Large scale determination of glucosinolates in brussels sprouts samples after degradation of endogenous glucose.

A method was developed for the determination of the glucosinolate content in glucose-rich samples of Brassica vegetables such as Brussels sprouts. Glucose in the samples was enzymatically degraded by the enzyme glucose oxidase (GOD). The resulting hydrogen peroxide and the enzyme GOD were thereafter respectively dissociated and inactivated by a heat treatment at 100 degrees C. After the degradation of endogenous glucose the glucosinolates were converted into glucose and related metabolites with the enzyme thioglucosidase originating from Brussels sprouts seeds. Glucose released was determined enzymatically with a glucose oxidase/peroxidase assay as a measure for the glucosinolate content of samples. The method was used to study the influence of harvest time, crop production location, and the choice of parental lines on the glucosinolate content of Brussels sprouts F1-hybrids. The sum of sinigrin and progoitrin of F1-hybrids was found to be significantly correlated to the glucosinolate content.

Organ-Specific Arabinogalactan-Proteins of Lycopersicon peruvianum (Mill) Demonstrated by Crossed Electrophoresis.

Extracts of style, petal, leaf, petiole, stem, and callus derived from stems of wild tomato (Lycopersicon peruvianum) contain characteristic sets of arabinogalactan-proteins. This is demonstrated by crossed electrophoresis in which Yariv reagent, which specifically binds to and precipitates arabinogalactan-proteins, is incorporated into the second gel.

Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism.

The structure of potato (Solanum tuberosum) lectin, which is a hydroxyproline-rich glycoprotein, has been investigated by circular dichroism. The spectra of the native lectin, and of the oxidized, reduced and carboxymethylated and deglycosylated derivatives were examined, as was a hydroxyproline-rich glycopeptide and its deglycosylated derivative. It is concluded that the lectin contains about 35% polyproline II conformation, 34% type II beta-turn and 31% irregular conformation. No indications were found for the presence of alpha-helix or beta-sheet conformations. The polyproline II conformation is heat-stable, but is markedly destabilized by deglycosylation. The type II beta-turn is destabilized by cleavage of disulphide bonds.

Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion.

The amount of arabinogalactan-protein in whole plant extracts can be quantified by single radial diffusion in agarose gels containing a dye known as the beta-glucosyl-Yariv reagent which specifically interacts with and precipitates arabinogalactan-proteins. The lower limit of quantification is 0.04 microgram of arabinogalactan-protein; gum arabic is used as a standard reference arabinogalactan-protein. In principle, this method can be adapted to measure levels of any dye-precipitating macromolecule; for example, acidic polysaccharides can be estimated by their binding to the cationic dye Alcian blue.

A developmentally regulated hydroxyproline-rich glycoprotein from the cell walls of soybean seed coats.

In soybean seeds the level of hydroxyproline is regulated in a developmental and tissue-specific manner. The seed coat contains approximately 77% of the total hydroxyproline in the seed at all stages of development. We determined the ratio of hydroxyproline to dry weight in a number of tissues within the seed; however, only the seed coat shows an increase in this ratio during development. Within the many cell layers of the seed coat, hydroxyproline is most abundant in the external layer. The hydroxyproline is present as an hydroxyproline-rich cell wall glycoprotein. The protein is rich in hydroxyproline (36%), lysine (11%), proline (10%), histidine (9%), tyrosine (9%), and serine (8%). The carbohydrate portion is 90 mole% arabinose and 10 mole% galactose. The arabinose residues are attached to hydroxyproline mostly in the form of trisaccharides. The apparent molecular weight of this glycoprotein is 100,000 daltons.

Polyproline II Confirmation in the Protein Component of Arabinogalactan-Protein from Lolium multiflorum.

Circular dichroism spectra of the arabinogalactan-protein from suspension-cultured Lolium multiflorum (ryegrass) endosperm cells demonstrate the presence of polyproline II conformation in the protein moiety of the proteoglycan. Subject to a number of theoretical and practical constraints of the method, it can be estimated that at least 30% of the protein component is in this conformation.

Reinforced Polyproline II Conformation in a Hydroxyproline-Rich Cell Wall Glycoprotein from Carrot Root.

The salt-extractable hydroxyproline-rich cell wall glycoprotein from carrot (Daucus carota L.) roots is composed of 35% (w/w) protein, 3% (w/w) galactose, and 62% (w/w) arabinose. The arabinose is attached to hydroxyproline as tetra- and trisaccharides. The circular dichroism of the glycoprotein shows that it is completely in the polyproline II conformation. After deglycosylation of the glycoprotein, the polyproline II conformation of the peptide backbone was lost. This indicates that the carbohydrate reinforces the polyproline II conformation.

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L.

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 x 10(5) daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with beta-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.

Hydroxyproline Glycosides in Secretory Arabinogalactan-Protein of Phaseolus vulgaris L.

The carbohydrate moiety of secretory arabinogalactan protein in bean seedlings (Phaseolus vulgaris L. cv. Prélude) is attached to the peptide backbone through hydroxyproline, serine, and threonine. Hydroxyproline-linked side chains, consisting of arabinose, galactose, glucose, and rhamnose, comprise the major part of the sugar residues. These hydroxyproline glycosides differ from those in non-extractable cell wall protein but show similarities with those in wall protein of the alga Chlamydomonas.

Changing cell-wall compositions in hypocotyls of dark-grown bean seedlings.

Hypocotyls of dark-grown 6-day-old seedlings of Phaseolus vulgaris L. proved to be sufficiently homogeneous to permit studies relating the rate of cell elongation to the composition of the primary cell walls. Whereas the levels of cellulose and uronic acids remained practically constant during and after cell extension, all other components showed major or minor changes. Cell-wall protein, as such, decreased by more than 50%, but indications are that hydroxyproline-rich glycoprotein increased with a decreasing rate of cell elongation, concomitant with a rise in the degree of arabinosylation of wall-bound hydroxyproline. As cell elongation slowed down, non-cellulosic glucose accumulated, presumably in the form of a ß-(1-4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.