Supplementary Table S1. By-products formed from [3H]XXXG by xyloglucan
endotransglucosylase (XET) activity during trans--xylosylation assays
The dual-labelling method described in Figures 4 and 5, with 37 µM [3H]XXXG, was used
in TX 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 XTHcatalysed 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.
Yield of [3H]xyloglucan (corrected cpm) a during
Source of
extract
20-h trans--xylosidase assay b in presence of
3-h standard XET assay
with tamarind xyloglucan c
No XGO-NH2
1 mM XGO-NH2
10 mM XGO-NH2
Cauliflower d
14
9
10
130
Parsley
13
14
10
512
Asparagus
32
16
15
1272
Chicory
9
nd
10
880
Snowdrop
7
6
8
254
a
Theoretical maximum = 13,700 cpm at 30% counting efficiency.
b
With no deliberately added xyloglucan.
c
With added tamarind xyloglucan but no XGO-NH2.
d
Ammonium sulphate pellet.
nd = not determined.
1
Supplementary 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.
Polysaccharide tested as
acceptor substrate
none*
Xyloglucan
Water-soluble cellulose
acetate
Mixed-linkage (13),
(14)--glucan
Glucomannan
Arabinoxylan
Radioactive polymeric products (cpm‡) formed after
16 h incubation
with [Xyl-3H]with [1-3H]XXFG as donor
XXFGol as mock
donor
at acceptor conc
at acceptor conc
0.1%
0.5%
0.1%
0.5%
2.8
6.6
1.5
2.2
±1.4
±1.6
±1.4
±1.4
854.4 1650.1 1143.5 1460.1
±11.5
±16.0
±13.3
±15.0
64.6
367.4
13.3
35.0
±3.4
±7.6
±1.9
±2.6
74.8
282.2
3.6
8.5
±3.6
±6.7
±1.5
±1.7
27.2
95.0
8.8
22.1
±2.4
±4.0
±1.7
±2.2
19.8
107.7
0.6
5.4
±2.2
±4.3
±1.3
±1.6
40 h incubation
with [Xyl-3H]with [1-3H]XXFG as donor
XXFGol as mock
donor
at acceptor conc
at acceptor conc
0.1%
0.5%
0.1%
0.5%
1.0
3.3
0.3
2.0
±1.3
±1.5
±1.1
±1.3
810.3 1624.9 1149.4 1888.1
±11.2
±15.9
±13.4
±17.1
47.6
325.4
14.3
41.2
±3.0
±7.2
±2.0
±2.8
60.7
208.1
10.0
7.8
±3.3
±5.8
±1.8
±1.7
22.5
78.4
8.1
20.4
±2.3
±3.7
±1.7
±2.2
15.0
52.3
1.0
1.1
±2.0
±3.1
±1.3
±1.3
[Xyl-3H]XXFG : [1-3H]XXFGol
products ratio
16 h incubation
40 h incubation
at acceptor conc
0.1%
0.5%
†
†
at acceptor conc
0.1%
0.5%
†
†
0.75
±0.01
4.86
±0.74
20.8
±8.6
3.09
±0.66
>10
0.70
±0.01
3.33
±0.50
6.07
±1.13
2.78
±0.64
>10
1.13
±0.02
10.5
±0.8
33.2
±6.7
4.30
±0.47
20
±6
0.86
±0.01
7.90
±0.57
26.7
±5.8
3.84
±0.45
>10
0.5% acceptor : 0.1% acceptor products ratio
16 h incubation
[Xyl-3H][1-3H]XXFG XXFGol
40 h incubation
[Xyl-3H][1-3H]XXFG
XXFGol
†
†
†
†
1.93
±0.03
5.69
±0.32
3.77
±0.20
3.49
±0.34
5.44
±0.63
1.28
±0.02
2.63
±0.43
†
2.01
±0.03
6.84
±0.46
3.43
±0.21
3.48
±0.39
3.49
±0.50
1.64
±0.02
2.88
±0.44
†
†
†
*In this control, no polysaccharide was present during the incubation with cauliflower protein, but xyloglucan was added to the concentration stated after the reaction had been
stopped with formic acid.
†Values too low for a meaningful ratio.
‡Corrected for background radiation of 10.5 cpm; theoretical maximum for 100% incorporation of 3H into polymeric products  6800 cpm; counting time was 26 min. The 95%
confidence-interval counting errors are given, and these have been compounded in the ratios.
2
†
†