An attempt was made to isolate hypotensive substances from a hot water extract of Citrus unshiu. Six flavonoid glycosides were isolated by repeated chromatography and gel nitration after extraction with butanol and treatment with lead subacetate. Their structures were established to be limocitrin 3-β-D-glucose (1), limocitrin-3-α-L-rhamnose (2), 3, 6-di-C-glucosylapigenin (3), narirutin (4), rutin (5) and narcissin (6) by UV, ¹H-NMR and ¹³C-NMR spectroscopy and sugar analysis. Each component was intravenously injected into SHR-SP rats (1 mg/100 g body weight), and 3 and 5 were found to lower their blood pressure.
Among these compounds, 2 and 3 were a new flavonol and flavone glycoside, respectively.

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The effect of ionic strength during heat denaturation was investigated on the surface hydrophobicity and surface functional properties of soybean 11S globulins. The change in surface hydrophobicity of the protein was examined during heating at different ionic strengths, and the value increased with heating temperature over the range of ionic strengths used. When the protein solution was heated at a low ionic strength, the surface hydrophobicity changed more with heating temperature and the thermal transition point was lower than that of the protein heated at a high ionic strength. This suggests that 1 IS globulins are more sensitive to heat at low ionic strengths than at high ionic strengths. The changes in surface functional properties of 11S globulins by heating were also investigated. The emulsion stability increased greatly with heating temperature in proportion to the increase in surface hydrophobicity. The emulsion of the protein heated at a low ionic strength was more stable than that of the protein heated at a high ionic strength, depending on the higher surface hydrophobicity. The emulsion stability of heated 11S globulins correlated linearly with the surface hydrophobicity at different ionic strengths. On the other hand, the foaming power produced a curvilinear correlation with the surface hydrophobicity.

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A method for the determination of allethrin and other pesticides in mosquito coils was developed by the combination of shaking extraction and gas chromatography (GC). Allethrin and other pesticides were adequately extracted by shaking for only half an hour with a mixture of toluene and 99% formic acid (5:1). This shaking extraction method was more effective for shortening the extraction time compared with the Soxhlet extraction method, and accurate determination was achieved without the interference from inert materials in the mosquito coils. The recovery of allethrin in various contents from the coils was 96.6 to 97.1%, with a 1.2 to 1.5% coefficient of variation. Furthermore, the recoveries of other pesticides and synergists from the coils were 94 to 102% by this shaking extraction method.

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Studies were carried out to elucidate the chemical nature of the desmutagenic factor of Burdock (Arctium lappa LINNE), Which is shown to be a complex polymer having a molecular weight of about 300, 000.⁵⁾ Its desmutagenic effects are demonstrated on indirect mutagens such as Trp-P-1 and Trp-P-2, as well as on direct mutagens such as 2-nitro-l, 4-diamino benzene and 4-nitro-l, 2-diamino benzene. The present findings suggest that this factor is not a protein because of the low content of nitrogen and the non-color reaction of its hydrolysate with ninhydrin. The desmutagenic factor probably possesses carboxyl groups as indicated by a ¹³C-NMR spectrum, potentiometric titration and analysis of its pyrolysates. This analysis of the factor's pyrolysates and its reductive degradation substances on the GC-mass spectrum provided evidence that the factor also has an aromatic ring as a basic structure. The factor may be a lignin-like compound having a 10% sugar content.

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The composition of low molecular weight carbohydrates in 26 samples of cultivated and wild soybeans were determined by gas liquid chromatography. Sucrose and stachyose were found to be major sugars in their seeds. The wild and cultivated types were different in their carbohydrate compositions. The cultivated types showed higher sucrose, galactopinitol A, galactinol and digalactosylglycerol contents and a lower stachyose content than the wild types.

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The effects of cellulose on the production and stimulation of β-transglycosylase were studied. The β-transglycosylase of Trichoderma longibrachiatum was produced specifically in the presence of cellulose in Czapeck-Dox medium containing sucrose as a sole carbon source. The enzyme activity was stimulated by the addition of cellulose in the reaction mixture, where the transfer reaction product (a water-insoluble glucan) was apparently synthesized on the surface of the added cellulose fibers.
The hyphal wall fraction of the fungus had the same stimulatory effect on β-transglycosylase as the cellulose fibers. A cellulose-like material in this fraction was found by partial acid hydrolysis and gas chromatography. Cellotriose was the smallest substrate effective for the synthesis of a water-insoluble glucan in the presence of cellulose by the β-transglycosylase, though a significant amount of glucan could not be synthesized without the addition of cellulose.

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An in vitro method, using proteolytic enzymes for protein hydrolysis, was used to evaluate the effect of extrusion cooking on protein quality. The products studied were protein-enriched biscuits processed in a twin-screw extruder under different conditions. In the product processed at low temperature (170°C), the digestibility was comparable with that of the raw material. With increasing temperature (170 to 210°C), the in vitro availability of amino acids decreased. This decrease was most pronounced in the case of lysine, but the recovery of methionine, arginine, glycine, aspartic acid and aspargine was also affected. A loss of lysine, methionine and arginine could also be detected after acid hydrolysis. The total amount of lysine present in the raw material was not completely available, as judged from the in vitro assay as well as from balance experiments in rats. With increasing severity of heat treatment, the loss of biologically available lysine was more pronounced than the loss of total lysine. However, the loss of enzymatically available lysine was in good agreement with the results obtained in animal experiments. An increase in moisture content of the feed during processing, from 13 to 18%, improved the availability of lysine in particular.

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The retrogradation process of gelatinized potato starch was studied by DSC as a function of the aging temperature and water content. Differences in the retrogradation states dependent on these parameters were revealed by the pattern of the DSC curves. Potato starch aged at a low temperature after gelatinization gave a low endothermic onset temperature. As the starch content increased, retrogradation proceeded over a wider temperature range. The retrogradation proceeded even at 50°C for a starch content of 50%. The gelatinization enthalpy for granular starch was found to be 4-4.5cal/g of dry starch. By contrast, the maximum gelatinization enthalpy obtained for retrograded starch was found to be as low as 2.5cal/g of dry starch.

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An acetic acid bacterium which produces a new type of extracellular soluble polysaccharide was isolated from vinegar mash. The isolated strain, NBI 1022, was tentatively identified as Acetobacter aceti subsp. xylinum. The polysaccharide, named AM-2, was composed of D-glucose, L-rhamnose, D-mannose, D-glucuronic acid, and O-acetyl in a molar ratio of approximately 4:1:1:1:1. From the results of methylation, Smith degradation, and partial acid hydrolysis of the polysaccharide and its derivatives, the polysaccharide AM-2 may have a branched structure containing a backbone chain of β-(1→4)-linked D-glucose residues and a side chain shown as L-rhamnosyl-(1→6)-β-D-glucosyl-(1→6)-D-glucosyl-(1→4)-D-glucuronosyl-(1→2)-D-mannose.

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A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220, 000 by gel filtration, and a molecular weight of 32, 000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.

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The substrate specificity of a new enzyme, N^α-benzyloxycarbonyl amino acid urethane hydrolase, was investigated. The enzyme hydrolyzed N^α-benzyloxycarbonyl-glycine and -alanine, and N^α-benzoyl-glycine and -alanine. N^α-benzyloxycarbonyl-glycine was hydrolyzed to give equimolar benzyl alcohol and glycine. Equimolar benzoic acid and glycine were produced from N^α-benzoyl-glycine by the enzyme reaction.
The Km, k_o, and k_o/Km values were measured for several substrates. The k_o values varied widely with the amino acid residues. N^α-benzyloxycarbonyl-glycine and N^α-benzoyl-glycine produced relatively small changes in the Km values (0.36-0.10mM) and the k_o/Km values (99440-2000M^-1 sec^-1). The rate of hydrolysis is significantly affected by electron-supplying substituents on the benzene ring.

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Laccase from Polyporus versicolor was found to develop a strong blue or red color with phenol in the presence of coupling compounds such as 2, 6-dibromo-4-aminophenol or 4-aminoantipyrine. Based on this phenomenon, concentrations of phenol (0.05-0.4 HIM) could be determined from the density of the color developed with laccase. Thus, assay methods for α-glucosidase and α-amylase were designed. α-Glucosidase (0.02-0.2 units) hydrolyzed phenyl α-glucoside and the phenol released was converted to a colored substance by laccase. Human α-amylase could also be determined using phenyl maltotetraoside or pheny maltopentaoside as the substrate. The phenyl maltooligosaccharide was hydrolyzed to phenol and glucose by the combined action of α-amylase and α-glucosidase, and the resultant phenol was assayed by the same method as described above. The proposed method may be used in conjunction with a conventional photometer, because the color developed is reddish or blue.

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An α-amylase inhibitor, Paim, obtained from the culture filtrate of Streptomyces corchorushii inhibited mammalian α-amylases except for human and rabbit, that is, Paim distinguished clearly between pig and human α-amylases, even though enzymatic properties of two amylases were very similar. El complex formation between pig pancreatic amylase and Paim was demonstrated by gel filtration and UV difference spectroscopy. Time required for complex formation was only 60 sec. Stoichiometry between I and E was 2:1. Paim combined with human salivary amylase in a ratio of 2:1, even though Paim had no inhibitory activity against human amylase.

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About 7000 microbial culture filtrates have been screened for inhibition of α₂-macroglobulin, one of the plasma protease inhibitors. One strain, identified as Streptomyces canus, was found to produce a potent inhibitor named API-13782. The inhibitor was purified from the culture filtrate by column chromatographies on Daiaion HP-20 and Dowex 1×2, and then by high performance liquid chromatography. API-13782 was an acidic compound of low molecular weight of, 441. It showed time-independent, fairly specific inhibition against α₂-macroglobulin, and required some kinds of divalent metal ions such as Ca⁺⁺, Mn⁺⁺, or Co⁺⁺ for development of its inhibitory action. In serum the activity of plasmin formed by urokinase, a plasminogen activator, was increased by preteatment with API-13782. This suggests that API-13782 is clinically useful as a potentiator of urokinase.

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Membrane-bound glycerol dehydrogenase was solubilized and purified about 100-fold from the membrane of Gluconobacter industrius IFO 3260 grown on a glycerol-glutamate medium. Solubilization of the enzyme was successfully achieved by use of 0.5% dimethyldodecylamineoxide in 0.05M Tris-HCl, pH 8.0. Alcohol dehydrogenase and D-glucose dehydrogenase, which were abundantly formed in the same bacterial membrane, were eliminated on Solubilization. Glycerol dehydrogenase was further purified through fractionation with polyethylene glycol 6000. The enzyme showed a broad substrate specificity and various kinds of polyhydroxyl alcohols, in addition to glycerol, were rapidly oxidized in the presence of 2, 6-dichlorophenolindophenol and phenazine methosulfate as the electron acceptor but NAD and NADP were inert. The enzyme was proved to be a quinoprotein in which pyrroloquinoline quinone functioned as the prosthetic group.

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The proline auxotrophic strain of Acetobacter aceti No. 1023 treated with CaCl₂ solution was transformed to the Pro⁺ phenotype at a frequency of up to 10²/μg DNA using chromosomal DNA prepared from the wild type prototrophic strain. The CaCl₂-treated cells of A. aceti No. 1023 could also be rendered competent for uptake of plasmid DNA. In an attempt to produce an appropriate cloning vector for A. aceti, the restriction patterns of the cryptic plasmids, pTA5001A (23.5 Kb) and pTA5001B (23Kb), found in A. aceti No. 1023 were determined. A selectable marker (ampicillin resistance) was introduced onto these cryptic plasmids by fusing them to vector pACYC177 from E. coli, using their single restriction site for XhoI. The hybrid plasmids generated could replicate in and confer ampicillin resistance to both A. aceti and E. coli. The maximum transformation frequency for A. aceti No. 1023 with these vectors was 10³/μg DNA.

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In order to elucidate the relationship between bitter taste and chemical structure in peptides, various kinds of model bitter peptides containing arginine, proline and phenylalanine were synthesized, and the contribution of the individual amino acids to the bitter taste was made clear. It was confirmed that, in order to strengthen the bitterness in di- and tripeptides, the hydrophobic amino acid needs to be located at the C-terminal and, conversely, the basic amino acid should be located at the N-terminal. Furthermore, a strong bitter taste was observed when arginine was contiguous to proline such as Arg-Pro, Gly-Arg-Pro and Arg-Pro-Gly. A synergistic effect for bitter taste was observed in the peptides whose structure is (Arg)_l, -(Pro)_m-(Phe)_n (l=1, 2; m, n=1-3) by increasing the number of amino acids. Among them, the octapeptide (Arg-Arg-Pro-Pro-Pro-Phe-Phe-Phe) possessed an extremely bitter taste with its threshold value of 0.002 HIM and was found to be the most bitter among the peptides.

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The amounts of free polyamines in Hiproly barley callus increased when the callus was deprived of 2, 4-D. At the 60th day after auxin withdrawal, the putrescine level was especially elevated in the callus. At the 90th day after auxin withdrawal, the levels of putrescine and spermine were elevated. The spermine level drastically increased with root formation, and the putrescine level in bound polyamines increased in the callus after auxin withdrawal. These results showed that the amounts of polyamines in Hiproly callus increased with root formation on the callus. In relation to changes in the levels of polyamines of Hiproly callus, arginine decarboxylase activity remained almost constant, but ornithine decarboxylase activity increased in the callus after 2, 4-D withdrawal. Ornithine decarboxylase activity was about 1.7-fold higher than arginine decarboxylase activity when Hiproly callus was deprived of 2, 4-D. It is suggested that the enhancement of ornithine decarboxylase activity is involved in the mechanism leading to elevated polyamine levels in the callus.

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DNA breakage by hydrolyzable tannins of known structures was investigated by agarose or polyacrylamide gel electrophoretic analysis. Hydrolyzable tannins could cause both double-strand and single-strand breakages in λDNA in the presence of Cu²⁺. The breaking reaction was strongly suppressed at concentrations higher than 100μM of hydrolyzable tannins. DNA breakage by various tannins was dependent upon their sorts, numbers, and binding sites of the constituent acids and polyalcohols. Metallic ions other than Cu²⁺ had little effect on the breaking reaction.

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Synthesis of γ-glutamyl-L-3, 4-dihydroxyphenylalanine from a γ-glutamyl donor (glutathione or glutamine) and DOPA (L-3, 4-dihydroxyphenylalanine) was examined making use of the partially purified γ-glutamyltranspeptidase from a bacterium, Proteus mirabilis. The optimum pH for γ-glutamyl-DOPA synthesis from glutathione was 9.5 to 11 and the amount of peptide produced increased with the glutathione concentration. The best conditions for γ-glutamyl-DOPA synthesis were investigated and 13mg/ml of γ-glutamyl-DOPA was synthesized with a yield of 6.7% of the substrate, glutathione. The product was isolated from a large scale reaction mixture and the structure determined by physicochemical analyses; PMR spectrometry, mass spectrometry and IR spectrometry. γ-Glutamyl-DOPA synthesis was also performed with glutamine as the γ-glutamyl donor.

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The distribution of microorganisms in 10 samples of salted dried fish and the effects of irradiation of them were studied. The total aerobic bacteria in commercial dried fish were determined to be from 2 × 10⁴ to 3 × 10⁶ per gram. Mold counts were 1 × 10² to 7 × 10³ per gram with a lower amount of yeasts. In spoiled dried fish, total aerobic bacteria were determined to be 4 × 10⁶ or 1 × 10⁷ per gram with a few yeasts. Coliforms were not isolated on MacConkey agar plates from any of the samples. The predominant bacteria occurring in spoiled dried fish were Pediococcus halophilus, Vibrio costicola and Planococcus sp. More than 50% of the molds consisted of the Aspergillus niger group, whereas lower amounts of the A. flavus, A. fumigatus and A. ochraceus groups, Penicillium chrysogenum series, etc. were also isolated from many samples of dried fish. All kinds of putrefactive microorganisms were radiation sensitive, and a dose of ca. 500 krad appears to be sufficient for extension of the shelf-life of dried fish from 2 to 4 times.

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The microbiological synthesis of 9-β-D-arabinofuranosyl adenine (ara-A, an antiviral drug) from adenine and arabinofuranosyluracil (ara-U) is described. Various bacteria, especially Enterobacter aerogenes, Escherichia coli, Erwinia herbicola and Aeromonas salmonicida, were found to be able to transfer the arabinofuranosyl moiety of ara-U to adenine (transarabinosylation) in the presence of inorganic phosphate. The optimum conditions for the transarabinosylation were pH 7.0 and 60°C. No reaction was observed in the absence of inorganic phosphate and its optimum concentration was around 30 mM. Six grams of ara-A was produced in liter of reaction mixture in the presence of wet cell paste of Enterobacter aerogenes AJ 11125. Ara-A formed was precipitated in the reaction mixture and isolated with an 87% yield. Physicochemical data for the compound agreed with those of authentic ara-A.

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UDP-N-acetyl-D-gaiactosamine: κ-casein polypeptide N-acetylgalactosaminyltransferase was purified from a crude Golgi apparatus of lactating bovine mammary gland after solubilization with Triton X-100. Through chromatography on DEAE-Sephadex A-50, apomucin-Sepharose 4B, FPLC mono S, and Sephacryl S-200, and then electrofocusing, the enzyme was purified up to 7500-fold from the homogenate.
The molecular weight of the enzyme was estimated at 200, 000 from gel filtration. The pI value of the enzyme was 6.4 on electrofocusing. The purified enzyme transferred GalNAc from UDP-GalNAc, not to the carbohydrate chains but to the polypeptide chains of the substrates, κ-casein and mucin. The enzyme required Mn²⁺, DTT, and Triton X-100 for maximal activity. The Km value for UDP-GalNAc was 16.2μM. Km values for κ-subcomponents 1 and 7, and apomucin were 1.15, 5.10, and 0.192mg/ml, and V_max values were 254, 259, and 581 nmol/hr/mg, respectively. Thermal stability and the effects of pH, milk components, lectins, and nucleotides were examined.
α_sl-Casein strongly inhibited GalNAc transfer to κ-casein. The inhibitory effect of α_sl-casein was canceled by the addition of Ca²⁺, which causes casein micelle formation. This means that the glycosylation of κ-casein occurs after casein micelle formation triggered by the accumulation of Ca²⁺ in vivo.

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The purified polyethylene glycol (PEG) dehydrogenase from cells of a synergistic mixed culture of Flavobacterium and Pseudomonas species showed a similar absorption spectrum to those of other quinoproteins reported so far. The prosthetic group of the PEG dehydrogenase after extraction with cold methanol and purification by DEAE-Sephadex A-25 column chromatography and Sephadex G-25 gel filtration showed the same elution profiles as those of authentic pyrroloquinoline quinone (PQQ). Absorption and fluorescence spectra of the purified prosthetic group and its prosthetic group capability for glucose dehydrogenase indicated that it was identical with authentic PQQ.
The enzyme was induced during bacterial cell growth on a medium containing.PEG 6000 as a sole source of carbon. The purified enzyme oxidized primary alcohols of C₂-C₁₆ and the corresponding aldehydes of C₄-C₇. The enzyme also reacted with nonionic surfactants containing PEG residues. The enzyme reduced 2, 6-dichlorophenolindophenol (DCIP) and the Km value for DCIP was calculated to be 1.4×10^-4M. The DCIP reductase activity was inhibited by carbonyl reagents like semicarbazide, hydrazine, hydroxylamine and 1, 4-benzoquinone. 1, 4-Benzoquinone inhibited the DCIP reductase activity competitively as to DCIP.

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An in vitro DNA-directed protein-synthesizing system of Bacillus subtilis was developed using vegetative and sporulating cell extracts. Protease activity was inhibited by the addition of three kinds of protease inhibitors and removed from the extracts by hemoglobin-Sepharose treatment. Endogenous RNA synthesis was very low because of elimination of endogenous DNA by polyethylene glycol 6000 treatment of the supernatant. Protein synthesis was dependent on the DNA template, ribosomes and supernatant. When pUB110 DNA was used as a template, three proteins (K1, K2 and K5) which have the same molecular weights as those synthesized in vivo were synthesized in vitro with vegetative, T₂ (2hr after the end of logarithmic growth) and T₄ cell extracts.

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Phospholipase B from baker's yeast (Saccharomyces cerevisiae) was purified by acid treatment of the crude extract, ammonium sulfate fractionation, and column chromatographies on DEAE-Sepharose CL-6B, Sepharose 4B, and Bio-Gel HTP. The purified preparation had lysophospholipase activity and phospholipase B activity in a ratio of 16:1. The optimum pH of both activities was 3.5-4.0. The enzyme was a glycoprotein and its molecular size was somewhat heterogeneous, ranged from about 280, 000 to 420, 000 by gel nitration. Phospholipase B activity was strongly stimulated by 0.1% DOC, but lysophospholipase activity was completely inhibited by the detergent. Neither activity was stimulated by Ca²⁺ and both were inhibited by SDS, Triton X-100, and Fe³⁺. The enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. The Km values for phosphatidylcholine and lysophosphatidylcholine were 0.63 mM and 0.05 mM, respectively.

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An amylase which produces maltotriose from starch as the main product was found in the culture filtrate of a strain of Bacillus subtilis newly isolated from soil. The enzyme was purified to almost complete homogeneity, as judged by disc electrophoresis in polyacrylamide gel, by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex gel filtration.
The optimum pH and temperature of the enzyme were around 6-7 and 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ strongly inhibitied the enzyme activity. The molecular weight was found to be about 25, 000 by gel filtration. The yields of maltotriose from short-chain amylose (DP 17), amylopectin, soluble starch and glycogen were about 69, 56, 56 and 40%, respectively.

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Nucleotide sequences of mRNAs were compared between major calcium-sensitive caseins of cow (α_s1-casein) and rat (α-casein). A best fit alignment of the two sequences showed homology of 81% and 69% for the 5'- and 3'-untranslated regions, respectively. Homology in the comparable coding region of the mature α_s1-casein (76% of total codons) was remarkably lower at amino acid level (46%) than at nucleotide level (69%). The low conservation at amino acid level is explained by the unusual nucleotide substitution pattern (random at all three positions of codons) in contrast to synonymous substitutions at the third position revealed on comparison of other related proteins. The evolutionary distances among the number of the casein family were estimated by comparing known nucleotide sequences of the signal peptides which were the most conserved coding regions in the family. The divergence time for most distantly related caseins (both rat α-casein/rat β-casein and rat α-casein/mouse ε-casein) was estimated to be about 170 million years.

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Endopolygalacturonase I [EC 3.2.1.15], the major component of endopolygalacturonases causing silver-leaf symptoms, was purified from culture liquids of Stereum purpureum by column chromatographies on CM-52 and Sephadex G-100. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient (S_{20, w}) was determined to be 3.21 S, and the molecular weight was estimated to be 40, 000 by gel filtration, 41, 000 by SDS-polyacrylamide gel electrophoresis and 44, 000 by sedimentation equilibrium. The enzyme had an isoelectric point of pH 8.5. The optimal pH of the enzyme was 3.5 for trigalacturonic acid, 4.0 for tetragalacturonic acid, and 4.5 for pectic acid. The enzyme was stable in the range of pH 4.0 to 9.0 and up to 70°C for 30min. The amount of the enzyme which was required to induce silver-leaf symptoms on apple trees was 20μg/tree.

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Moranoline (1-deoxynojirimycin) is a strong α-glucosidase inhibitor. A rapid screening method for isolation of moranoline-producing Streptomyces (oblate agar plate method) was developed. A glucoamylase inhibitor was isolated from the culture filtrate of Streptomyces lavendulae GC-148, a mutant strain obtained from Streptomyces lavendulae MB-733, a soil isolate; its spectral data were identical with those of moranoline from Morus species.
Increased moranoline production was achieved through media improvement and mutagenic treatments (ultraviolet irradiation and N-methyl-N'-nitro-N'-nitrosoguanidine treatment): 27-33-fold augmentation (150 to 4000-5000 μg/ml) was obtained.

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Isoprothiolane sulfoxide, an oxidative metabolite of isoprothiolane, was reduced to isoprothiolane in living rats and in an in vitro system containing rat liver supernatant. No stimulatory effect of a flavin cofactor (FAD) was observed on the sulfoxide reduction in vitro.

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A rice blast controlling agent, isoprothiolane (diisopropyl 1, 3-dithiolan-2-ylidenemalonate), stimulated the reduction of methionine sulfoxide to methionine by the rice plant. In the presence of isoprothiolane, the methionine/(methionine + its sulfoxide) ratio was increased to 129 - 208% of the control. The ethylene production by the plant was also enhanced by isoprothiolane, probably because methionine is an important precursor of ethylene. The non-parasitic damping-off caused by chilling stress on rice seedlings was effectively prevented with the application of isoprothiolane as well as ethephon, which easily decomposes to ethylene and acids. Therefore, the ethylene level modified by isoprothiolane and ethephon can contribute to their protective activity against the non-parasitic damping-off of rice seedlings. Indeed, a close relationship between the ethylene level and the protective activity against damping-off was obtained with isoprothiolane, but not with ethephon. Endogenous ethylene seems to be more effective in controlling the damping-off than exogenous ethylene from ethephon.

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Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in L-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80-90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no D-leucine transaminase activity, but five-times higher D-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for D-amino acid dehydrogenase, a key enzyme in D-amino acid metabolism.

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We describe here the synthesis of selenium araino acids with O-acetylhomoserine Sulfhydrylase, partially purified from baker's yeast. The enzyme was found to catalyze the synthesis of L-selenocystine and L-selenohomocystine from Na₂Se₂ with the corresponding acetyl-derivatives of serine and homoserine, respectively. L-Serine-O-sulfate also serves as a substrate of the β-replacement reaction. Na₂Se₂ is less efficient as a substituent donor than the physiological substrate, NaHS, and inhibits the enzyme at high concentrations. Therefore, limited amounts of Na₂Se₂ were added to the reaction mixture to increase the yield (50 to 60%). This provides a facile method to produce optically active Selenocystine and Selenohomocystine.

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The production of anthranilic acid (AnA) was investigated for 40 bacterial strains in the presence and absence of aniline. Resting cells of all aniline-assimilating bacteria tested produced AnA with aniline, but not without aniline. The cells of aniline-assimilating Rhodococcus erythropolis strains produced more AnA than those of other aniline-assimilating bacteria. Resting cells of several non-aniline-assimilating strains produced AnA in the absence of aniline. However, its production by these strains was much lower than that by the Rhodococcus strains. The production of AnA by cells of aniline-assimilating R. erythropolis AN-13 was promoted by aliphatic monocarboxylates, ATP, biotin and coenzyme A, and repressed by catechol analogues, N-ethylmaleimide and iodoacetate. On the other hand, its production by non-aniline-assimilating Pseudomonas sp. AN-21 was repressed by glucose, mannose and some amino acids.

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A β-xyloside hydrolytic enzyme of the fungus Chaetomium trilaterale was further purified by a modification of Kawaminami's procedure (DEAE-Sephadex A-25 and Sephadex G-75 column chromatography), followed by isoelectric focusing. The purified preparation was homogeneous by polyacrylamide disc gel electrophoreses at pH 4.3 and pH 8.3. The purified enzyme hydrolyzed β-D-glucopyranosides as well as β-D-xylopyranosides, and the ratio of β-glucosidase activity against β-xylosidase activity increased about 3 fold during the purification steps. The molecular weight of this preparation was estimated to be about 240, 000 by Sephadex G-200 gel filtration and 118, 000 by SDS-polyacrylamide slab gel electrophoresis. The isoelectric point was 4.86 and the amino acid composition was also determined.
The optimum pH was at 4.2 for phenyl β-D-glucoside and around 4.5 for phenyl β-D-xyloside. The β-xylosidase activity was relatively stable but β-glucosidase activity was rapidly inactivated, at the alkaline pH range above 11. The heating of the preparation at 60°C didn't show a parallel inactivation of the two activities. N-Bromosuccinimide strongly inactivated both enzyme activities. Nojirimycin and glucono-l, 5-lactone showed a stronger inhibition on β-xylosidase activity than on β-glucosidase activity. The maximal velocities decreased in the order; phenyl β-D-glucoside > cellobiose > phenyl β-D-xyloside > xylobiose; the value with phenyl β-D-glucoside was about 28-fold higher than that with phenyl β-D-xyloside.

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The nature of the active site of Chaetomium trilaterale β-xylosidase catalyzing the hydrolysis of β-D-glucopyranoside and β-D-xylopyranoside was investigated by kinetic methods. On experiments with mixed substrates, such as phenyl β-D-xylopyranoside and phenyl β-D-glucopyranoside, the kinetic features agreed very closely with those features theoretically predicted for a single active site of the same enzyme catalyzing the hydrolysis of these two kinds of substrates.
Both the β-glucosidase and β-xylosidase activities were strongly inhibited by glucono-1, 5-lactone and nojirimycin (5-amino-5-deoxy-D-glucopyranose). β-Xylosidase activity was inhibited non-competitively by the two inhibitors, but β-glucosidase activity was competitive. Methyl β-Dxylopyranoside, methyl β-D-glucopyranoside, 1-thiophenyl β-D-xylopyranoside, and 1-thiophenyl β-D-glucopyranoside poorly inhibited both activities. Methyl β-D-xylopyranoside inhibited the β-xylosidase activity competitively but the β-glucosidase activity was non-competitive, whereas methyl β-D-glucopyranoside inhibited the β-xylosidase activity non-competitively but the β-glucosidase activity was competitive. 1-Thiophenyl β-D-xylopyranoside and 1-thiophenyl β-D-glucopyranoside behaved as competitive inhibitors.
From these results, it was concluded that the β-xylosidase and β-glucosidase activities reside in one catalytic site, and this suggests that there might be two kinetically distinct binding sites in the active center of the same enzyme.

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Iodinations of two Ricinus communis lectins, ricin D and hemagglutinin (CBH), with potassium iodide at pH 7.0 and 0°C led to inactivation of the cytoagglutinating activity on sarcoma 180 ascites tumor cells as well as the toxicity to HeLa cells of ricin D, whereas the cytoagglutinating activity of CBH was affected slightly. In the presence of lactose, which binds to ricin D, one tyrosyl residue in the B-chain of ricin D was protected from iodination and 40% of the cytoagglutinating activity was retained. This protection against iodination was not observed in the presence of glucose, which does not bind to ricin D. This suggested that the protected tyrosyl residue in the B-chain of ricin D may be situated at or near the saccharide binding site and directly involved in the binding to the saccharide moieties of the cellular receptors.
Adsorption of the iodinated ricin D to Sepharose 4B indicated that one of the two saccharide binding sites in ricin D is still intact and participates in the binding to saccharide: ricin D was altered from divalent to monovalent by the iodination.
We found from binding experiments with ¹²⁵I-labeled iodinated ricin D to HeLa cells, that the low toxicity of the iodinated ricin D may be attributed mainly to its decreased internalization into the cells and that the divalent binding of ricin D to the cellular receptors is important for this internalization.

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Two triterpenoid saponins were isolated from an aqueous or a methanolic extract of camellia (Camellia japonica) leaf. They had an antifungal activity characterized by abnormal germination of conidia. These saponins were composed of 3β-hydroxy-18β-acetoxy-28-norolean-12-en-16-one or 3β 8β-dihydroxy-28-norolean-12-en-16-one as aglycon, and D-glucuronic acid, D-glucose and two moles of D-galactose as the sugar moiety. The authors have named these new saponins "Camellidin, " which might have value for studies in the fields of phytopathology and biochemistry.

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