Transcript TPJ_4966_sm_FigS1-S6

Xyl
Man
Glc
Gal
Lam2
Cell2
Lam3
Cell3
Lam4
Nitrophenyl glycoside substrate
Markers
Enzyme only
NP--Man
NP--Man
NP--Gal
NP--Gal
NP--Glc
NP--Glc
NP--Xyl
NP--Xyl
NP--Man
NP--Man
NP--Gal
NP--Gal
NP--Glc
NP--Glc
NP--Xyl
NP--Xyl
Cell5
Cell6
Substrate + enzyme
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--Xyl, which is not hydrolysed by cauliflower extracts).
Incubation times were: NP--Xyl, 24 h; NP--Xyl, 8 h; NP--Glc, 24 h; NP--Glc, 4 min; NP-Gal, 2 min; NP--Gal, 6 min; NP--Man, 4 min; NP--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--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 (-xylosidase and -glucosidase), there is evidence for the formation
of an 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 contrastenhanced as the rest of the image.
a
b
Xyl
Xyl
Glc
Glc
Lam2
DP2
XG
Isopr
Cello2
Lam3
Cello3
DP4
XXG
Lam4
Cello4
DP6
DP7
XXXG
Cello6
XXXG
DP8
DP9
DP10
XXLG
XLLG
0h
¼h
1h
4h
16h
Low activity enzyme
0h
1h
2h
4h
8h 18h 24h 32h 48h C0h C48h
High activity enzyme
No enzyme Markers
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.
0
0
1
6
0
0
1
2
0
DP>10 0.6%
0
8
0
0
4
0
0
Glc-ol 0.2%
0
DP2 1.1%
2
XGol 17.5%
0
DP4 2.5%
0
XXGol 30.1%
4
XXXGol 19.1%
2
DP7 2.1%
DP6 5.5%
0
DP8 13.3%
0
DP10 1.2%
DP9 5.7%
8
Polymer 1.0%
Radioactivity (counts per 85 m channel)
2
0
-
0
1
1
2
3
4
5
6
7
8
9
1
10
11
12
13
14
15
16 1 7 8
Distance migrated (cm from origin)
Figure S3 A representative quantitative scan of radioactive oligosaccharides as
resolved by thin-layer chromatography
The products formed by TX 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.
Xyl
Glc
Gal
IP
GG
XG
Glc
Gal
XG
GGG
XXG
? DP6
? XLG
? GXXG
XXXG
DP8
XXLG
DP9–10
XLLG
DP10–11
GGGG
GGGGG
XXXG
XXLG
midrib
markers
70% AS ppt
60% AS ppt
50% AS ppt
40% AS ppt
30% AS ppt
20% AS ppt
no AS pptn
crude
70% AS ppt
60% AS ppt
50% AS ppt
40% AS ppt
30% AS ppt
20% AS ppt
no AS pptn
crude
XLLG
lamina
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.
a
b Ninhydrin
c AHPh
d AgNO3
DNPLys
GlcN
XGONH2
DNP-Lys
XGO-NH2
Glc +
XXXG
Glc
origin
origin
MM 1
2
3
4 MM 1
2
3
4 MM 1
2
3
4 MM
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: markermixture (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.
Radioactivity (cpm/cm)
(a) 0.25 h
50
0
(b) 1.5 h
50
0
150
(c) 7.2 h
100
50
0
(d) 24 h
250
200
150
DP14–18
Xyl
DP7–9
100
50
0
(e) Marker
4000
2000
0
0
2
4
6
8
10
12
14
16
Distance migrated (cm from origin)
Figure S6. XEG digestion products of TX-treated xyloglucan.
Xyloglucan was incubated with [Xyl-3H]XXFG in the presence of cauliflower-leaf enzymes
(containing TX 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 NaB 3H4. The
positions of non-radioactive markers are shown on (d): XGOs with the indicated DPs and free
xylose.