Phleinase induced in stem base of orchardgrass (Dactylis glomerata L.) after defoliation was partially purified with ammonium sulfate precipitation, DEAE-Sephadex chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The molecular weight of phleinase was 57,000 as determined by gel chromatography. The kg/ha) was applied after the second cutting. On July 25, the stubbles were dug out and the vegetative stems of 2.5 cm from the base were collected.Preparation of Crude Enzyme. Two hundred and fifty g of the stem bases were cut into slices (about 2 mm in thickness) and homogenized in 4 volumes of cold 0.1 M K-phosphate (pH 7.2) containing 5 mM ,3-mercaptoethanol with 10% insoluble PVP for 2 min using a Waring Blendor. The homogenate was squeezed through four layers of gauze and centrifuged at 14,000g at 4C for 20 min. Solid (NH4)2SO4 was added to the supernatant and the precipitate between 40 and 70% saturation was collected by centrifugation. The precipitate was dissolved in 20 ml of 20 mm K-phosphate (pH 7.2) and filtrated on a column of Sephadex G-25 (2 x 26 cm) with the same buffer as eluant. All subsequent procedures were carried out at about 4C. The protein fractions were combined and dialyzed against two changes of 20 volumes of 50 mM Na-acetate buffer (pH 5.4) for 4 h with stirring. The protein precipitated during dialysis was removed by centrifugation and the supernatant was used as the starting material for further purification.Preparation of Phlein and Triticin. Two hundred g ofthe stem bases of orchardgrass collected in late November were cut into slices and homogenized in 3 volumes of cold 10 mm ,B-mercaptoethanol solution. The homogenate was squeezed through gauze, and the filtrate was centrifuged at 14,000g at 4°C for 15 min. Each of 50 ml of 5% ZnSO4-7H20 and 0.3 N Ba(OH)2 was successively added to the supernatant, and the resulting precipitate was removed by centrifugation. Four volumes of ethanol were added to the supernatant to collect the precipitate by centrifugation. The precipitate was dissolved in 200 ml of distilled H20 in a boiling water bath for 3 min and cooled in running tap water, followed by centrifugation. The supernatant was treated with 20 g of the mixture of Amberlite and IRA45(OH-), and shaken occasionally for 30 min at room temperature, followed by filtration through a filter paper in vacuo. Four volumes of ethanol were added to the filtrate and the resulting precipitate was collected by centrifugation. The precipitate was suspended in 150 ml of 80% ethanol (v/v)

The new phytosphingosine-type ceramide asteriaceramide A (1) and glucocerebroside asteriacerebroside G (2), together with two known cerebrosides, asteriacerebrosides A and B, were isolated from lipophilic fractions of the whole bodies of the Northern Pacific starfish Asterias amurensis Lütken. The water-soluble fraction afforded two known asterosaponins, glycoside B(2) and asterosaponin-1. The structures of 1 and 2 were determined on the basis of chemical and spectroscopic evidence as (2S,3S,4R,13Z)-2-[(2'R)-2-hydroxyhexadecanoylamino]-13-docosene-1,3,4-triol (1) and 1-O-(beta-d-glucopyranosyl)-(2S,3R,4E,13Z)-2-[(2'R)-2-hydroxytetradecanoylamino]-4,13-docosadiene-1,3-diol (2). Compounds 1, 2, and asteriacerebrosides A and B promoted plant growth in sprouts of Brassica campestris.

Stereoselective conversion of a selenoglycoside to a beta-bromoglycoside in the absence of a glycosyl acceptor followed by the coupling with another selenoglycoside affords the corresponding glycosylated selenoglycoside, which could be directly used for the next glycosylation. The iteration of this sequence allows the synthesis of a variety of oligosaccharides including an elicitor active heptasaccharide. [reaction: see text]

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A new method for constructing an oligosaccharide library composed of structurally defined oligosaccharides is presented based on an iterative glycosylation of selenoglycosides. Treatment of 2-acyl-protected selenoglycosides with bromine selectively generates beta-bromoglycosides, which serve as glycosyl cation equivalents in the oligosaccharide synthesis. Thus, the coupling of the bromoglycosides with another selenoglycoside affords the corresponding glycosylated selenoglycosides, which can be directly used to next glycosylation. The iteration of this sequence allows the synthesis of a variety of oligosaccharides including an elicitor active heptasaccharide. A characteristic feature of the iterative glycosylation is that glycosyl donors and acceptors with the same anomeric reactivity can be selectively coupled by activation of the glycosyl donor prior to coupling with the glycosyl acceptor. Therefore, same selenoglycosides can be used for both the glycosyl donors and the acceptors. This feature has been exemplified by a construction of an oligosaccharide library directed to elicitor-active oligosaccharides. The library composed of stereochemically defined oligoglucosides with considerable structural diversity can be constructed starting from simple selenoglycosides.

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The optimal pH and temperature of p-coumarate decarboxylase were 6.0 and 23 degrees C respectively. The enzyme activity was reduced to three quarters by heat treatment at 35 degrees C for 5 min and by half at 25 degrees C in 24 h, but kept almost unchanged at -20 degrees C at least for 10 days. The activity was not inhibited by potassium cyanide, sodium diethyldithiocarbamate, ethylenediaminetetraacetic acid disodium salt, or sodium citrate at 10 mM concentration, but was inhibited by p-chloromercuribenzoate or iodoacetate at 0.1 mM, the inhibition by the former being prevented to a great extent by the presence of reduced glutathione or dithiothreitol. The activity was inhibited by maleic acid cinnamic acid, or p-methoxycinnamic acid, but not by fumaric acid, acrylic acid, p-hydroxystyrene, furcatin p-hydroxyphenylacetic acid, or phloretic acid. An unsubstituted p-hydroxy group on the benzene ring and an acrylic acid side chain were required for the enzyme activity. Km value for trans-p-coumaric acid was about 6.5 X 10(-4) M.