Characteristics of an α-galactosidase associated with grape flesh

Article abstract of DOI:10.1016/S0031-9422(01)00207-2

α-Galactosidase activity in grape flesh (Vitis venifera L. Muscat of Alexandria) was characterized by a marked increase in its activity 4 weeks after fruit bearing. After 12 weeks the specific activity of the enzyme had increased 15-fold. Several other gl

Full text of DOI:10.1016/S0031-9422(01)00207-2

Phytochemistry 58 (2001) 213–219
www.elsevier.com/locate/phytochem
Characteristics of an a-galactosidase associated with grape flesh
Han-Chul Kang *, Seon-Hwa Leeb
a,
aDepartment of Plant Nutrition, National Institute of Agricultural Science and Technology, Rural Development Administration,
Suwon 441-707, South Korea
bNational Horticultural Research Institute, Rural Development Administration, Suwon 440-310, South Korea
Received 1 September 2000; received in revised form 4 April 2001
Abstract
a-Galactosidase activity in grape flesh (Vitis venifera L. Muscat of Alexandria) was characterized by a marked increase in its
activity 4 weeks after fruit bearing. After 12weeks the specific activity of the enzyme had increased 15-fold. Several other glycosi-
dases were measured at different stages of fruit development but none showed the increased levels of activity displayed by this a-
.
galactosidase. a-Galactosidase activity (unit/g fresh wt) increased by 52% during postharvest storage, whereas the unripe grape
showed a ‘‘stagnancy’’ for 10–15 days prior to the increase. An a-galactosidase was partially purified ca. 103-fold from grape flesh of
Vitis labruscana Honey black, by a procedure involving ammonium sulfate fractionation, Biogel P-60, melibiose-agarose, and Sepha-
cryl S-200 chromatographic separations. The enzyme was effectively separated by affinity chromatography on melibiose-agarose, and
was a monomer of 40–45 kDa as determined by SDS-PAGE and Sephacryl S-200 chromatographic analysis. The hydrolysis rate of
p-nitrophenyl-a-d-Gal (PNP-a-d-Gal) was 4.2times higher than that of PNP- b-d-Gal, implying an apparent a-anomer specificity,
and natural oligosaccharides such as melibiose, stachyose, and raffinose were also considerably hydrolyzed. The enzyme was active
over a narrow pH range with an optimal hydrolysis of stachyose and PNP-a-d-Gal at pH 6.0 and 7.0, respectively. EDTA or 1,10-
phenanthroline did not substantially affect enzyme activity. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Vitis veniferae; Vitaceae; Grape; a-Galactosidase
1
. Introduction
1980; Smart and Pharr, 1980; Dey et al., 1983; Mujer et
al., 1984; Haibath et al., 1991; Chrost and Schmitz,
1997; Irving et al., 1997). a-Galactosidases are generally
involved in metabolic utilization of oligosaccharides
such as raffinose, stachyose, melibiose, and galacto-
mannan, that are even more widely distributed in sto-
rage organs such as seeds, roots, and tubers (McCleary
and Matheson, 1974; Kandler and Hopf, 1980). a-
Galactosidases have been implicated with the metabo-
lism of galactolipids (Sastry and Kates, 1964; Pridam
and Dey, 1974). In addition to these hydrolysis reac-
tions, transglycosylation is catalyzed by the enzyme
with somewhat broad acceptor specificity (Dey and Pri-
dam, 1972).
Despite this wide distribution and diversity, a-galac-
tosidases seem to be less abundant in fruits compared
with other plant organelles. Biochemical/physiological
aspects in fruits differ from those of other tissues or
organelles in plants, and hence other types of a-galac-
tosidase may be present in grape. Characterization of a-
galactosidase in grape flesh would be helpful in under-
standing the physiological role of the enzyme. In this
a-Galactosidase (E.C. 3.2.1.22, a-d-galactoside galac-
tohydrolase) is one of the exoglycosidases, capable of
hydrolysing a-1,6 linked a-galactoside residues. a-Galac-
tosidases are widely distributed in intra- or extracellular
forms in microorganisms, plants and animals. The
enzyme in plants, especially, has been greatly investigated
from seeds in relation to germination (Dey and Pridam,
1
1
969; Barham et al., 1971; McCleary and Matheson,
974; Dey et al., 1983; Corchette and Guerra, 1987) and
is also found in leaves and other tissues (Gatt and
Baker, 1970; Thomas and Webb, 1978; Smart and
Pharr, 1980; Gaudreault and Webb, 1983; Burns, 1990).
Diverse forms of a-galactosidase occur occasionally in
many plants, having molecular weights ranging from 25
to 270 kDa and different properties (Dey and Pridam,
1
969, 1972; Thomas and Webb, 1977; Hankins et al.,
*
Corresponding author. Tel.: +82-31-290-0260; fax: +82-31-290-
261.
E-mail address: hckang3436@hanmail.net (H.-C. Kang).
0
0
031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00207-2

214
H.-C. Kang, S.-H. Lee / Phytochemistry 58 (2001) 213–219
paper, we partially purified an a-galactosidase asso-
ciated with grape flesh and present some of its bio-
chemical characteristics.
stage from 4 to 12weeks. The most prominent increase
was found at the mid-term of ripening stage (4–8 weeks
from fruit bearing). The expression of a-galactosidase in
the flesh was characterised by an increase in activity per
.
.
g fresh wt as well as in the activity per mg protein along
with the ripening stage. These results suggest that a-
galactosidase might play an important role in grape flesh
during fruit development. The marked expression of a-
galactosidase synchronizing with the ripening stage could
be correlated with the report of Gross (1986) that applied
galactose stimulated ethylene production and promoted
ripening of mature green tomatoes. But the results pre-
sented here do not elucidate any role for the a-galactosi-
dase in the grape flesh. Putative variation of a-galacto
2
. Results and discussion
2
.1. ꢀ-Galactosidase activity from grape flesh
The glycosidase activities of flesh (mesocarp plus pla-
cental tissue) from Vitis venifera L. Muscat of Alexan-
dria are shown in Table 1. Of the various glycosidases
assayed, a-galactosidase was the most active, followed
by a-mannosidase, as measured by the assays at pH 5.0
or 7.0. Significant activities of a- and b-glucosidase, and
b-galactosidase were found at acidic or neutral pH.
Glycosidases were also evaluated from the flesh of other
grape cultivars, Honey black, Marguerite, Edelweiss,
Agawan, Egiodola, Green hungarian, and Takasumi at
pH 7.0. For all of the strains tested, a-galactosidase was
the most active, although other glycosidases also showed
significant activities (data not shown). Fig. 1 shows the
variation in the specific activities of galactosidase, glu-
cosidase, and a-mannosidase in flesh from Muscat of
Alexandria at different stages of fruit development.
These glycosidase activities were scarcely found at the
unripe stage (approximately up to 4 weeks from fruit
bearing). After this stage, the activities increased rapidly
to different extents. Approximately 15 times increase in
a-galactosidase activity was observed during the ripening
ꢀ
sidase during postharvest storage at 4 C was assessed
from the flesh of Honey black harvested after 9 or 12
weeks from fruit bearing (Fig. 2). In the latter grape, the
.
a-galactosidase activity (unit/g flesh wt) increased by
52% of control after 15 days of storage and then
declined slowly. The unripe grapes harvested after 9
weeks, however, showed a ‘‘stagnancy’’ for 10–15 days
of storage before an increase in activity occurred, and
then the activity slowly increased albeit to a lesser extent
than the more ripe grape.
2.2. Purification of ꢀ-galactosidase
Purification of a-galactosidase was performed using
flesh of Vitis labruscana Honey black. With regard to
Table 1
Distribution of glycosidases in grape flesh (Vitis venifera L. Muscat of
Alexandria). The activity was measured for the grape harvested after 3
months from fruit bearing. The enzyme assay was run by hydrolysis of
PNP-glycosides (2mM) in 50 mM Na-acetate (pH 5.0) or K-phos-
phate (pH 7.0) containing 0.5% glycerol. Glycosidase activities were
.
represented as units per g fresh wt. Soluble solids content of the grape
ꢀ
berry was 17.1 Bx. Activity values were expressed as means Æ S.E. of
three independent experiments
.
Glycosidase activity (unit/g flesh wt)
Substrate
pH 5.0
pH 7.0
PNP-a-d-Gal
PNP-b-d-Gal
PNP-a-d-Glc
PNP-b-d-Glc
PNP-a-l-Fuc
PNP-b-d-Fuc
PNP-a-d-Man
PNP-a-l-Ara
545.6Æ26.5
206.4Æ18.4
195.4Æ12.0
201.9Æ10.2309.0
49.2Æ 4.3
31.3Æ 2.5
398.3Æ22.3
8.9Æ 0.26.1
0.6Æ 0.1
814.2Æ31.6
190.8Æ 9.5
374.1Æ11.7
Æ22.1
101.4Æ 2.5
55.4Æ 3.1
569.8Æ18.4
Æ 0.6
Fig. 1. Occurrence of glycosidases at different stages of fruit develop-
ment. Glycosidase activities in grape flesh (Vitis venifera L. Muscat of
Alexandria) was assessed up to 3 months after fruit bearing. a-Galac-
tosidase (-*-), b-galactosidase (-!-), a-mannosidase (-*-), a-gluco-
sidase (-&-), and b-glucosidase (-!-) activities were measured using
PNP-glycosides and represented as unit per mg.protein. Activities per
PNP-a-l-Rha
0.8Æ 0.2
0.5Æ 0.1
Æ 0.2
PNP-b-d-GlcUA
PNP-a-d-GlcNAc
PNP-b-d-GlcNAc
PNP-b-d-GalNAc
Polygalacturonic acid
0.4Æ 0.1
2.4Æ 0.20.9
1.6Æ 0.1
0.7Æ 0.1
63.3Æ 4.5
7.7Æ 0.2
57.4Æ 2.4
5.9Æ 0.5
a
.
g fresh wt increased also gradually along with the ripening stage
a
The polygalacturonase activity was measured using 2-cyanoace-
tamide (Gross, 1982).
(These data are not represented in the figure). Each value was the
average of three replicate assays.

H.-C. Kang, S.-H. Lee / Phytochemistry 58 (2001) 213–219
215
the a-galactosidase extraction from grape flesh, we note
The fractions obtained from the affinity chromato-
graphy were further purified using Sephacryl S-200
chromatography (Fig. 3). This gel chromatography was
the final step in the purification of a-galactosidase
because its instability impeded any further chromato-
graphy. Specific activity of the enzyme was increased
more than 103-fold by the purification procedure
(Table 2). SDS-PAGE analysis of the main fractions
with peak activity from the Sephacryl S-200 chromato-
graphy showed one major band with a monomeric
molecular mass of 45 kDa and some faint bands (Fig. 4).
Affinity chromatography on melibiose-agarose was cru-
cial for enzyme purification. Even though melibiose (6-
O-a-d-galactopyranosyl-d-glucose) might not be perfect
as an affinity ligand for chromatography, this carbohy-
drate seems to be effective for the enzyme purification in
that homogenization of grape flesh with 0.2M Na CO
resulted in an increase in a-galactosidase activity (unit/
2
3
.
g fresh wt) by 3.1 times compared with the extraction
without Na CO . Alternatively, addition of 0.05 N
2
3
NaOH caused the enzyme activity to increase as much
as 2.8 times. Thus, the increase of ionic strength and/or
the augmentation of pH (pH was elevated to 5.1 from
3
.9.) appeared to release more a-galactosidase from the
grape flesh. The a-galactosidase from grape flesh was
stable during the initial steps of the purification proce-
dures. The stability gradually decreased as the purifica-
tion steps proceeded and dilution of the enzyme also
accelerated a decrease in activity (data not shown). Sta-
bilization or restoration of activity was attempted by
adding dithiothreitol (2and 10 mM) or EDTA (1 and 5
mM), but the stability of a-galactosidase was not affec-
ted by these compounds. The activity loss was con-
siderably prevented by addition of glycerol in the
equilibration/elution buffers during the purification
procedure.
The enzyme was partially purified by salting out with
ammonium sulfate and using chromatographic steps
involving Biogel P-60, melibiose-agarose, and Sephacryl S-
200 columns respectively. A peak of a-galactosidase activ-
ity was resolved on melibiose-agarose chromatography.
Fig. 3. Elution profile of a-galactosidase from Sephacryl S-200 chro-
matography. The active fractions from melibiose-agarose chromato-
graphy were loaded onto a Sephacryl S-200 column that was
equilibrated with K-phosphate buffer (pH 7.0, 50 mM) containing 2%
glycerol. Proteins were eluted using the same buffer along with
measurement of absorbance at 280 nm (—). The a-galactosidase
activity (unit/ml, -*-) was monitored using PNP-a-d-Gal as a sub-
strate. One fraction volume was 5 ml.
Table 2
Purification summary of a-galactosidase from grape flesh of Vitis lab-
ruscana Honey black
Fig. 2. Activity variation of a-galactosidase during postharvest sto-
rage. Grapes (Honey black) were collected after 9 weeks (-*-) and 12
a
ꢀ
weeks (-*-) and stored at 4 C. The fruits reached each storage day
Purification step
Total
Total
Specific activity Purification
ꢀ
were stored at À70 C. The a-galactosidase activity in flesh was mea-
protein activity (m/mg protein) fold
sured at the same time. One hundred per cent activity of a-galactosi-
(
mg)
(m)
.
dase was 109 unit/g fresh wt. The a-galactosidase activity increased
more rapidly at room temperature than at 4 C. Difficulties in storage
at room temperature hampered the test for more prolonged intervals
ꢀ
Crude extract 980.1
Ammonium sulfate 275.4
490.1
294.7
201.9
140.5
98.0
0.50
1.07
1.00
2.14
(data not shown). Protein concentration was roughly consistent during
postharvest storage. Soluble solid contents increased parallel with the
Biogel P-60
Melibiose-agarose
Sephacryl S-200
64.2
5.8
3.14
24.22
51.58
6.28
48.44
103.16
ꢀ
storage days as follows: from 12.1 to 14.5 and from 14.7 to 18.1 Bx in
grapes harvested after 9 and 12weeks, respectively. Each value was the
average of three replicate assays.
1.9
a
a-Galactosidase assay used 2mM PNP- a-d-Gal as a substrate.

216
H.-C. Kang, S.-H. Lee / Phytochemistry 58 (2001) 213–219
Table 3a
Substrate specificity of a-galactosidase assayed using PNP-glycosides.
The purified a-galactosidase was assayed for its activity using different
PNP-glycosides (2mM) in K-phosphate (50 mM, pH 7.0) containing
0
.5% glycerol. The activities measured by the standard assay method
were compared with the hydrolysis of PNP-a-d-Gal (100%), which
.
was 49 units/mg protein. All values were the mean of two replicate
assays
Substrates
Relative activity
%)
(
PNP-a-d-Gal
ONP-a-d-Gal
PNP-b-d-Gal
PNP-b-d-Lac
100.0
78.2
16.0
4.3
a
PNP-Lac-N-bioside
PNP-b-d-GalUA
PNP-6-O-b-d-Gal-b-d-Gal
PNP-a-d-Glc
PNP-b-d-Glc
PNP-a-l-Fuc
PNP-b-d-Fuc
PNP-a-d-Man
PNP-a-l-Ara
PNP-a-l-Rha
4.7
12.6
16.1
7.0
Fig. 4. SDS-polyacrylamide gel electrophoresis of purified a-galactosi-
dase from the grape flesh. Outer left lane: reference proteins consisted of
6.2
0
phosphorylase b (M
bonic anhydrase (29,000), trypsin inhibitor (20,100), and lysozyme
14,400). Other lanes: M, the active fraction from melibiose-agarose
chromatography; 28–36, the fraction numbers from the Sephacryl S-
00 chromatography. The gel was stained with Coomassie Brilliant
r
; 97,400), BSA (66,200), ovalbumin (43,000), car-
0
3.7
(
<1.0
0
2
PNP-b-d-GlcUA
PNP-a-d-GlcNAc
PNP-b-d-GlcNAc
PNP-b-d-GalNAc
<1.0
<1.0
<1.0
<1.0
Blue R-250 and then with silver nitrate. Some faint bands (35 and 38
kDa) were observed in lanes 34 and 36 (not very visible in gel photo).
view of 7.7 times increase in specific activity during the
stage from Biogel P-60 to affinity chromatography. The
a
ONP-a-d-Gal is o-nitrophenyl a-d-galactopyranoside.
ꢀ
enzyme was stored in 20% glycerol at À70 C, and over
Table 3b
8
5% of the initial activity was retained after 3 months.
Substrate specificity of a-galactosidase for natural or synthetic carbo-
hydrates. The purified a-galactosidase was incubated with galactose
related carbohydrates (2mM) in K-phosphate (50 mM, pH 6.0) con-
taining 0.5% glycerol, and estimation of galactose released was per-
formed as described in Experimental. Polygalacturonic acid was used
as a substrate for polygalacturonase assay, and the activity determined
2
.3. Substrate specificity of ꢀ-galactosidase
The purified a-galactosidase was assayed using differ-
ent PNP-glycosides (Table 3a). The PNP-a-d-Gal was
hydrolyzed more efficiently by the purified galactosidase
than any other substrate tested, and o-nitrophenyl a-d-
Gal (ONP-a-d-Gal) was also easily hydrolyzed. b-d-gal
was scarcely hydrolyzed, showing 16% of the a-d-Gal
hydrolysis (control), and the extent of 6-O-b-d-Gal-b-d-
Gal was similarly low. Another galactose-related com-
pounds such as b-d-Lac, Lac-N-bioside, and b-d-
GalUA were rarely hydrolyzed, resulting in 4.3–12.6%
activity of the control, and b-d-GalNAc was not effi-
ciently hydrolyzed. The rapid hydrolysis of a-d-Gal and
low hydrolysis of b-d-Gal suggest that the galactosidase
is a-anomer specific.
using 2-cyanoacetamide. The activities were compared with the
hydrolysis of ONP-a-d-Gal (100%), which was 42units/mg . protein.
All values were the mean of three replicate assays
Substrates
Free galactose
released (%)
ONP-a-d-Gal
Melibiose
100.0
17.1
7.6
d-Lactose
Lactulose
2.0
2.7
Lacto-N-tetraose
Lactobionic acid
Melibionic acid
7.1
3.0
3
-O-b-d-Gal-d-Ara
-O-a-d-Gal-d-Gal
<1.0
26.7
12.7
11.3
32.4
19.8
<1.0
4
Table 3b shows the hydrolysis of a range of natural or
synthetic di-polysaccharides related with galactose. The
activity represented as amount of galactose liberated
was compared with the hydrolysis of ONP-a-d-Gal
4-O-b-d-Gal-d-Man
4-O-b-d-Gal-d-Glc
Stachyose
Raffinose
Polygalacturonic acid
(
control). ONP-a-d-Gal was used as a control substrate
in place of PNP-a-d-Gal since p-nitrophenol was an
inhibitor of the galactose dehydrogenase used to measure
the galactose released (Bhalla and Dalling, 1984). Con-
siderable liberations of galactose were obtained from
natural oligosaccharides such as melibiose, stachyose,
and raffinose, and the hydrolysis extent from stachyose
was the most, showing 32.4% of control. A synthetic
disaccharide, 4-O-a-d-Gal-d-Gal, was also significantly
hydrolyzed. Poly-galacturonic acid was not effectively

H.-C. Kang, S.-H. Lee / Phytochemistry 58 (2001) 213–219
217
hydrolyzed, suggesting that the purified enzyme is not a
polygalacturonase. Rapid hydrolysis of the synthetic
substrate PNP-a-d-Gal and relatively slow hydrolysis of
the oligosaccharides were similar to the a-galactosidase
from Cucurbita pepo leaves (Thomas and Webb, 1977).
2
.4. Other characteristics
Apparent molecular mass of the a-galactosidase was
also determined by a gel filtration chromatography. The
4th fraction obtained from the final chromatographic
step was co-injected with size marker onto a Sephacryl S-
00 column. This protein mixture was eluted along with
3
2
monitoring of the activity (unit/ml) peak, and the mole-
cular mass was calculated to be ca. 40 kDa. On the ana-
logy of the result from this gel chromatography, the 45
kDa band in the SDS-PAGE (Fig. 4) was considered as
an a-galactosidase since 40 kDa of molecular mass
determined from the chromatography approached to
this value and the intensities of the 45 kDa bands were
roughly proportional to the activity profile.
The a-galactosidase was further characterized by
measuring its activity in buffers of varying pH using 2
mM PNP-a-d-Gal or stachyose as substrates (Fig. 5-A
and B). When PNP-a-d-Gal was used as a substrate, the
optimum pH for the activity was estimated to be around
7
.0. But the pH profile assayed with a natural substrate,
stachyose, resulted in a lowered pH optimum of 6.0.
The pH for half maximal activity was ca. 4.5–5.2and
the activity rapidly increased from pH 4.0 to 6.0–7.0.
This rapid increase in the activity up to pH 6.0–7.0
could have a correlation with the environment of grape
since pH in flesh increases into a weaker acidity as
ripening proceeds. Thus, increase in pH during ripening
might be an important factor of a-galactosidase regula-
tion in the grape flesh. However, it was not confirmed in
this experiment whether such pH variation play an
exclusive role in the enzyme activation.
Fig. 5. Effect of pH on the hydrolysis of synthetic and natural sub-
strates by a-galactosidase. The purified a-galactosidase was assayed
using the following two series of buffers (1 ml) supplemented with
The requirement of divalent cations for the a-galac-
0
.5% glycerol. (1) A three-buffer mixture (25 mM acetic acid, 25 mM
2
+
tosidase activity was examined using 1 mM Mg
2+
,
Mes, and 50 mM Tris, -*-) titrated to the indicated pH values with
KOH or HCl. (2) Acetate buffer 50 mM, pH 4.0–6.0; K-phosphate 50
mM, pH 6.0–8.0; Tris–HCl 50 mM, pH 8.0–9.0 (-*-). The pH effect
was examined using PNP-a-d-Gal (A) and stachyose (B) as substrates.
The relative activities calculated by two replicate assays were com-
pared with the maximum activities (100%) that were equivalent to 39
Co2+, Zn2+, Fe2+, Cu2+, Mn2+, Cd2+, and Ca with
2
+
chloride ion as a counter part ion. Addition of Fe ion
to the assay mixture in the absence of EDTA elevated
the a-galactosidase activity by 46%. Other cations did
not significantly increase or decrease the activity, except
.
and 14 units/mg protein for (A) and (B), respectively.
4
9% inhibitory effect by Cd2+ (detailed data not
shown). 1, 10-Phenanthroline or EDTA (0.5 and 2.0 mM)
resulted in only a slight decrease in activity ranging
from 90 to 98% of the initial activity (detailed data not
shown).
3. Experimental
3.1. Plant materials
Heat stability was examined with the 36th fraction
from the Sephacryl S-200 chromatography. The enzyme
Grapevines of 4–5 year old Vitis cultivars (Muscat of
Alexandria, Agawan, Marguerite, Edelweiss, Egiodola,
Green hungarian, Honey black, and Takasumi) were
cultivated in a greenhouse under ca. 14 h of daylight
and 10 h of darkness at temperatures ranging from 18 to
ꢀ
was preincubated at 30, 45, or 60 C for 10 min and
assayed using PNP-a-d-Gal. The activity decreased to
ꢀ
6
(
2% of the initial activity when preincubated at 60 C
detailed data not shown).
ꢀ
31 C. Healthy, unblemished grapes were harvested up

218
H.-C. Kang, S.-H. Lee / Phytochemistry 58 (2001) 213–219
to 3 months at intervals of 2weeks after fruit bearing.
After surface sterilization with 60% ethanol, fruits were
rinsed with sterile water and immediately stored at
was released in place of p-nitrophenol. Separate blanks
were used for each enzyme and substrate preparation.
The pH effect of a-galactosidase was examined with 2
mM PNP-a-d-Gal or stachyose using the following
buffers of similar ionic strengths: a three-buffer mixture
(25 mM acetic acid, 25 mM Mes, and 50 mM Tris; Ellis
and Morrisson, 1982) titrated to different pH values
with KOH or HCl. Another buffer series was also used
for this pH experiment: acetate buffer 50 mM, pH 4.0–
6.0; K-phosphate 50 mM, pH 6.0–8.0; Tris/HCl 50 mM,
pH 8.0–9.0. All buffers contained 0.5% glycerol.
Hydrolysis of PNP-a-d-Gal in these buffers was deter-
mined by the standard assay method as described. The
enzyme assay with stachyose was carried out in 500 ml of
the two series buffers. After incubation for 1 h, each
reaction mixture (200 ml) was taken to minimize the pH
effect of these buffers on the subsequent galactose de-
hydrogenase. These samples were each added to 800 ml of
the reaction mixtures for the galactose dehydrogenase
assay. Further incubation and determination of galactose
were performed as described. Non-enzymatic hydrolysis
of PNP-a-d-Gal occurred slightly at alkaline pH, and
these values were subtracted.
ꢀ
À70 C until needed.
In order to test variations in a-galactosidase activities
during postharvest storage, several bunches of grapes
(
Vitis labruscana Honey black) harvested after 9 or 12
ꢀ
weeks after fruit bearing were stored for 5 weeks at 4 C.
Infection was minimized by repetitive rinses using 60%
aq. ethanol and sterile water every 5 days during sto-
rage. At intervals of 5 days, the grapes were randomly
picked from the bunches and immediately stored at
ꢀ
À70 C until a simultaneous assay of a-galactosidase
activity in flesh could be carried out. The grapes stored
ꢀ
at À70 C just after harvest were used as controls for
measuring the initial activity of the flesh.
3
.2. Measurement of glycosidases
Grape flesh, separated from whole grapes, was mixed
with 2% glycerol, and then directly homogenized for 30
s. The resulting paste was filtered through two layers of
nylon cloth, and the filtrate was centrifuged at 8000 g
for 20 min. After eliminating pellets, the supernatant
was used for measurement of glycosidase activity in
flesh. The standard assay of glycosidase was performed
using PNP-glycosides as substrate. Enzyme samples
were added to K-phosphate (50 mM, pH 7.0, in 1 ml of
final volume) containing 0.5% glycerol. Reactions were
initiated by adding 2mM (final concentration) of sub-
3.3. Extraction and purification of ꢀ-galactosidase
ꢀ
All purification procedures were carried out at 4 C.
Grape flesh (2.6 kg) from Vitis labruscana Honey black
was homogenized for 30 s in the presence of 0.2M
Na CO , 1% (w/v) insoluble polyvinylpolypyrrolidone,
2
3
ꢀ
strate. After 30 min of incubation at 37 C, release of p-
and 5% glycerol. The homogenate was filtered through
two layers of nylon cloth. The resulting filtrate was cen-
trifuged at 8000 g for 20 min and the pellet discarded. The
supernatant was fractionated with ammonium sulfate at
30–55% saturation. The precipitate was recovered by
centrifugation at 10,000 g for 20 min and resuspended in
100 ml of 40 mM potassium phosphate buffer (pH 7.0)
containing 2% glycerol (buffer A). Clarification was
done by dialysis with the same buffer and insoluble
materials were removed by centrifugation at 10,000 g
for 20 min.
The clarified supernatant was applied to a Biogel P-60
(Bio-Rad) column equilibrated with buffer A. Proteins
were eluted with the same buffer at a flow rate of 12ml/
h. An additional aim of this step prior to affinity chro-
matography was to minimize various small substances
such as pigments, which may interfere with chromato-
graphic behavior by nonspecific adsorption. a-Galactosi-
dase activity was monitored using PNP-a-d-Gal as a
substrate throughout the purification procedures includ-
ing this step.
nitrophenol from the hydrolysis of substrate was detec-
ted spectrophotometrically by measuring the increase in
absorbance at 410 nm (molar extinction coefficient=
4
À1 À1
1
.84Â10
M
cm ). Eventually, hydrolysis of di-,
oligo-, and polysaccharides by a-galactosidase was
examined by measurement of the galactose content
using galactose dehydrogenase according to the method
of Kurz and Kurt (1974). Enzyme sample was added to
4
0
00 ml of K-phosphate (50 mM, pH 6.0) containing
.5% glycerol and 2mM substrates. After incubation
ꢀ
for 60 min at 37 C, the reaction was stopped by boiling
for 5 min. The hydrolysed sample was cooled and put
together with 570 ml Tris/HCl (0.1 M, pH 8.6), 20 ml of
15 mM NAD, and 10 ml galactose dehydrogenase solu-
tion (Sigma. from Pseudomonas fluorescens, 8.5 units/
ml), with the incubation then further continued for 60
ꢀ
min at 37 C. The difference in Abs
was measured
40nm
3
from zero to the final incubation time. Quantitative
analyses were performed from a calibration curve of
galactose from 0 to 100 nmol. One unit of enzyme activ-
ity, in the method using PNP-glycosides, was expressed
as the release of one mmol of free p-nitrophenol/min and
The active fractions obtained were loaded onto a
melibiose-agarose (Sigma) column (2 Â 10 cm) equili-
brated with buffer A. After washing the column, proteins
were eluted with 200 ml of buffer A containing a linear
gradient of K-phosphate (0–500 mM) at a flow rate of 15
.
specific activity as the amount released/min/mg protein.
Activity units from the method using galactose dehy-
drogenase were defined in the same way, except galactose

H.-C. Kang, S.-H. Lee / Phytochemistry 58 (2001) 213–219
219
ml/h. Fractions with a-galactosidase activity were col-
lected and clarified by centrifugation for next step.
The pooled fractions from the affinity chromato-
graphy were further purified using Sephacryl S-200
a-galac-tosidases and acid invertase during muskmelon (Cucumis
melo L.) fruit development. J. Plant Physiol. 151, 41–50.
Corchette, M., Guerra, H., 1987. a- And b-galactosidase activities in
protein bodies and cell walls of lentil seed cotyledons. Phytochem-
istry 26, 927–932.
(
5
Pharmacia) chromatography. Proteins were eluted with
0 mM K-phosphate buffer (pH 7.0) containing 2% gly-
Dey, P.M., Del Campillo, E.M., Lezica, R.P., 1983. Characterization
of a glycoprotein a-galactosidase from lentil seeds (Lens culinaris). J.
Biol. Chem. 258, 923–929.
cerol at a flow rate of 12ml/h. Purification was followed
by monitoring the absorbance at 280 nm and assaying
enzyme activity. Except for physiological aspects, all of
the experiments on a-galactosidase were performed
using this sample obtained from this chromatography.
Dey, P.M., Pridam, J.B., 1969. Purification and properties of a-galac-
tosidases from Vicia faba seeds. Biochem. J. 113, 49–55.
Dey, P.M., Pridam, J.B., 1972. Biochemistry of a-d-galactosidase.. In:
Meister, A. (Ed.), Advances in Enzymology, Vol. 36. Academic,
New York, pp. 91–130.
Ellis, K.J., Morrisson, J.F., 1982. Buffers of constant ionic strength
for studying pH-dependent processes. Methods Enzymol. 87, 405–
3
.4. Native molecular mass determination
4
26.
Gatt, S., Baker, E.A., 1970. Purification and separation of a- and b-
galactosidases from spinach leaves. Biochim. Biophys. Acta 206,
125–135.
The apparent molecular mass of the purified a-galac-
tosidase was estimated by gel filtration chromatography
using a Sephacryl S-200 column (2 Â 90 cm), which was
equilibrated with 50 mM K-phosphate buffer (pH 7.0)
containing 2% glycerol. The following proteins were co-
injected with the enzyme to the column for the calibra-
Gaudreault, P.R., Webb, J.A., 1983. Partial purification and proper-
ties of an alkaline a-galactosidase from mature leaves of Cucurbita
pepo. Plant Physiol. 71, 662–668.
Gross, K.C., 1982. A rapid and sensitive spectrophotometric method
for assaying polygalacturonase using 2-cyanoacetamide. HortScience
17, 933–934.
tion: aldolase (M ; 158,000), phosphorylase b (97,400),
r
Gross, K.C., 1986. Promotion of ethylene evolution and ripening of
tomato fruit by galactose. Plant Physiol. 79, 306–307.
Haibath, F., Hata, J., Mitra, M., Dhar, M., Harmata, M., Sun, P.,
Smith, D., 1991. Purification and characterization of a Coffea cane-
phora a-d-galactosidase isozyme. Biochem. Biophys. Res. Commun
181, 1564–1571.
BSA (66,200), ovalbumin (43,000), and cytochrome c
(12,000). The proteins were eluted with the same buffer
at a flow rate of 12ml/h. Along with measuring Abs 280nm
,
the activity peak was monitored by assaying the enzyme
activity using PNP-a-d-Gal as substrate.
Hankins, C.N., Kindinger, J.I., Shannon, L.M., 1980. Legume a-
galactosidases which have hemagglutinin properties. Plant Physiol.
3
.5. Other analytical methods
6
5, 618–622.
Irving, D.E., Hurst, P.L., Ragg, J.S., 1997. Changes in carbohydrates
and carbohydrate metabolism enzymes during the development,
maturation, and ripening of buttercup squash (Cucurbita maxima
D. Delica). J. Am. Soc. Hort. Sci. 122, 310–314.
Soluble solid contents were measured with a refract-
ometer using 50 ml of flesh juice, that was prepared by
slightly grinding and subsequent centrifugation at 10,000
g for 5 min. Reducing sugars produced from the hydro-
lysis of polygalacturonic acid were measured using 2-
cyanoacetamide (Gross, 1982). SDS-PAGE was per-
formed according to the method of Laemmli (1970). A
slab gel that consisted of 12.5% acrylamide resolving gel
and 5% acrylamide stacking gel was used, with the gel
stained with Coomassie Brilliant Blue R-250 and then
with silver nitrate (Wray et al., 1981). Protein con-
centrations were determined by the dye-binding assay of
Bradford (1976) using BSA as a standard protein.
Kandler, O., Hopf, H., 1980. Occurrence, metabolism and function of
oligosaccharides. In: Stumpf, P.K., Conn, E.E. (Eds.), The Bio-
chemistry of Plants. A Comprehensive Treatise, Vol. 3. Academic,
New York, pp. 221–270.
Kurz, G., Kurt, W., 1974. d-Galactose UV-assay with galactose
dehydrogenase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic
Analysis, Vol. 3. Academic, New York, pp. 1279–1287.
Laemmli, U.K., 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage. Nature 227, 680–685.
McCleary, B.V., Matheson, N.K., 1974. a-d-galactosidase activity and
galactomannan and galactosylsucrose oligosaccharide depletion in
germinating legume seeds. Phytochemistry 13, 1747–1757.
Mujer, C.V., Ramirez, D.A., Mendoza, E.M.T., 1984. Coconut a-d-
galactosidase isoenzymes: isolation, purification and characteriza-
tion. Phytochemistry 23, 1251–1254.
References
Pridam, J.B., Dey, P.M., 1974. The nature and function of higher
plant a-galactosidases. In: Pridam, J.B. (Ed.), Plant Carbohydrate
Biochemistry. Academic, New York, pp. 83–96.
Barham, D., Dey, P.M., Griffiths, D., Pridam, J.B., 1971. Studies on
the distribution of a-galactosidases in seeds. Phytochemistry 10,
Sastry, P.S., Kates, M., 1964. Hydrolysis of monogalactosyl and
digalactosyl diglycerides by specific enzymes in runner-bean leaves.
Biochemistry 3, 1280–1287.
1759–1763.
Bhalla, P.L., Dalling, M.J., 1984. Characteristics of a b-galactosidase
associated with the stroma of chloroplast prepared from mesophyll
protoplast of the primary leaf of wheat. Plant Physiol. 76, 92–95.
Bradford, M.M., 1976. A rapid and sensitive method for the quanti-
tation of microgram quantities of proteins utilizing the principle of
protein-dye binding. Anal. Biochem. 72, 248–254.
Smart, E.L., Pharr, D.M., 1980. Characterization of a-galactosidase
from cucumber leaves. Plant Physiol. 66, 731–734.
Thomas, B., Webb, J.A., 1977. Multiple forms of a-galactosidase in
mature leaves of Cucurbita pepo. Phytochemistry 16, 203–206.
Thomas, B., Webb, J.A., 1978. Distribution of a-galactosidase I in
Cucurbita pepo. Plant Physiol. 62, 713–717.
Burns, J.K., 1990. a- And b-galactosidase activities in juice vesicles of
stored Valencia oranges. Phytochemistry 29, 2425–2429.
Chrost, B., Schmitz, K., 1997. Changes in soluble sugar and activity of
Wray, W., Boulikas, T., Wray, V.P., Hancock, B., 1981. Silver staining
of proteins in polyacrylamide gels. Anal. Biochem. 118, 197–203.