SEARCH

SEARCH BY CITATION

Figure S1. Differences in the occurrence of transglycosylation during hydrolysis of nitrophenyl glycosides by cauliflower-leaf extract. Each nitrophenyl (NP) glycoside substrate was incubated at 1.4 mM with cauliflower lamina enzyme (50-60%-saturated ammonium sulphate cut; final concentration 1.2 mg protein ml-1) for an appropriate time, determined in preliminary tests to permit the partial hydrolysis of the substrate (except in the case of NP-a-Xyl, which is not hydrolysed by cauliflower extracts). Incubation times were: NP-a-Xyl, 24 h; NP-b-Xyl, 8 h; NP-a-Glc, 24 h; NP-b-Glc, 4 min; NP-a-Gal, 2 min; NP-b-Gal, 6 min; NP-a-Man, 4 min; NP-b-Man, 8 min. Products were run by TLC in butan-1-ol/acetic acid/H2O, 4:1:1, and stained with thymol/H2SO4. The NP-glycoside substrates were all para- except NP-b-Gal, which was the ortho-isomer; they are all of high chromatographic mobility (green arrows), and their monosaccharide products are indicated by blue arrows. In two cases (b-xylosidase and b-glucosidase), there is evidence for the formation of a faint intermediary transglycosylation product (NP-disaccharide; yellow arrows). The markers (right lane) were run and stained on the same plate, but have not been as highly contrast-enhanced as the rest of the image.

Figure S2. Time-courses for action of cauliflower leaf enzymes on XXXG. Two dialysed preparations of a 50-60%-saturated (NH4)2SO4 cut from cauliflower lamina were incubated with 1.4 mM XXXG for various times and the products were analysed by TLC in BAW. (a) Standard enzyme preparation (as used in other experiments), TLC with two ascents; (b) high-activity enzyme preparation, TLC with three ascents. C = enzyme-free controls incubated for 0 or 48 h. Other details, including the colour-coding of labels, are as in Figure 2.

Figure S3. A representative quantitative scan of radioactive oligosaccharides as resolved by thin-layer chromatography. The products formed by TaX after 12 h with 2048 µM [3H]XXXGol were resolved by TLC (as shown in Fig. 3d) and then quantified with a LabLogic AR2000 radioisotope imaging scanner.

Figure S4. Products formed by cauliflower leaf (NH4)2SO4 fractions on galactosylated XGOs. Substrate: a mixture of XGOs (principally XLLG > XXLG > XXXG > XLXG; final concentration 1.5 mg/ml » 1.2 mM). Other details, including protein loadings, as for Figure 2.

Figure S5. Electrophoretic analysis of the cationic acceptor substrate, XGO-NH2. (a) Non-radioactive markers that were electrophoresed adjacent to lanes containing radioactive reaction-products and then stained with ninhydrin (top) or aniline hydrogen-phthalate (bottom). Electrophoresis run-time: 45 min. The dashed rectangle indicates the zone corresponding to that cut out from neighbouring lanes for assaying cationic radiolabelled reaction-products. (b)-(d) Four independent XGO-NH2 preparations (1-4) and electrophoresed for quality control. Electrophoresis run-time: 30 min. Replicate loadings were stained with: (b) ninhydrin to reveal amino compounds, (c) aniline hydrogen-phthalate (AHPh) to reveal reducing sugars, or (d) AgNO3 to reveal total sugars. Each sample had 2,4-dinitrophenyl-lysine added as a visible (yellow) internal marker, which was circled in pencil before the sheet was stained. MM: marker-mixture (GlcN, glucosamine; DNP-Lys, 2,4-dinitrophenyl-lysine; Glc, glucose). Preparation 3 [the material in the blue rectangles; not visible in (c) because it is not a reducing sugar] was the XGO-NH2 used in ‘dual labelling’ transglycosidase assays.

Figure S6. XEG digestion products of TaX-treated xyloglucan. Xyloglucan was incubated with [Xyl-3H]XXFG in the presence of cauliflower-leaf enzymes (containing TaX activity) for 0.25-24 h (a-d), and the radiolabelled polymers formed were digested with xyloglucan endo-glucanase (XEG). The fragments thus generated were run by TLC in propan-1-ol/acetic acid/H2O (2:1:1) and each lane was dissected into strips which were assayed for radioactivity. (e) Shows a marker: a Glc8-based oligosaccharide pool generated by partial hydrolysis of tamarind xyloglucan with XEG and then radiolabelled with NaB3H4. The positions of non-radioactive markers are shown on (d): XGOs with the indicated DPs and free xylose.

Table S1. By-products formed from [3H]XXXG by xyloglucan endotransglucosylase (XET) activity during trans-a-xylosylation assays. The dual-labelling method described in Figures 4 and 5, with 37 µM [3H]XXXG, was used in TaX assays, and the 20-hour products were electrophoresed; then the origin of the paper electrophoretogram was cut out, water-washed to remove mono- and oligosaccharides, and assayed for remaining bound radioactivity (potentially [3H]xyloglucan, formed by XTH-catalysed endo-transglycosylation with traces of polysaccharide co-extracted along with the plant enzymes). The extracts were also tested for XET activity by a standard 3-hour assay with 37 µM [3H]XXXG plus 4.2 mg/ml tamarind xyloglucan.

Table S2. Quantification of radioactive products formed by xylosyl-transfer with polysaccharides as acceptor substrates. Reaction mixtures contained—Xylosyl donor: 47 µM [Xyl-3H]-XXFG (or [1-3H]-XXFGol as a ‘mock’ donor to check for XET activity). Xylosyl acceptor: 0.1 or 0.5% (w/v) polysaccharides as listed.

Enzyme: 0.62 mg/ml cauliflower leaf protein. Incubation was stopped after 16 or 40 h, and high-Mr products were assayed for 3H.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
TPJ_4966_sm_FigS1-S6.ppt1423KSupporting info item
TPJ_4966_sm_suppl-Fig-legends.doc69KSupporting info item
TPJ_4966_sm_TableS1-S2.doc116KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.