Organophosphorus (OP) compounds have a great variety of biological activities. The functions of the phosphorus moiety in OP agrochemicals may be classified as follows; 1) the principal of phosphorylation; 2) the leaving group in alkylation; 3) a building block to maintain the Shape or physical properties of an active molecule; 4) the analog of a carboxyl group or its tetrahedral transition state in enzyme reactions; 5) a moiety of anti-metabolites mimicking physiological phosphates; 6) a carrier or protective group making a prodrug that produces an active principle after biotransformation; and 7) other unknown functions including as stressors. To have a definite, selective biological activity, the OP molecule should have a specified structure suitable in biodynamic, biokinetic, and environmental aspects.

View full abstract

Alternaria solani is a causal fungus of early blight disease of potato and tomato. Two phytotoxins, alternaric acid and solanapyrones, were isolated from the different strains of the fungus. Biosynthetic studies on these two phytotoxins have been done, and in the biosynthesis of solanapyrones, it was proved that a biological Diels-Alder reaction is involved through the polyketide pathway and the participation of the enzyme Diels-Alderase has been proved for the first time.

View full abstract

The pyruvate kinase of Microbispora thermodiastatica was purified to homogeneity and some properties of the enzyme were characterized. The molecular weight of the enzyme by gel filtration is 277, 000. The subunit molecular weight is 55, 000 by SDS-polyacrylamide gel electrophoresis, and only one N-terminal amino acid sequence was obtained. It had d pH optimum around pH 4.5 to 7.0 and was stable over the range of pH 4.0-8.0. The enzyme is thermostable and no activity was lost after heat treatment at 55°C for 60 min. AMP activated this enzyme and the saturation curve of the enzyme for PEP changed from sigmoidal type to hyperbolic type in the presence of AMP.

View full abstract

Three exopolygalacturonases (exoPG) were purified from the culture filtrate of Alternaria mali and characterized; Three exoPGs were distinguished by chromatographic properties. They contained a large amount of carbohydrates, and the molecular masses were estimated to be 51-80kDa (exoPG I) and 51-58 kDa (exoPG II and III) by SDS-PAGE. After treatment with endo-β-N-acetylglucosaminidase, their molecular masses decreased equally to 43 kDa. In addition, the amino acid sequences of the N-terminal 20 residues of the three enzymes were identical except for a few amino acid residues. The pH- and thermal stabilities, optimum pHs, and K_ms for unsatd. oligoGAs among the three exoPGs were very similar. However their substrate specificities were clearly different. ExoPG I hydrolyzed satd. oligoGAs faster than 4, 5-unsatd. oligoGAs. On the contrary, exoPG II and III preferred to hydrolyze 4, 5-unsatd. oligoGAs. No enzyme with a substrate specificity like exoPG II and III has so far been reported. It was found that A. mali also produced pectate lyase (PL), pectin lyase (PNL), and pectinesterase (PE) but no endoPG under these growth conditions.

View full abstract

Kinetic analyses have been done on the hydrolysis of p-nitrophenyl β-D-glucoside (PNPG) and P-nitrophenyl β-D-fucoside (PNPF) by the β-D-glucosidase/β-D-fucosidase enzyme from Thai Rosewood (Dalbergia cochinchinensis Pierre). PNPF showed a competitive inhibition of PNPG hydrolysis with a K_i of 0.42 mM. Hydrolysis of mixtures of PNPG and PNPF at fractional ratios ranging from 0 to 1 showed Lineweaver-Burk plots intermediate between the two extremes. The apparent K_m and apparent V_max at each fractional ratio showed good correspondence with the theoretical curves predicted for the existence of a single common active site for the hydrolysis of the two substrates.

View full abstract

We found two types of novel alkaline metalloendopeptidases (AP1 and AP2) from a marine bacterium, isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) and identified as Vibrio sp. (NUF-BPP1). We studied the structure-function relationship of these marine bacterial proteases. The electrophoretically homogeneous proteases had a molecular mass of 48 kDa for AP1 and 36 kDa for AP2 on SDS-PAGE, and showed optimum activity at around pH 9.5-10.0 (casein as substrate). Calcium chloride (5 mM) stabilized the activities over pH 6-11, but o-phenanthroline and EDTA inhibited the activities of both AP1 and AP2. The EDTA-inactivated proteases were partly restored to activity by addition of either zinc or calcium. Sodium chloride (3.5M) increased the activities toward Z-Gly-Leu-NH₂. N-Terminal sites of hydrophobic amino acid residues (Leu, Ile, Phe, Tyr, and Trp) of the peptide substrates were cleaved by AP1 and by AP2. Autolysis of AP1 in the absence of calcium ion probably produced AP2 by releasing a fragment (molecular mass of about 12kDa) from the C-terminal end of AP1.

View full abstract

In the course of our screening work for new types of lipase inhibitors, hesperidin was identified as a simple and small molecular weight inhibitor in the peels of Citrus unshiu. Hesperidin inhibited lipase activity from porcine pancreas and that from Pseudomonas, and their IC₅₀ was 32 and 132 μg/ml, respectively. Hesperidin, neohesperidin, narirutin, and naringin are known as the main flavonoids in Citrus unshiu. Neohesperidin also inhibited the lipase from procine pancreas, but did not have any effect on Pseudomonas. Narirutin and naringin did not show this effect on lipases from porcine pancreas and Pseudomonas. In animal experiments, the concentration of plasma triglyceride in rats fed a diet containing 1O% hesperidin were significantly lower than that fed the control diet.

View full abstract

When suspension cells of French bean (Phaseolus vulgaris L. cv. Goldstar) were cultured in Murashige and Skoog (MS) liquid medium containing 10^-6 M 1-naphthaleneacetic acid (NAA) and 10^-6 M 6-benzylaminopurine (BAP) (control medium), endopeptidase activity was expressed throughout the cell growth. In the medium containing 10^-5 M gibberellic acid (GA₃), the endopeptidase activity increased notably during the cell culture, but in the medium containing amino acids the activity decreased. Protein immunoblotting with an antiserum raised against a French bean cysteine endopeptidase (EP-C1) showed that a 34-kDa polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from cultured cells. Five forms of endopeptidase in extracts of cells cultured in the control medium and the four forms in the medium containing GA₃ were detected by activity staining after non-denaturing polyacrylamide gel electrophoresis. Experiments with various protease inhibitors indicated that a serine endopeptidase was expressed at high levels in cultured cells in the control medium and the activity of a cysteine endopeptidase was increased by GA₃.

View full abstract

We developed a rapid and simple staining method for lyases catalyzing cleavage of a C-S bond in sulfur-containing compounds after polyacrylamide gel electrophoresis. This method was based on the finding that 3-(4', 5'-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was readily reduced by various thiol compounds in the presence of phenazine methosulfate to yield formazan. Cystathionine y-lyase [EC 4.4.1.1] and cystine lyase were quickly detected by this staining method after polyacrylamide gel electrophoresis. The staining method developed here may be applicable to a wide range of lyases catalyzing cleavage of a C-S bond as a quick and simple method for detection of their activities on polyacrylamide gels.

View full abstract

A short (3-5 steps) synthesis of a racemic and diastereomeric mixture of Matsucoccus sex pheromones (1-3) is described. The key reaction is Lewis acid-mediated cyanation of symmetric tertiary alcohol 6 to afford common intermediate 7.

View full abstract

A Gram negative bacterial strain that can use cholate or deoxycholate as the sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida. The strain formed 3α-hydroxysteroid dehydrogenase (EC 1.1.1.50) in the cytosolic fraction. The enzyme was purified and crystallized from the cell-free extract by a procedure involving chromatographic separation and ammonium sulfate fractionation. The crystallized enzyme showed a single protein band on both polyacrylamide gel electrophoresis and SDS-PAGE, giving a molecular weight of 25, 000. An alternative molecular weight measurement by gel filtration also indicated that the enzyme has such a low molecular weight. This is the lowest molecular weight among the related enzymes so far reported. In addition to high enzyme content in the cell-free extract, since the enzyme was stable in solution for a long period and tended to be crystallized readily, a simple purification method for the enzyme with high recovery was proposed that is convenient for a large scale rapid enzyme preparation. The enzyme required NAD specifically as the primary electron acceptor but not NADP or other compounds. The apparent Michaelis constant, K_m, for cholate was 15.0μM. The enzyme also showed high substrate specificity to other 3α-hydroxysteroids. Besides cholate, deoxycholate, androsterone, glycocholate, taurocholate, and ursodeoxycholate were readily oxidized by the enzyme and showed a similar order of kinetic parameters. Judging from the enzymatic properties obtained, it is reasonable to propose that the enzyme can be available for enzymatic diagnosis of bile acids, though the enzyme reaction is virtually reversible.

View full abstract

We isolated a bacterial strain with an enzyme which releases dipeptide from Gly-Arg-p-nitroanilide. The bacterium was tentatively identified as Aureobacterium sp. The enzyme, named AuDAP, was purified and characterized. It was homogenous by SDS-PAGE and IEF, and had a molecular mass of 90, 000 Da by SDS-PAGE and 88, 000 Da by gel filtration, so it may be a monomer. The isoelectric point was 3.8 and the optimum pH was 10.0. The purified enzyme hydrolyzed Gly-Arg-pNA, a model substrate for DAP I, and Arg-Arg-MNA, a model substrate for DAP III. However, this enzyme did not hydrolyze Gly-Phe pNA, also a model substrate for DAP I. These results suggested that this enzyme did not fall under the classification of mammalian DAPs and was similar to DAP BI from Pseudomonas sp. WO24 and dDAP from Dictyostelium discoideum, although several differences Were observed between them. The N-terminal amino acid sequence of this enzyme showed no significant homology to any enzyme and protein, except only for DAP BI.

View full abstract

NADP-dependent serine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of serine to form 2-aminomalonate semialdehyde, was purified to homogeneity from a crude extract of Agrobacterium tumefaciens ICR 1600. The enzyme had a molecular mass of about 100 kDa and consisted of four identical subunits. In addition to L-serine, D-serine, L-glycerate, D-glycerate, and 2-methyl-DL-serine were substrates. However, O-methyl-DL-serine and L-threonine were inert. The enzyme showed maximal activity at about pH 9 for the oxidation of L-serine. The enzyme required NADP as a coenzyme, NAD was inert. The enzyme was not inhibited by EDTA, o-phenanthroline, or α, α'-dipyridyl, but was inhibited by HgCI₂, p-chloromercuribenzoate, L-cysteine, D-cysteine, malonate, 2-methylmalonate, and tartronate. The Michaelis constants for L-serine, D-serine, and NADP were 42, 44, and 0.029 mM, respectively.

View full abstract

We searched hop extract for active component(s) that inhibited bone resorption in the pit formation assay, and isolated xanthohumol and humulone as active ingredients. Especially humulone had extraordinarily strong inhibitory activity and the IC₅₀ (concentration of 50% inhibition) value was 5.9 x 10^-9 M.

View full abstract

The most serious damage to cells exposed to radiation is attributed mostly to effects on the structure of cellular DNA. We found that trehalose protects DNA from irradiation. In the presence of 10 mM trehalose, DNA can be protected from about 4 timed higher doses of β-and y-ray irradiation. The protective effect increases with the amount of the sugar. Other disaccharides, sucrose, and maltose had similar effects.

View full abstract

Neoagarobiose, a disaccharide, showed a higher hygroscopic ability than glycerol or hyaluronic acid, typical moisturizing reagents. Beside, neoagarobiose whitened B16 murine melanoma cells, and showed low cytotoxicity. Therefore neoagarobiose was a rare reagent showing both moisturizing and whitening effects.

View full abstract

β-1, 4-Linked mannooligosaccharides were obtained regioselectively when mannose was incubated with β-mannosidase from Rhizopus niveus. By use of the obtained β-1, 4-linked mannobiose as a donor, the transglycosylation reaction was done with the same enzyme, and β-linked alkyl glycosides were obtained from various alcohols. In the reaction using the β-1, 4-linked mannobiose as a donor and acceptor, β-1, 4-linked mannooligosaccharides were obtained by a transglycosylation mechanism.

View full abstract

Oat seed lipase was extracted with 0.01 M calcium chloride solution containing 0.5% Triton X-100 and precipitated with ammonium sulfate. The precipitate was dissolved in phosphate buffer at pH 6.0 and the supernatant was used as the lipase preparation. The lipase was very selective in the ester positions of 1, 2, 3-trihexanoylglycerol, hydrolyzing sn-3 most quickly, sn-1 moderately, and sn-2 hardly at all.

View full abstract

Four arginine/glutamate rich polypeptides referred to as 5k-, 6.5k-, 12.5k-, and 14k-AGRPs were purified to homogeneity by gel filtration on Sephadex G-75 followed by CM-cellulose, butyl-Toyopearl 650M, and reverse-phase HPLC from the seed of sponge gourd (Luffa cylindrica). Tricine SDS-PAGE indicated that 5k-and 6.5k-AGRPs are single polypeptides, but 12.5k- and 14k-AGRPs consist of two polypeptide chains which are linked by disulfide bond(s). The N-terminal amino acid sequences of four AGRPs were analyzed by a gas-phase sequencer, and the result indicated that they are distinct molecules. Comparison of the sequences with those of proteins in the protein data base demonstrates that 5k- and 6.5k-AGRPs have a significant homology with a basic peptide from pumpkin seeds and with cocoa seed vicilin, respectively, and that 12.5k- and 14k-AGRPs are related to 2S seed storage proteins. Furthermore, it was assumed that the four AGRPs might occur in the protein bodies within cells of the seed.

View full abstract

A flake-chitin degrading marine bacterium was isolated and identified as Aeromonas hydrophila. This strain secreted five chitinases and an β-N-acetylglucosaminidase. The main chitinase (Chi-A) was purified and characterized. The optimum pH of Chi-A was 5-8, and the activity was inhibited by Hg²⁺ and Fe³⁺. Chi-A was different from chitinases of other Aeromonas species with respect to molecular weight (62, 000) and insensitivity to monoiodoacetate. The amino-terminal amino acid sequence showed extensive similarity with chitinases from Gram-negative bacteria.

View full abstract

Depolymerization of hyaluronic acid (HA) by low-molecular-weight Amadori-rearrangement products in the presence of Cu²⁺ was studied as an in vitro model for the glycated protein-mediated degradation of biopolymers. This oxygen radical-mediated depolymerization was found to be specifically accelerated by Cu²⁺, and significantly inhibited by catalase, hydroxyl radical scavengers, and metal ion chelators. Glycated polylysine also depolymerized HA. The difference in depolymerization rate between low-and high-molecular-weight Amadori products is discussed.

View full abstract

The optical resolution of (±)-2, 2, 2-trifluoro-1-(1-pyrenyl) ethanol was achieved by using lipases. In particular, Pseudomonas aeruginosa lipase (lipase LIP) showed high enantioselectivity (E= >100) and reactivity in the alcoholysis of the chloroacetyl ester of the title compound. The reactivity of the lipase LIP-catalyzed enantioselective alcoholysis of the chloroacetate with 1-hexanol was much higher than that of the acetylating the alcohol with vinyl acetate.

View full abstract

Human urokinase (UK) was easily degraded during the incubation at 37°C in a time-dependent manner. The degradation was also observed in the presence of trypsin inhibitors, suggesting that UK was not degraded by exogeneous trypsin-like serine protease but by autolysis. In this cases, the A-chain of UK was selectively degraded. Polyethylene glycol-polypropylene glycol conjugated urokinase (PEG-PPG-UK) was not degraded after prolonged incubation at 37°C. These results demonstrated that PEG-PPG modification completely blocked the degradation of UK by autolysis.

View full abstract

A specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), was found to inhibit the regulated exocytosis of pancreatic acinar AR42J cells. When AR42J cells were stimulated with cholecystokinin octapeptide (CCK-8), the regulated exocytosis monitored by amylase release was rapidly activated and increased by 2.5-fold during one hour. After AR42J cells were treated with AACOCF₃, amylase release by CCK-8 remained at the basal level. Thus, changes in the composition of membrane phospholipids before and after stimulation were investigated. Within 1 min after CCK-8 stimulation, lysophosphatidylethanolamine (lysoPE) in the cellular membranes of AR42J was increased while lysophosphatidylcholine stayed unchanged. In the presence of AACOCF₃, lysoPE was not increased by CCK-8. Those results indicate that the increment of lysoPE is linked to the regulated exocytosis.

View full abstract