Studies on Nicotinoids as an Insecticide

In search for the mechanism of insecticidal action of nicotinoids, the kinetics of house fly head cholinesterase inhibition by nicotine were studied to determine the type of inhibition. The pH dependency of inhibition was interpreted in terms of protonation of nitrogen atom in the molecule and the inhibition was shown to be the mixed type closing to competitive type. The Michaelis constants are 3.5 × 10^?4 M and 4.1 × 10^?4 M, while the apparent inhibition constants obtained are 1.0 × 10^?3 M and 2.3 × 10^?3 M at pH 7.4 and 8.4, respectively. The type of the inhibition by nicotine monomethiodide carring univalent cation was competitive and the apparent inhibition constant is 1.5 × 10^?4 M. These data indicated that the cationic head of nicotinium ion interacts with the anionic site in the active center of cholinesterase.     

α-Glucosidase Inhibitor from the Seeds of Balsam Pear (Momordica charantia) and the Fruit Bodies of Grifola frondosa

α-Glucosidase inhibitory activities were found in aqueous methanol extracts of the seeds of Momordica charantia and the fruit bodies of Grifola frondosa. An active principle against the enzyme prepared from rat small intestine acetone powders was isolated and characterized. The structure of the isolated compound was identified as D-(+)-trehalose by FDMS, ¹H-, ¹³C-NMR, and [α]_D measurements. The inhibitory activity of trehalose was compared with 1-deoxynojirimycin. Trehalose showed 45% inhibitory activity at the concentration of 2×10^?3 M, but 1-deoxynojirimycin had 52% inhibitory activity at 1×10^?7 M.     

Simple Determination of Trace Amounts of Anionic Surfactants in River Water by Spectrophotometry Combined with Solid-phase Extraction

A simple and sensitive specrophotometric method combined with solid-phase extraction (SPE) for the simultaneous determination of sodium linear-dodecylbenzenesulfonate (DBS) and sodium dodecyl sulfate (SDS) is described. The C2 (ethyl group bonded silicagel) cartridge could be repeatedly used more than 500 times for SPE, and it enabled the anionic surfactants to be concentrated by 50-fold. The calibration graph for DBS was linear in the range from 1.6×10^?8 M to 5.0×10^?7 M and for SDS from 2.0×10^?9 M to 3.0×10^?7 M. The relative standard deviation (n=5) for 5.0×10^?7 M DBS was 3.1% and for 2.5×10^?7 M SDS was 1.7%. The proposed method was applied to the simultaneous determination of DBS and SDS in river-water samples.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

UDP-glucose Pyrophosphorylase from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-1-phosphate and UTP were determined to be 4.45 × 10^?4 M, 2.33 × 10^?4 M, 9.38 × 10^?4 M and 2.98 × 10^?4 M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.     

Kinetic Studies on Crystalline Yeast Phosphoglyceric Acid Mutase

Effects of the substrate and the coenzyme on the crystalline yeast phosphoglyceric acid mutase activity have been investigated. Lineweaver-Burk plots at different concentrations of the substrate (d-3-phosphoglyceric acid: 3×10^?7 to 8×10^?3m) and the coenzyme (d-2, 3-diphosphoglyceric acid: 8×10^?7 to 10^?5m) change in such a way to indicate the involvement of an enzyme-substrate-coenzyme ternary complex as an active intermediate in the enzymic reaction process. It is concluded that the reaction catalyzed by the yeast enzyme follows the sequential pathway and that a phosphorylated enzyme does not participate as an obligatory intermediate in the reaction mechanism, if it occurs. Kinetic studies indicate Km values of 6×10^?4m for d-3-phosphoglyceric acid and 8×10^?7m for d-2, 3-diphosphoglyceric acid. The substrate is a competitive inhibitor of the coenzyme with a Ksi (inhibition constant) of 3.2×10^?3m. The coenzyme inhibition is not observed at concentration tested. A kinetic treatment to determine the mechanism of the enzyme reaction from the experimental data which are obtaind in the range of inhibitory substrate concentrations is presented.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Inhibitory Activities of 3-Thiophenecarboxylic Acid and Related Compounds on Plant Growth

3-Thiophenecarboxylic acid (1) showed strong growth-inhibitory activity toward the following plants but not Glycine max Merrill; Brassica campestris subsp. rapa Hook. fil. et Anders, Sesamum indicum L., Lactuca sativa L. var. longifolia Lam, Echinochloa utilis Ohwi et Yabuno and Allium tuberosum Rottler. Compound 1 strongly inhibited the growth of roots of S. indicum and L. sativa even at the low concentration of 5.0 × 10^?5 m. The growth-inhibitory activity of 1-related compounds (2–6) on S. indicum was also studied. Among the compounds, 3-thiopheneacetic acid (6) showed the strongest inhibitory activity, but 3-thiophenecarboxaldehyde (2), 3-thiophenemethanol (3), and 3-thiophenecarboxamide (5) showed no activity. The radicles of plants treated with these active compounds showed negative geotropism.     

Amino Acid Metabolism in Microorganisms

3-Methylthiopropylamine (MTPA) formation from l-methionine in Streptomyces sp. K37 was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of l-methionine. The properties of the enzyme were studied in detail using acetone dried cells or cell-free extract. The enzyme was specific for l-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The optimum pH for the reaction was 6.0~8.0 and the optimum temperature was about 40°C. Carbonyl reagents (10^?2~10^?3 m) inhibited the reaction completely, and silver nitrate and mercuric chloride (10^?3~10^?4 m) markedly inhibited the reaction. Km value for the reaction was 1.21 × 10^?5 m. l-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use.     

Depolymerization of Hyaluronic Acid by D-Fructose 6-Phosphate

Depolymerization of hyaluronic acid obtained from Streptococcus zooepidemicus by D-fructose 6-phosphate was investigated for characterization of reducing sugar-mediated degradation of biopolymers under physiological conditions. The extent of depolymerization was monitored by the decrease of viscosity of a reaction mixture containing 1.0% hyaluronic acid, D-fructose 6-phosphate, and 1.0 × 10^?2 mM of Cu²⁺ in phosphate buffer, pH 7.4. It was found that the depolymerization of hyaluronic acid was dependent on the concentration of the reducing sugar and was specifically accelerated by the presence of Cu²⁺. The reaction was found to be significantly inhibited by catalase, superoxide dismutase (SOD), 1,2-dihy­ droxybenzene 3,5-disulfonic acid (Tiron), and chelating agents such as EDTA and diethylene triamine penta­ acetic acid (DETAPAC), although the inhibition by SOD was low. Almost the same depolymerization rates were observed in hyaluronic acid preparations of different molecular weight (1.1 × 10⁶, 8.8 × 10⁵, and 6.8 × 10⁵). The rates, however, were different for hyaluronic acids obtained from S. zooepidemicus, rooster comb, and umbilical cord. It was concluded that depolymerization of the polysaccharide was caused by active oxygen species generated by the autoxidation of D-fructose 6-phosphate in the presence of Cu²⁺, in a mechanism similar to that previously reported for the degradation of DNA and inactivation of virus in vitro.     

Production of l-aspartic acid by biotransformation and recovery using reverse micelle and gas hydrate methods

l-Aspartic acid (l-Asp) was produced using Escherichia coli (ATCC 11303), and its recovery from the reaction mixture was studied using reverse micelle and gas hydrate methods. The effect of initial substrate concentration on l-Asp production was also investigated, and inhibition was shown to occur above 0.75 mol L^?1. The values of the kinetic constants were determined as r_max=2.33×10^?4 mol L^?1 min^?1, K_M=0.19 mol L^?1, and K_ss=3.98 mol L^?1. The reverse micelle phase used for extraction contained Aliquat-336, 1-decanol and isooctane, and a micro-injection technique was used for extraction of l-Asp. The reverse micelle system is a useful technique for obtaining small particle sizes, which can be used for the synthesis of nanoparticle biomolecules. Recovery of l-Asp from reverse micelles using CO₂ hydrates was carried out, giving a recovery of 55%. The formation of CO₂ hydrate from the reverse micelle solution breaks the micelle by reducing the amount of water in the micelle structure, thus precipitating the l-Asp.     

Fukiic Acid Isolated from the Hydrolysate of a Polyphenol in Petasites japonicus

d-Glucose-isomerizing enzyme was purified in a crystalline form with a good yield from the cells of Bacillus coagulans, strain HN-68, and some phsicochemical properties were investigated.

The purified enzyme was homogeneous on both ultracentrifugal and disc-electrophoretical analyses. The molecular weight of the enzyme was determined to be 175,000 and 160,000 from the sedimentation-viscosity method and the gel filtration method, respectively.

The sedimentation coefficient , partial specific volume, at 280 mμ, and the nitrogen content of the enzyme were determined to be 10.2×10^?13 sec, 0.705 cm³g^?1, 10.6 and 16.2%, respectively. The integral numbers of amino acid residues per molecule calculated on the basis of 160,000 were as follows; Lys₁₂₀, His₄₉, Arg₆₁, Asp₁₈₂, Thr₈₇, Ser₇₀, Glu₁₃₆, Pro₄₄, Gly₁₀₆, Ala₁₄₀, Half-Cys₀, Val₅₃, Met₂₇, Ileu₅₁, Leu₁₃₄, Tyr₅₈, Phe₉₆, Try₁₃, and amide-ammonia₈₀.

Purified enzyme preparation obtained from Bacillus coagulans, strain HN-68 requires Co²⁺ for d-glucose- and d-ribose-isomerizing activities and Mn²⁺ for d-xylose-isomerizing activity. The values of Km for d-glucose, d-xylose and d-ribose were 9×10^?2, 1.1×10^?3, 7.7×1O^?m and of the relative V_max were 0.52, 1.1 and 0.25 mg/min at 40°C, respectively. d-Glucose-isomerizing activity was inhibited by d-xylose and d-ribose. However, there was not a difference among three activities of the enzyme with respect to following properties: Activation energy was 14,600 cal per mol. The enzyme was inhibited in a competitive manner by tris(hydroxymethyl)aminomethane, d-xylitol, d-sorbitol and d-mannitol, and the Ki values for these inhibitor were 3×10^?4, 2.5×10^?3, 2.9×10^?2 and 7×10^?2m, respectively. The ratio of three activities did not change by heat- and pH-treatments. Mn²⁺, Co²⁺ and Ni²⁺ protected strongly the enzyme from heat denaturation. The enzyme can isomerize d-glucose, d-xylose and d-ribose to their corresponding ketose, but the kinetic constants and induction studies indicated that ^d-xylose is the natural substrate for the enzyme.     

The Production and Stability of Intracellular Myrosinase from Aspergillus niger

After Screening 100 micro-organisms to detect intracellular myrosinase, only Aspergillus niger produced myrosinase.

Enzyme production was induced by the addition of ten percent of a mustard extract^* to the culture medium. The enzyme was produced in considerable amounts on the first and second day of cultivation. L-Ascorbic acid was an excellent carbon source.

The enzyme was unstable but was stabilized by coexistence with 2-mercaptoethanol (10^?2 M) and ascorbic acid (10^?3 M).     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Substrate Specificity of an α-Glucosidase in Sugar Beet Seed

The substrate specificity of sugar beet α-giucosidase was investigated. The enzyme showed a relatively wide specificity upon various substrates, having α-1,2-, α-1,3-, α-1,4- and α-l,6-glucosidic linkages.

The relative hydrolysis velocity for maltose (G2), nigerose (N), kojibiose (K), isomaltose (I), panose (P), phenyl-a-maltoside (?M) and soluble starch (SS) was estimated to be 100:130: 10.7: 22.6: 54.6: 55.8: 120 in this order; that for malto-triose (G3), -tetraose (G4), -pentaose (G5), -hexaose (G6), -heptaose (G7), -octaose (G8), amyloses (G13) and (G17), 91: 91: 91: 91: 80: 57: 75: 73. The Km values for N, K, I, P, and SS were 16.7 mM, 1.25 mM, 10.8 mM, 8.00 mM, 4.12 mM and 1.90 mg/ml, respectively; that for G2, G3, G4, G5, G6, G7, G8, G13 and G17 were 20.0 mM, 3.67 mM, 2.34 mM, 0,64 mM, 0.42 mM, 0.32 mM, 0.23 mM, 0.36 mM and 0.26 mM, respectively.

The enzyme, though showed higher affinity and activity toward soluble starch than toward maltose, was considered essentially to be an α-glucosidase.