M. Gustafsson et al. / Phytochemistry 58 (2001) 243–248
247
alcohol were added to the treatment bottles. The cul-
tures were allowed to grow for 4 days (5-day culture in
total) after which the nutrient medium was collected.
4.7. Isoelectric focusing (IEF)
IEF was accomplished with a Multiphor II apparatus
(Pharmacia) in a 4% polyacrylamide gel containing
Ampholine with a pH range of 3.5–10.0 according to the
manufacturer’s instructions. Peroxidase was visualized
in the gel by staining with 50 mM Na-citrate buffer, pH
5.0 containing 0.03% 3,3-diaminobenzidine, 10 mM
CaCl2 and 10 mM H2O2 (Sato et al., 1993). After pre-
incubation for 20 min in staining buffer without H2O2
the reaction was started by addition of H2O2.
4.5. Isolation of proteins from culture medium
The nutrient medium was collected after 5 or 6 days in
culture. Because on day 6 some of the suspension cul-
tures showed signs of ageing, a 5-day culture period was
used in the following replicate cultures. The nutrient
medium was decanted, filtered through glass wool and
the extracellular precipitate collected using centrifugation
ꢀ
(12,000 g, 15 min at 4 C). The pellet was washed twice
with distilled water, freeze-dried, quantified and stored at
Acknowledgements
À20 ꢀC for NMR studies. pH of the nutrient medium was
ꢀ
measured, the medium was frozen and stored at À80 C
The tissue culture work was funded by the Finnish
Forest Cluster Research Programme WOOD WISDOM
supported by The National Technology Agency, Tekes;
and the Finnish Academy of Sciences. We thank Ms
Maaret Mustonen for skillful technical assistance.
until used. The cells were washed in a Buchner funnel
with distilled water and the fresh weight of the cells was
measured. The viability of cells was checked by staining
with Evans Blue (Yeoman, 1986).
After quick thawing the nutrient medium was cen-
trifuged for 45 min at 4 ꢀC at 12,000 g. The supernatant
was concentrated over 10-fold using an Amicon ultra-fil-
tration apparatus (YM 10 membrane) and passed through
Sephadex G-25 columns (PD10, Pharmacia) pre-equili-
brated with 50 mM Tris–HCl buffer, pH 7.5 containing 0.5
mM CaCl2, 5 mM Na2B4O7, 30 mM NaCl and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF; modified from
Fagerstedt et al., 1998). Peroxidase activity measure-
ments were made using concentrated bulk proteins of
the nutrient medium.
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