1466
N. WAKIUCHI et al.
dithiothreitol, and 1.16 kBq UDP-[U-14C]-galactose
in a total volume of 55 l. The mixture was incubated
at 40 C for 20 min. The reaction was started by addi-
tion of myo-inositol, and stopped by addition of cold
m
Materials and Methods
9
Chemicals. UDP-
D
-[U-14C]galactose, lithium salt
(10.9 GBq mmol) was purchased from Amersham
ethanol (200
m
l). One ml of DE-52 suspension (DE-
W
=
Pharmacia Biotech. UDP-Gal 4-epimerase from
52:H2O 0.3:1.0, w v) was added, and the mixture
W
galactose adopted yeast, and
a
-galactosidase from
was shaken for 20 min at room temperature. After
centrifugation, 500 ml of the supernatant was trans-
green coŠee beans were obtained from Sigma, and
UDP-Gal was from Calbiochem.
ferred to a vial with scintillator cocktail (Scintisol
EX-H, Dojindo Laboratories), and the radioactivity
was measured with a liquid scintillation counter
(LSC-5100, Aloka). The same volume of water in
place of myo-inositol was used as a control. One unit
Plant materials. Cucumber plants (Cucumis sati-
vus L. cv. Suyo) were grown in an experimental ˆeld
of the Faculty of Agriculture of Kobe University.
The mature leaves were harvested, washed, frozen in
of enzyme activity was deˆned as 1
tinol formed per min.
mmol of galac-
liquid nitrogen, and stored at
-
80 C before use for
9
GS puriˆcation. Sweet potato roots were obtained
from a farm in Kobe, and stored under the same
conditions.
Assay for SS activity. The activity of SS was meas-
ured by the method of Murata,8) and Delmer9) with a
modiˆcation. The reaction mixture contained SS
enzyme solution, 50 m
50 m sucrose, and 25 m
200 l. The mixture was incubated at 37
9
M
Tris-acetate buŠer (pH 6.0),
UDP in a total volume of
C for
Partial puriˆcation of plant enzymes.
M
M
Preparation of GS enzyme solution.6,7) Cucumber
m
leaves (400 g) were homogenized in 50 m
M
Hepes-
dithiothreitol
phenylmethanesulphonyl ‰uoride.
120 min. The reaction was stopped by heating in boil-
ing water. The mixture was clariˆed by centrifuga-
tion, and chromatographed on Whatman 17CHR
NaOH (pH 7.0) buŠer containing 1 m
mixed with 1 m
M
M
After centrifugation of the homogenate, the super-
natant solution was fractionated with solid
paper with 1
M
z
ammonium acetate-95 ethanol
(3:7), descendingly. Spots were detected under a UV
(252 nm) lamp. Standards of UDP-Glc and UDP
were run simultaneously. A strip containing UDP-
Glc was eluted with water. The UDP-Glc content was
estimated by the absorbance at 260 nm. One unit of
the enzyme activity was deˆned as the amount of
(NH4)2SO4. The fraction precipitated in 35–55
z
saturation was dissolved in the Hepes buŠer, dialyzed
against the buŠer overnight, and the solution was
clariˆed by centrifugation.
The supernatant was chromatographed on a
DEAE-cellulose column (DE-52, Whatman), eluting
enzyme required to form 1
UDP for 1 min.
mmol of UDP-Glc from
stepwise with the Hepes buŠer containing 110 m
M
and 175 m NaCl. The active fractions of GS were
M
collected. The activity was stable at
least two months.
-
10
9
C for at
Production of galactinol. Using the GS enzyme so-
lution, galactinol was produced from UDP-Gal and
myo-inositol. The production was investigated under
various conditions to identify the optimal conditions.
According to the results, the GS enzyme assay
mixture was modiˆed, and the reaction mixture
Preparation of SS enzyme solution. The partial
puriˆcation of SS was done by the method of
Murata.8) Brie‰y, peeled sweet potato roots (800 g)
were homogenized in 50 m
After centrifugation, the supernatant solution was
precipitated by addition of solid (NH4)2SO4 to 50
M
Tris-acetate buŠer.
contained GS enzyme solution, 50 m
buŠer (pH 7.5), 10 m UDP-Gal, 50 m
inositol, 3 m dithiothreitol, and 2 m MnCl2 in a
total volume of 200 l. The mixture was incubated at
40 C for 120 min. The reaction was stopped by addi-
M
Hepes-NaOH
z
M
M
myo-
saturation, followed by dialysis. The dialysate was
applied on a DE-52 column. The column was washed
M
M
m
with 5 m
0.1 Na acetate, and then eluted with a linear Na
acetate gradient ranging from 0.1 to 0.6 . Active
M
Tris-acetate (pH7.2) buŠer containing
9
M
tion of cold ethanol (1 ml). The solution was evapo-
rated in vacuo to dryness, and analyzed by HPLC
using a Sugar-Pak column.
To produce galactinol from UDP-Glc as a starting
material, UDP-Gal 4-epimerase and UDP-Glc were
added to the GS reaction mixture in place of UDP-
Gal. After reaction, the mixture was analyzed by
HPLC.
For galactinol production from sucrose as a start-
ing material, SS, sucrose, and UDP were added to
the epimerase-GS reaction mixture in place of UDP-
Glc. However, galactinol production was not detect-
ed on an HPLC chromatogram.
M
fractions were collected, and precipitated by
(NH4)2SO4 again. The dialyzed solution was used as
SS enzyme solution for UDP-Glc synthesis.
Enzyme assay.
Assay for GS activity. The activity of GS was
6)
measured by the method of Smith et al
.
with a
modiˆcation. The reaction mixture contained GS
enzyme solution (protein concentration, 27.5 mg
W
ml), 50 m
M
Hepes-NaOH buŠer (pH 7.5), 10 m
myo-inositol, 5 m MnCl2, 3 m
M
UDP-Gal, 20 m
M
M
M