. A total of five amino acid substitutions was detected in these sequences, which represent about 10% of the whole sequences of the waxy proteins. A comparison of these sequences in polyploid wheats with those in related diploid species revealed the following: (i) waxy proteins encoded by the A genome of polyploid wheats were identical to that of Triticum monococcum, (ii) the waxy protein encoded by the B genome of T. turgidum was identical to that of T. searsii, but differed from those of T. speltoides and T. longissimum by one amino acid substitution, (iii) the waxy protein encoded by the B genome of T. aestivum differed from that encoded by the B genome of T. turgidum by one amino acid substitution, and (iv) the waxy protein encoded by the D genome of T. aestivum was identical to that of T. tauschii.

Modeling of continuously and directly analyzed biphasic reaction courses of ribulose 1,5-bisphosphate carboxylase/oxygenase.

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.

Purification and characterization of phosphoribulokinase from the cyanobacterium Synechococcus PCC7942.

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.

Variation in the primary structure of waxy proteins (granule-bound starch synthase) in diploid cereals.

The molecular weights of waxy proteins, by SDS-PAGE, and the N-terminal amino acid sequences of mature protein and of V8 protease-induced fragments were determined in diploid cereals. The homology of the primary structure was relatively high among cereals examined here, and there appeared to be a common sequence, V-F-V-G-A-E-M-A, in the vicinity of the N terminus. Based on the amino acid sequences, these cereals could be divided into two groups, including corn and rice in one and diploid wheat, four Aegilops species, rye, and barley in the other. In diploid wheat and Aegilops species there were substitutions of amino acids in the primary structure. Variations of this sort suggest that the primary structure of waxy proteins would provide clues to the phylogenetic relations in the wheat group.

Specific Toxicity of δ-Endotoxins from Bacillus thuringiensis to Bombyx mori.

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.

Regulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase through cooperative binding of 6-phosphogluconate to its regulatory sites.

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.

Cooperative binding of carboxyarabinitol bisphosphate to the regulatory sites of ribulose bisphosphate carboxylase/oxygenase from spinach.

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.

3-Hydroxykynurenine as O2-. scavenger in the blowfly, Aldrichina grahami.

We studied the distribution of O2-.-scavenging activity in 6-day-old larvae of Aldrichina grahami. Total activity was highest in the muscle. The specific activity per milligram of protein in the Malpighian tubules was highest, 10 times the highest elsewhere. Most of the O2-. scavenging activity in muscle depended on superoxide dismutase. However, the activity in the Malpighian tubules mostly depended on 3-hydroxykynurenine.

Action of a cysteine proteinase from pupae of the blowfly Aldrichina grahami on the oxidized B-chain of bovine insulin.

A cysteine proteinase purified from pupae of the blowfly (A. grahami) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Fifteen peptides were separated on HPLC using both gradient and isocratic elution methods. Analyses of amino acid content and N-terminal amino acids indicated that these were eleven homogeneous peptides produced by digestion and undigested insulin B-chain. Glu13-Ala14 and Tyr26-Thr27 were the major cleavage sites, and Asn3-Gln4, Cys7-Gly8, Tyr16-Leu17, Leu17-Val18 and Cys19-Gly20 were also often cleaved. These findings show the similarity between this enzyme and cathepsin L.

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena.

The nonenzymatic reaction of glyoxylate and H2O2 was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H2O2 was secondorder, with a rate constant of 2.27 l mol(-1) s(-1) at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn(2+) and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H2O2. From a comparison of the rates of H2O2 decomposition by catalase and the reaction with glyoxylate, we conclude that H2O2 produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO2 originates from glycine, but not from glyoxylate, in C3 plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO2. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H2O2 to produce photorespiratory CO2 during the glycolate metabolism of this organism.

Proline synthesis from glutamate in the mitochondria isolated from a blowfly, Aldrichina grahami.

Proline biosynthesis from glutamate was demonstrated in a cell free system prepared from blowfly abdomen. The biosynthetic activity was found mainly in the mitochondrial fraction. The biosynthesis of proline from glutamate required ATP, NADPH and Mg++ as cofactors.

Inactivation of DNase by 2-nitro-5-thiocyanobenzoic acid. II. Serine and threonine are the sites of reaction on the DNase molecule.

The amino acid compositions of various fragments isolated from DNase treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) show peptide bond cleavages to be at Thr14, Ser40, and Ser135. Isolation and characterization of radioactive tryptic and chymotryptic peptides of [14C]cyano-DNase reveal four points of peptide bond cleavage; in addition to Thr14, Ser40, and Ser135, cleavage occurs at the amino end of Ser72. Approximately 2.8 mol of [14C]cyano group are incorporated in the completely inactivated enzyme, in which 0.6 residue of Thr14, 0.8 of Ser40, and approximately 0.3 each of Ser72 and Ser135 are modified. The inactivation by NTCB can also be obtained by reacting the enzyme with a mixture of 5,5'-dithiobis(2-nitrobenzoic acid), KCN, and iodoacetate which generates NTCB. The mixture facilitates the uses of K[14C]N, which is readily incorporated into the enzyme as the [14C]cyano derivative. The reaction of NTCB with serine or threonine resembles that with cysteine.

Isolation and characterization of multiple forms of ovine pancreatic deoxyribonuclease. Chromatograhpic behavior of the enzyme on concanavalin A-agarose and carboxymethylcellulose columns.

A new procedure has been devised for the purification of ovine DNase, including (NH/4)2SO4 fractionation, two steps of CM-cellulose chromatography, concanavalin A-agarose chromatography, and gel filtration on Sephadex G--100. The enzyme, like bovine DNase, exhibits multiplicity due to changes in the primary structure and the sugar structure of the carbohydrate moiety. Unlike bovine DNase, ovine DNase does not have sialic acid in any of its multiple forms. Concanavalin A-agarose is useful in the purification of not only ovine but also bovine DNase. For ovine DNase, it is a necessary and key step of purification; for bovine DNase, it can be used to purify commercial preparations of DNase free from proteases in a single step as judged by its stability in Ca2+-free media at pH 8.0. The purified enzyme has a specific activity equal to that of a highly purified DNase and presumably contains predominantly DNases A and C. Two of the four forms of ovine DNase have been purified to apparent homogeneity and subjected to chemical analysis. The present results show that bovine and ovine DNases have indistinguishable molecular weights and identical end groups, suggesting that they may have the same number of amino acid residues. The amino acid composition indicates that two enzymes may have six residues of amino acids subject to substitution which can be explained by single base changes in their genetic code words. Amino acid analyses also indicate that the most likely difference between two forms of ovine DNase is the substitution of Leu for Arg.