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.Hydroxyproline-containing insoluble cell wall glycoproteins (extensins) occur in a wide variety of plants. One of the main problems in studying these extensins has been the difficulty in extracting the proteins from the wall (see 22 for review). They are usually covalently linked in the wall, presumably by isodityrosine cross-links (7,14). Wounded carrots accumulate a hydroxyproline-rich cell wall glycoprotein (see 30 for review) that slowly becomes insolubilized (4, 7). A considerable amount of this protein not yet covalently linked to the wall can be extracted under nondenaturing conditions with salts (4, 31). The biosynthesis of this protein has been studied extensively (30) but a reliable amino acid composition was not available until 1980 (31). The amino acid composition is similar to that of the hydroxyproline-rich potato agglutinin (24) which is localized in the wall (25). The major amino acids found in the carrot glycoprotein are the same amino acids which preferentially accumulate in the cell walls of elongating pea and bean stems (19,34).The hydroxyproline-rich cell wall glycoprotein is thought to be a structural wall component (22). We studied the structure of the hydroxyproline-rich cell wall glycoprotein from carrot roots (which we propose to call 'extensin precursor' in concordance with Lamport's usage of the term).MATERIALS AND METHODS Plant Material. Carrot (Daucus carota L.) roots were obtained from a local merchant and stored at 5°C until needed. For incubation of carrot slices, the roots were washed, surface sterilized with 1% (w/w) NaClO for 10 min and sliced (about 1 mm thick in a food processor). The slices were put in racks in a plastic box and incubated for 5 d in moist air at room temperature.Cell Wall Isolation. The fresh or incubated tissue (about 1.5 kg) was homogenized in a blender in a solution containing 0.5% ' Supported by grants from the National Science Foundation (PCM 7923550 and PCM 8104516) and from the Monsanto Co. (an unrestricted postdoctoral fellowship grant).(v/v) Nonidet P-40 (Sigma) plus 2 mM Na2S205 and filtered through Miracloth (Calbiochem). The cell walls were extensively washed with 2 mm-Na2S205 and finally with 20 mM Tris-HCl buffer, pH 8.0, plus 2 mM Na2S20.Isolation of Hydroxyproline-Rich Cell Wall Glycoprotein. In the following fractionation procedures, we determined the salt concentration of the fractions with the refractometer, the protein content by light absorption at 280 nm, and the hydroxyproline content-aft...

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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 β-(1-4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.

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.

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.