This study was done to investigate the effects of modified nucleotide sequences around the initiation codon on gene expression efficiency in Escherichia coli. A human tumor necrosis factor-β-galactosidase fused gene was used to randomly modify both the upstream nucleotide sequence from the initiation codon (- 4 to - 1) and the silent third letter of the codons corresponding to the 4 amino acid residues of the N-terminal region. It was observed that the frequency of adenine nucleotide usage in this region was higher in the high expression group than in the low expression group.

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A new thermophilic and anaerobic bacterium, Thermobacteroides leptospartum sp. now., which produces extracellular proteases was isolated from cattle compost. This organism is a Gramnegative, non-sporulating, non-motile and obligately anaerobic filamentous bacterium. Ultra-thin sections of the cells reveal a multilayered envelope structure. The major cellular fatty acids are n-C_16:0 (27.7%), iso-C_17:0 (18.5%) and iso-C_15:0 (16.4%). No hydroxy fatty acids were detected. The temperature range for growth is between 45°C and 71°C, with optimum growth at 60°C; the pH range for growth is between 6.7 and 8.9 at 60°C with a doubling time of 8.6hr. Various carbohydrates, but not fructose or sucrose, are fermented. The major fermentation products from glucose are ethanol (14.1 mM) and acetate (8.8 mM). Growth is inhibited by polymyxin B, neomycin and gentamicin. The DNA base composition is 43±2mol% G+C.

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Various glucobioses were synthesized enzymatically by two procedures, an incubation of a highly concentrated solution of D-glucose in the presence of glycosidases (batch method reaction), and the other is circulation of highly concentrated D-glucose solution through an immobilized glycosidase column and an activated carbon column connected in series (column method reaction). The composition of disaccharides differed with the difference of reaction methods and with the kind of glycosidases.
A similar reaction was also done with glucoamylase and the factors controlling the regioselectivity of the reaction with the differences of enzyme or with the differences of the procedures are discussed.

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Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl.). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.

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Superoxide dismutase (SOD) was purified from Bacillus circulans IFO 3329 to an electrophoretically homogeneous state and partially characterized. The purification procedure involved (NH₄)₂SO₄ fractionation, column chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-100. The molecular weight and subunit molecular weight of the purified enzyme were estimated to be 46, 000 and 26, 000, respectively. The isoelectric point of the enzyme was about pH 5.2. The purified enzyme remained stable at pH 7.0 and 20°C for 36 hr, but rapidly inactivated above pH 8.0 and below pH 5.0. SOD was stable at pH 7.0 up to 40°C but was rapidly inactivated at temperatures above that. Metal ions had little effect on the SOD activity. The absorption maximum in the visible range was found at 475 nm, and the enzyme was red-purple in color and cyanide-insensitive. These results suggest that the enzyme is a Mn-containing SOD.

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The effect was examined of aqueous dialyzates from 16 kinds of vegetables and fruits on the mutagenicity of some mutagens toward Salmonella typhimurium TA 100. Each dialyzate inhibited the mutagenicity of Trp-P-2, and the antimutagenicity was retained even after heating at 100°C for 20min. Dialyzates of burdock, eggplant, spinach and apple also inhibited the mutagenicity of Trp-P-l, benzo[a]pyrene, sterigmatocystin, aflatoxin Bl, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and N-methyl-N'-nitroso-N-nitrosoguanidine. The dialyzates of apple reacted with S9 mix and Trp-P-2. A Sepharose CL 6B gel filtration study of the dialyzates of apple indicated that the antimutagenic activity of these dialyzates on Trp-P-2 and AF-2 was mainly detectable in the polyphenol-rich fractions.

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The distribution of glutathione S-transferase (GST) (EC 2.5.1.18) in yeasts was investigated. High enzyme activity was found in some strains of Issatchenkia and Candida. Of 168 strains tested, Iss. orientalis showed the highest activity. The enzyme activity exists constitutively in the yeast cells but it increased with the addition of an enzyme substrate, o-dinitrobenzene, to the culture medium. Moreover, the addition of L-cysteine and glycine to the medium also increased the enzyme activity. This enzyme was so unstable that it lost almost all its activity on ammonium sulfate precipitation and 93% of its activity was lost when it was stored at 4°C for two weeks in a soluble state. We found that it was stabilized considerably in a solution containing 20% glycerol, 1 mM EDTA, 2 mM DTT and 10 mM sodium sulfite.

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Mirabilis jalapa cell strains producing high levels of anti-plant-viral protein (MAP) were selected. The MAP content of the strain H9-52 obtained in the 9th selection was 6.3mg/g dry wt. which was about 3 times that of the root and was about 20 times that of the original cells. The ratio of MAP to total soluble protein also increased from 0.3% in the original cells to 6.5% in the strain H8-50 obtained in the 8th selection. MAP was isolated from the strain H8-50 and its anti-plant-viral activity was the same level as that of the root. The MAP productivity of strain H8-50 was maintained during 14 successive subculturings.

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A cellulolytic enzyme was purified from a crude enzyme preparation from Robillarda sp. Y-20 by consecutive column chromatography. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and contained 10.5% carbohydrate as glucose. The enzyme had a molecular weight of 56, 000, an isoelectric point of 3.80, an optimum pH of 5.0, and an optimum temperature of 60°C. It was stable from pH 4.0-7.0 at 37°C for 1 hr and below 50°C for 30min.
The amino-terminal amino acid sequence of the enzyme was Tyr⁴-Ala⁵-Gly⁶-Val⁷-Ala⁸-Glu⁹-Ser¹⁰-Ser¹¹-Gly¹²-Glu¹³-Phe¹⁴-Gly¹⁵-Val¹⁶-Trp¹⁷-Ser¹⁸.
The enzyme was inactivated by 1 mM Fe³⁺, Hg²⁺, or N-bromosuccinimide. It was active on sodium carboxymethyl cellulose (CMC) and cellooligosaccharides (cellotriose to cellohexaose), but not on either cellobiose or microcrystalline cellulose (Avicel) under our assay conditions. The Km value of the enzyme for CMC was 0.060%.

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Eukaryotic initiation factor eIF-2 has key roles in the initiation of protein synthesis. This work describes a partial characterization of pig liver eIF-2 and the phosphorylation site sequence of the α subunit. Our pig liver eIF-2 was composed of α and γ subunits. The N-terminal sequences of these subunits were analyzed by an automated Edman degradation. Forty residues of the N-terminal sequences of the α subunit were identified; they were mostly hydrophobic amino acids. On the other hand, we identified only 19 residues of the N-terminal sequence of the γ subunit, since pig liver eIF-2γ contained a minor component having a sequence lacking the N-terminal Ala-Gly of the γ subunit. Exhaustive tryptic digestion of [³²P]-and [³H]carboxymethyl-labeled eIF-2α produced one major phosphopeptide. The peptide was purified by one dimensional thin-layer chromatography and high performance liquid chromatography. The sequence of the peptide was Ser-Glu-Leu-Ser(P)-Arg-Arg.

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The mechanism of polygodial antifungal action was studied using Saccharomyces cerevisiae. It inhibited the incorporation of radioactive precursors into macromolecules in whole cells. However, no specificity in the inhibition of precursor incorporation was observed among various species of macromolecules, i.e., DNA, RNA, protein, and polysaccharide. It also inhibited the exogenous but not endogenous respiration of the cells. Polygodial greatly enhanced the leakage of cellular constituents from the treated cells, and the examination under an electron microscope revealed structural damage of the cell membranes of treated cells. These results support the conclusion that polygodial acts primarily by damaging the permeability barrier of the yeast cells.

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Dodecadien-1-ols, tetradecadien-1-ols and hexadecadien-1-ols with a conjugated (E, E)- or (E)-diene system between the ω1, ω3- and ω5, ω7-positions, their acetates, and aldehyde derivatives (lepidopterous sex pheromones and candidates) were analyzed by electron impact mass spectrometry, which was operated at 70 eV ionization voltage. Three functional derivatives with a same diene system presented a similar spectral pattern, except for the molecular ions (M⁺), [M-H₂O]⁺ of the alcohols and [M-CH₃CO₂H]⁺ of the acetates. Each isomer showed a characteristic fragment ion series of C_nH_2n-2⁺-C_nH_2n-5⁺ (C₄-C₉), which reflected the double-bond position in the molecule, indicating a method for determining the position of a natural diene pheromone by comparing its mass spectrum with those of the synthetic dienes. By this method, the natural pheromone of Hellula undalis was confirmed to be a ω3, ω5-diene. Furthermore, the fitness indexes proposed by Kuwahara et al. were calculated for some pheromone components, using the mass spectra of synthetic dienes, in order to examine the possibility and limitation for applications of those mass spectra to natural pheromone studies.

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A strain of Bacillus that produced an alkaline carboxymethyl cellulase (CMCase) was isolated from a soil sample and was found to be taxonomically similar to Bacillus pumilus. The growth rate and production of CMCase were greater during cultivation in neutral medium than in alkaline medium. Glucose, sucrose, cellobiose, maltose, starch and xylan, in addition to carboxymethyl cellulose, induced the production of the CMCase.
The CMCase, partially purified by precipitation with ammonium sulfate, was very active over the pH range of 7 to 10 and was fairly stable over a broad pH range (pH 5-12). The reaction catalyzed by the CMCase showed an optimum temperature of about 50°C and the enzyme was stable at temperatures up to 50°C or higher at pH 9. The partially purified enzyme preparation exhibited essentially no activity toward insoluble cellulosic materials such as filter paper, Avicel, cellulose powder, or alkali- or H₃PO₄-swollen celluloses, nor was it active toward cellobiose or p-nitrophenyl-β-D-cellobioside.
The CMCase activity was characteristically stable in the presence of surfactants, chelating agents and proteolytic enzymes used as components of laundry detergents.

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The interspecific electrofusion between protoplasts of Streptomyces antibioticus a20 (Ala^-) and S. fradiae f2 (His^-) was examined. The pulse intensities required for the breakdown of half of the protoplasts were 7kV/cm for S. antibioticus a20 and 5kV/cm for S. fradiae f2. The optimum pulse intensity for the fusion was found to be 6 kV/cm. When the density of a protoplast suspension was 1 × 10⁸ protoplasts/ml in a 0.3M sucrose solution containing 2.5mM MgCl₂ and CaCl₂, the maximum fusion frequency of 2.7% was obtained on the application of one square field pulse of 6kV/cm for 20μsec after dielectrophoresis in an alternating field of 800V/cm (1 MHz) for 60 sec. The fusion frequency was much higher than that obtained on polyethyleneglycol-induced fusion (0.15%). This is the first case of electrofusion of Streptomyces protoplasts, which are as small as those of some bacteria.

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Since new methods to isolate connectin or titin in a native state have been published, it has become important to reinvestigate the role of connectin (titin) in meat tenderization during postmortem storage. In this paper, the isolation of native connectin from rabbit skeletal muscle (L. dorsi) was carried out, and postmortem changes in native connectin were studied quantitatively and qualitatively.
On the basis of the amino acid composition and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel, it is shown that the isolated protein was a highly pure connectin. An increase of the extractability of native connectin from the muscle during storage was observed, and an electron microscopic study of the purified native connectin indicates a qualitative change of connectin in the muscle during postmortem storage.
If connectin has some influence on meat tenderness, the quantitative and qualitative changes of native connectin in the muscle are possibly one of the cause of meat tenderization during conditioning.

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The systemic fungicide, flutolanil (α, α, α-trifluoro-3'-isopropoxy-o-toluanilide, Moncut^®), strongly inhibited the mycelial O₂ consumption of Rhizoctonia solani as well as the activity of succinate dehydrogenase complex (Complex II) in mitochondria, while the fungicide showed no effect on the NADH-cytochrome c reductase activity. Consequently, the site of action of flutolanil must be situated on the mitochondrial Complex II. Flutolanil was more potent in blocking the Complex II activity of R. solani than other toluanilide fungicides such as mebenil and mepronil. Neither the mycelial growth nor Complex II activity of fungi in other classes except Basidiomycetes was affected by flutolanil. Since the Complex II activity in rat liver mitochondria was also insensitive to flutolanil, a difference in enzyme susceptibility should contribute to the high selectivity in antifungal action and low mammalian toxicity of the fungicide.

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Lysozyme, ribonuclease A, ovalbumin and bovine serum albumin were reacted with reducing sugars under physiological conditions of 37°C and pH 7.4, and polymerization of proteins, changes in amino acid composition and carbonyl compounds formed during the reaction were investigated. Incubation of all the protein-sugar systems resulted in noticeable losses of arginine and lysine residues. Polymerization, as well as impairment of arginine residues of lysozyme, with fructose and aldopentose was higher than with aldohexoses. 3-Deoxyglucosone (3DG) was identified as one of the major carbonyl compounds generated in the reaction systems of proteins with glucose and fructose. The formation of 3DG in protein-fructose systems was 1.3-2 times that in protein-glucose systems. Glucose alone did not polymerize succinylated lysozyme, but fructose did and it impaired only arginine residues. These results indicate that 3DG was generated on the reaction between glucose and free amino groups in proteins, whereas it was generated from fructose alone with the modification of free amino groups. It is strongly suggested that 3DG was the cross-linker responsible for the polymerization of proteins and the attacker of arginine residues.

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The cell-associated β-mannosidase of an alkalophilic Bacillus sp. (AM-001) was purified to the electrophoretically homogenous state. The relative molecular mass of the purified enzyme was 94, 000 daltons by SDS-PAGE and the pi was 5.5 by isoelectric focusing. The enzyme was most active at pH 6.0 and 50°C. This β-mannosidase was inhibited by some metal ions and chemical reagents as follows: Ag⁺, Cd²⁺, Cu²⁺, Hg²⁺, Zn²⁺, PCMB, SDS, DBS, and NBS, but was not inhibited by D-mannose, D-mannitol, or D-mannonic acid-γ-lactone. Using synthetic substrates such as p-nitrophenyl glycopyranosides, this enzyme showed strong activity only with p-nitrophenyl β-D-mannopyranoside, and no activity with p-nitrophenyl α-D-mannopyranoside. The Michaelis constant (Km) of the enzyme for p-nitrophenyl β-D-mannopyranoside was 1.3mM. The enzyme hydrolyzed β-1, 4-mannooligosaccharides larger than mannobiose.

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A protein antigenic to the IgE antibody of allergic individuals was isolated from rice grain by ion-exchange and gel-filtration chromatographies. The molecular weight of the allergenic protein was estimated to be about 16, 000 by sodiumdodecylsulfate-gel electrophoresis. The protein contained 7mol of cystine residue per mole of protein and no cysteine residues, and its immunoreactivity was quite stable to heating at 100°C. This allergenic protein was mainly present in the endosperm portion of rice seeds.

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The distribution and some properties of polyphosphate kinase, that catalyzes the formation of polyphosphate from ATP, were investigated. High enzyme activity was found in Alcaligenes faecalis, Brevibacterium ammoniagenes, Escherichia coli, Micrococuss lysodeikticus and Pseudomonas aeruginosa. The enzyme required Mg²⁺ for maximum activity and was activated by basic proteins, polyamines and phosphate polymers of low molecular weight. The enzyme from E. coli B could catalyze the reverse reaction, and generated ATP from ADP and metaphosphate. The feasibility of the generation of ATP by the E. coli B enzyme was confirmed by means of the coupled reactions of polyphosphate kinase and hexokinase, which produces glucose-6-phosphate.

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This work was designed to obtain a microbial mutant suitable for the industrial production of uridine. The growth of a wild type strain of Bacillus subtilis was inhibited by uracil analogs, such as 2-thiouracil and 6-azauracil, and the inhibition was reversed by adding uracil. Mutants resistant to these analogs were isolated. Among them, strain No. 508 produced 46 mg/ml of uridine; however, 6mg/ml of uracil was accumulated concomitantly. When this strain was subjected to further mutation to obtain mutants deficient in uridine phosphorylase activity, another mutant, No. 556, was obtained that produced 55 mg/ml of uridine with no appreciable amount of uracil.

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A lectin was purified to homogeneity from the fruitbodies of Flammulina velutipes by conventional purification procedures. The purified lectin was demonstrated to be a dimeric protein consisting of two identical subunits with an apparent molecular mass of 11 kDa. The lectin was an acidic protein with a pi value of 5.4, and devoid of cysteine, methionine, and histidine as amino acid constituents. Its hemagglutinating activity was totally unaffected by mono- and oligosaccharides and glycosides, but inhibited by some desialylated glycoproteins. Immunological assays revealed that no protein cross-reacting with rabbit anti-F. velutipes lectin antibody was apparently present in vegetatively growing mycelia but was distributed throughout the fruitbody at different concentrations.

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Bisbenzylisoquinoline alkaloid production was studied in roots of Stephania cepharantha (Menispermaceae). Various cultural conditions influenced alkaloid production. Low concentrations of gibberellin suppressed browning of the cultured roots, and stimulated their growth and the production of bisbenzylisoquinoline alkaloids. Reducing the major element concentration of B5 medium by half increased root growth and alkaloid content. Elimination of the ammonium sulfate from B5 medium also increased the root alkaloid content, but it decreased growth as well. The optimal sucrose concentration was 3%. Cytokinin did not promote alkaloid production. As a result of these investigations, a modified B5 medium (SB5 medium) was developed for the production of bisbenzylisoquinoline alkaloids by cultured roots of S. cepharantha. The cultured roots grew well in this medium, with about a 20-fold dry weight increase in 30 days. The aromoline content in cultured roots was more than 1% and berbamine content more than 0.5% of the dry wt. The alkaloid levels of these cultured roots are much higher than those of the plants.

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Microorganisms that produce ribavirin (1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide; virazole^®) directly from orotidine and 1, 2, 4-triazole-3-carboxamide (TCA) were screened from our stock cultures. Of the 425 strains, Erwinia carotovora AJ 2992 was found to possess potent ribavirin-producing ability, from orotidine and TCA. In the presence of intact cells of E. carotovora AJ 2992, 183 mM ribavirin was produced from 300 mM orotidine and 300 mM TCA on 48 hr reaction.

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The amino acid sequence of barley rootlet trypsin inhibitor (BRTI) has been established. The reduced and S-carboxymethylated BRTI was cleaved with cyanogen bromide (CNBr), and three fragments (I, II, and III) were separated using gel filtration on a Bio-Gel P-30 column. Each CNBr-fragment was digested with trypsin and the resulting tryptic peptides were sequenced either by manual Edman degradation or by the DABITC/PITC double-coupling method. Alignment of the tryptic peptides from CNBr-fragment CB-I, which is the largest fragment, was done by analyzing the chymotrypic and thermolytic peptides derived from the CNBr-fragment CB-I. Thus, the amino acid sequence of BRTI consisting of 124 amino acid residues was established. BRTI proved to have intramolecular homology of 55% between the N-terminal half (positions 1-62) and the C-terminal half (positions 63-124); it also displays intermolecular homology with the trypsin inhibitors from wheat germ, rice bran, and soybeans. By comparing with soybean Bowman-Birk trypsin inhibitor, the reactive sites of BRTI are assumed to be Arg(17)-Ser(18) and Arg(75)-Ser(76).

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N-alkyl-N'-(α, α, dimethylbenzyl)-2, 4-diamino-6-chloro-s-triazines were synthesized, and their phytotoxicity against paddy weeds and rice plants was evaluated. The α-branched alkyl group played an important role in increasing the phytotoxicity against barnyardgrass. Among these compounds, N-sec-butyl-N'-(α, α-dimethylbenzyl)-2, 4-diamino-6-choloro-s-triazine (9) and N-(1-ethylpropyl)-N'-(α, α-dimethylbenzyl)-2, 4-diamino-6-chloro-1y-triazine (13) were found to have high herbicidal activity against barnyardgrass with safety for directly sown rice plants during pot testing. These two compounds (9 and 13) also showed high selectivity between rice plants and barnyard millet from the petri dish test, and scarcely inhibited the photosynthesis of leaf slices of pokeweed (Phytolacca americana L.). These compounds seem to have a different mode of action from that of atrazine.

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The L-leucine productivity of an L-leucine producer, Corynebacterium glutamicum H-1204, was unstable due to reversion, which was assumed to be caused by the depletion of intracellular L-valine. The genetic enhancement of L-valine biosynthesis was attempted for stabilization using L-valine accumulation as an index. Strain AB-47, derived as an α-aminobutyrate resistant mutant, accumulated L-valine as a by-product besides L-leucine. The L-leucine productivity of strain AB-47 was remarkably stabilized under conditions under which that of parental strain H-12Q4 became unstable. These results indicate that the balanced biosynthesis of metabolites is important for the breeding of a stable amino acid producer.

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The enzymatic background for the reversion or stabilization of an L-leucine producer, Corynebacterium glutamicum H-1204, was examined as to the activities of α-acetohydroxy acid synthetase (AHAS) and α-isopropylmalate synthetase (IPMS), which are the key enzymes for the L-valine and L-leucine biosynthetic pathways branching from a common precursor, respectively.IPMS was derepressed and desensitized to L-leucine in strain L-76 (having the same properties as H-1204) and a stabilized strain, AB-47. In contrast, AHAS was derepressed only in strain AB-47. These results clarified enzymatically that the specific enhancement of L-leucine biosynthesis induced the depletion of L-valine, which caused reversion, and also the balanced metabolism was of great importance for the breeding of a stable amino acid producer.

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The oxidative modification of bovine serum albumin with an ascorbic acid-copper ion system was studied. Under physiological conditions (pH 7.2, ambient temperature), this system mainly caused the modification of amino acid residues in the protein, and its polymerization was scarcely observed. The results of spectrophotometric assays and amino and analysis of the protein clearly suggested the selective damage to tryptophan and histidine residues. The reaction could be retarded by catalase and Cu(II)-chelating agents, while superoxide dismutase and hydroxyl radical scavengers showed little effect. These specific reactions were explained by the site-specific formation of the oxygen-derived free radical followed by its reaction with a specific site of the protein.

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Three novel dithiazine compounds in the aroma concentrate from cooked sakuraebi, Sergia lucens Hansen, were isolated. Their structures were confirmed as 4, 6-dimethyl-2-propyl-1, 3, 5-dihydrodithiazine (A), 4-butyl-2, 6-dimethyl-1, 3, 5-dihydrodithiazine (B) and pyrrolidino[1, 2-e]4H-2, 4-dimethyl-1, 3, 5-dithiazine (C) by spectroscopic analyses. The same compounds have also been found in the aroma concentrate from cooked krill. These three compounds were newly discovered as food volatiles, and among them, compound C seems to take an important role in the aroma of cooked small shrimp by its strong roasted aroma and its relatively high concentration.

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An enzymatic reaction with supercritical carbon dioxide (SCCO₂) as the reaction medium was investigated. Lipase could catalyze the batch type reaction of hydrolysis and interesterification (acidolysis) in SCCO₂ at 50°C and 29.4 MPa. The time course of interesterification was influenced by the water content as well as by the kind of reaction medium. The initial velocities of hydrolysis and interesterification were greater in SCCO₂ than in n-hexane when the water content increased. A part of this difference in reaction velocity was supposed to be due to water, a modifier of the solvent, the solubility of which in SCCO₂ was estimated to be a hundred times that in n-hexane.

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Glycinin-rich organelles were isolated from developing soybean cotyledons by two steps of sucrose gradient ultracentrifugation. The organelles had an average density of 1.24, and contained 63-68% protein, 2-14% sugar, and 2-7% lipid. The lipid was 21-24% phospholipid and 76 -79% neutral lipid. The polypeptide compositions of the isolated organelles were compared with those of developing cotyledons and mature protein bodies by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the cotyledons at mid-developing stage α and α' subunits of β-conglycinin were the major polypeptides, and many other polypeptides including small amount of glycinin were found. In the isolated organelles, most of the component polypeptides were glycinin and a slight amount of β-conglycinin was associated.

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D-Gluconate dehydrogenase (EC 1.1.99.3) from bacterial membranes was immobilized on the surface of a carbon paste electrode with mixed-in p-benzoquinone. The electrode surface was covered with a dialysis membrane. The current response to D-gluconate of electrodes with different amounts of the immobilized enzyme and different thicknesses of the dialysis membrane was studied, and their effects on the current response were quantitatively evaluated by the theory of biocatalyst electrodes with an entrapped mediator. On this basis, an electrode suited for a D-gluconate sensor was constructed and the current-response characteristics of the electrode (reponse time, linear range of the calibration curve, influences of pH, temperature, oxygen and so on) were investigated. The amperometric D-gluconate sensor required neither oxygen nor any reagents to be added to the test solution, and could measure D-gluconate in the range of 0.1 to 4 mM.

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Bacillus thuringiensis subsp. aizawai IPL7 produced 130-kDa and 135-kDa insecticidal proteins. The 135-kDa protein gene was cloned from a cured derivative of strain IPL7, which lacked the 130-kDa protein gene. The cloned 3.6-kb DNA fragment contained a 3, 623-bp protein-coding region with 72-bp 5'-flanking and 20-bp 3'-flanking regions. The open reading frame encoded a 133, 161-Da protein consisting of 1, 176 amino acid residues. When compared with the amino acid sequence of the 130-kDa protein reported previously, it was found that amino acid substitutions mainly occurred in three regions; amino acid residues 206 to 455, 788 to 819, and 1, 061 to 1, 137 of the 135-kDa protein. The cloned 135-kDa protein gene was expressed in Escherichia coli under the control of the tac promoter and rrnB terminator. The synthesized 135-kDa protein amounted to 30% of the total cellular protein. The purified 135-kDa protein had insecticidal activity against Plutella xylostella larvae.

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Serratia Mn-superoxide dismutase (SOD) was modified with dextran (Dx) of M.W. 80K and polyethyleneglycol (PEG) of M.W. 5K. SOD was modified at 34% of its free amino groups by Dx. DX-SOD retained 67% of the enzymatic activity of native SOD and had cross-reacted less with anti-SOD in vitro but higher immunogenicity against SOD in vivo. SOD was modified at 24% of the free amino groups by PEG. PEG-SOD retained 52% of the enzymatic activity, had lower antigenicity and immunogenicity, and induced immuno-tolerance to SOD. The half-life of PEG-SOD in the rat blood circulation was about ten times as long as that of SOD. PEG-modification of SOD enhanced its pharmacological activities as an anti-inflammatory and radioprotective agent.

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The absolute configuration of allosamizoline (1), a constituent aminocyclitol derivative of allosamidin (2), was elucidated by the exciton chirality method using its 3, 4-bis[p-(dimethylamino)benzoyl]-6-trityl derivative (7).

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