Plant CellPhysiol. 41(4): 408-414 (2000)
JSPP © 2000
Purification and Characterization of Two Sucrose Synthase Isoforms from
Japanese Pear Fruit
Koji Tanase and Shohei Yamaki'
Laboratory of Horticultural Science, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601
Japan
Two isoforms (SSI and SSII) of sucrose synthase (SS;
EC 2.54.1.13) were purified from Japanese pear fruit and
their properties were compared. SS I mainly appeared in
young fruit and SS II mainly in mature fruit. SS I and SS
II were purified to the specific activity of 3.37 and 4.26
(units (mg protein)" 1 ), respectively. The MW of native and
subunit proteins of SS I and SS II were almost the same
and both SSs seemed to be a tetramer composed of an 83
kDa polypeptide. However, the ionic charges of the native
proteins and the kinetic parameters of SSs were different.
Specifically, the Km value for UDP-glucose in SS I was the
same as that for UDP, while the Km value for UDP-glucose
in SS II was less than that for UDP. SS II easily reacted for
sucrose synthesis than sucrose cleavage compared with SS
I. Therefore, it is considered that SS I and SS II play
different roles in the utilization of carbohydrate in young
and mature fruit, respectively.
Key words: Enzyme purification and characterization —
Isoforms of sucrose synthase — Japanese pear (Pyrus
serotina) — Sucrose synthase (SS; EC 2.54.1.13)
kinetic parameters between SS isozymes were also the same
in sugarcane, wheat and cucumber (Buczynski et al. 1993,
Gross and Pharr 1982, Larsen et al. 1985). The two SS
isozymes mentioned above have been purified and the
genes encoding them have been isolated from many plants
such as maize, barley, rice, and Arabidopsis thaliana
(Huang et al. 1994, Martinez et al. 1993, Wang et al. 1992,
Werr et al. 1985).
Accumulation of sucrose in fruit is related to various
sucrose-metabolizing enzymes including SS. The mechanism of sucrose accumulation in fruit is different in various
species and varieties. In strawberry, melon, mango and
banana fruit, sucrose is synthesized by sucrose phosphate
synthase (SPS) rather than SS (Hubbard et al. 1991, 1989,
Nasciment et al. 1997). However, in peach fruit, the rise in
SS activity with fruit maturation contributed to sucrose
accumulation (Moriguchi and Yamaki 1988). In Japanese
pear fruit, the increase in both SS and SPS activities caused
the increase of sucrose content during maturation, but SS
activity contributed more than SPS activiy (Moriguchi et
al. 1992). SSs partially purified from young and mature
Japanese pear fruit had different characteristics (Suzuki et
al. 1996). It was suggested that SSs in Japanese pear fruit
exist as isoforms (or isozymes) and that SS appearing in
mature fruit played an important role in sucrose synthesis.
To clarify the properties of these SSs, we purified and
characterized both SSs from young and mature Japanese
pear fruit, and discussed their physiological roles in sucrose
metabolism.
Sucrose synthase (SS) (EC 2.54.1.13) catalyzes the reversible reaction (sucrose + UDP <
•— fructose + UDP-glucose). However, it is generally known that the role of SS is
to cleave sucrose to supply UDP-glucose for starch and
cell-wall synthesis (Amor et al. 1995, Chourey and Nelson
1976, Huber and Akazawa 1986). In maize, SSI, which is a
sucrose synthase isozyme and localized in the endosperm
Materials and Methods
seemed to take a major part in starch synthesis (Chourey et
al. 1986). SS2, another isozyme and present in many other
Plant materials—Young and mature fruits of Japanese pear
organs including the endosperm, seemed to provide sub- {Pyrus serotina Rehder var. culta Rehder cv. 'Hosui') were harstrates for respiration as well as for cell-wall synthesis vested on June 3 and September 10 in Nomura's orchard at Anjo
(Chourey et al. 1986, MaCarty et al. 1986). Immuno- in Aichi-pref. Japan, respectively. Flesh tissues were excised and
stored at — 80°C after freezing them in liquid nitrogen.
histochemical studies showed that SS2 is localized in both
Purification of sucrose synthase from Japanese pear fruitvascular bundle and sink tissue (Nolte and Koch 1993). In All procedures were carried out at 4°C. For purification of SS I,
spite of their different roles, the kinetic parameters of SSI
250 g of frozen flesh tissue from young Japanese pear flesh were
and SS2 were very similar (Nguyen-Quoc et al. 1990). The homogenized in 750 ml of 0.2 M K-phosphate buffer (pH 7.8)
containing l m M EDTA, 10 mM Na-ascorbate, 10 mM /?-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride (PMSF) and
10% insoluble polyvinylpolypyrolidone (PVPP). The homogenate
was filtered through 3 layers of cheesecloth and centrifuged at
10,000 xg for 30 min. The supernatant was adjusted to 40% ammonium sulfate saturation and centrifuged at 10,000 xg for 30
Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PVPP, polyvinylpolypyrrolidone; SS, sucrose synthase; SPS, sucrose phosphate synthase.
1
Corresponding author.
408
Sucrose synthase isoforms from Japanese pear Fruit
min. The precipitate was dissolved in a small volume of 20 mM
Tris-HCl buffer (pH8.5) containing 1 mM MgSO4 and 1 mM
dithiothreitol (DTT) (buffer A), dialyzed against buffer A and
adsorbed to 20 ml DEAE-Sephacel (Pharmacia, Uppsala, Sweden) pre-equilibrated with buffer A. The gel was washed with
buffer A, then the proteins were eluted with 200 ml of the elution
medium which was a linear gradient of 0 to 500 mM KC1 in buffer
A. The active fractions were precipitated by 70% ammonium
sulfate saturation and the precipitate was dissolved in 2 ml buffer
A. The sample was loaded onto a column of 200 ml of Sepharose CL-6B (Pharmacia, Uppsala, Sweden) pre-equilibrated with
buffer A containing 10% glycerol and run with the same buffer.
After centrifugation in a Centricon 30 microcentrifuge tube
(Amicon, California, U.S.A.), the active fractions were dissolved
in 5 mM K-phosphate buffer (pH 7.8) containing 1 mM DTT
(buffer B). The sample was adsorbed to 2 ml of hydroxyapatite
(Wako Pure Chemical Co., Japan) equilibrated with buffer B. The
gel was washed with buffer B, then the proteins were eluted with
a linear gradient of 5 to 300 mM K-phosphate buffer (pH 7.8)
containing 1 mM DTT. The active fractions were collected and
dissolved in a small volume of buffer A containing 5 M ammonium sulfate which had been centrifuged in a Microcon 30 microcentrifuge tube (Amicon, California, U.S.A.). The sample was
adsorbed to 1 ml Phenyl Sepharose (Pharmacia, Uppsala, Sweden) pre-equilibrated with buffer A containing 5 M ammonium
sulfate. The gel was washed with buffer A containing 5 M ammonium sulfate and further washed with buffer A. Then the
proteins were eluted with 15 ml of the elution medium of buffer A
containing 50% sucrose. The peak fractions were adjusted to 10%
glycerol and stored at — 80°C.
For purification of SS II, 900 g of the frozen tissue from
mature Japanese pear flesh were homogenized in 1,000 ml of 0.2
M K-phosphate buffer (pH 7.8) containing 1 mM EDTA, 10 mM
Na-ascorbate, 10 mM /?-mercaptoethanol, 2 mM PMSF and 10%
insoluble PVPP. The subsequent purification steps were the same
as described for purification of SS I except for the omission of
Phenyl Sepharose step. The active fractions eluted from hydroxyapatite gel were collected and dissolved in buffer A containing
10% glycerol which had been centrifuged in a Microcon 30 microcentrifuge tube (Amicon, California, U.S.A.). The sample was
loaded in a column of 20 ml Superose 6 (Pharmacia, Uppsala,
Sweden) pre-equilibrated with buffer A containing 10% glycerol
and run with the same buffer. The peak fractions were adjusted to
10% glycerol and stored at -80°C.
Estimation of molecular weight by Superose 6 gel filtration—
The molecular weight of SSs was estimated using a column of Superose 6 gel filtration. The standard protein samples with known
molecular weights (thyrogloblin, 669,000; yS-amylase, 200,000;
bovine serum albumin, 66,000 and blue dextran, 6,000,000) were
applied to a 20 ml Superose 6 column pre-equilibrated with buffer
A containing 300 mM KC1 and run with the same buffer. The
fraction was collected in 0.5 ml each and assayed for the activity
of SSs and the absorbance of standard protein samples at 280 nm.
Assay of sucrose synthase activity—Assay A (for sucrose
synthesis): The reaction mixture contained 15 mM HEPES-KOH
buffer (pH8.0 or 8.5), 50 mM fructose, 2 mM UDP-glucose, 5
mM MgCl2 and enzyme solution in a total volume of 1.2 ml. The
mixture was incubated for 30 or 60 min at 30°C and the reaction
was stopped by the addition of 50 /A of 2 M NaOH. Production of
sucrose was determined by Roe's method (1934).
Assay B (for sucrose synthesis): The production of UDP was
determined according to the enzyme-coupling method reported by
Morell and Copeland (1985). The reaction mixture (0.6 ml) com-
409
posed of 20 mM HEPES-KOH buffer (pH 8.0 or 8.5), 50 mM
fructose, 2 mM UDP-glucose, 5 mM MgCl2, 4 mM phosphoenolpyruvate, 0.15 mM NADH, 20 mM KC1, pyruvate kinase, lactate
dehydrogenase and the sample.
Assay C (for sucrose cleavage): The reaction mixture consisted of 30 mM HEPES-KOH buffer (pH 7.0 or 7.5), 200 mM
sucrose, 5 mM UDP and enzyme solution in a total volume of 1.5
ml. The reaction was stopped by heating the mixture in boiling
water for 3 min. The production of fructose was determined by
the enzyme-coupling method using ATP, NADP + , hexokinase,
phosphoglucose isomerase and glucose 6-phosphate dehydrogenase (Morell and Copeland 1985).
One unit is defined as the amount of enzyme that catalyzes
the formation of 1 /umol of product per min.
Determination of protein content—Protein content was
measured by Bensadoun and Weinstein's method (1976). BSA was
used as the standard.
SDS-PAGE and Immunoblotting—SDS-PAGE was carried
out as described by the method of Laemmli (1973). The gel was
stained with AgNO3 using Silver Stain Plus (BIO-RAD, California, U.S.A.). For immunoblotting, after SDS-PAGE, proteins
were transferred to a cellulose nitrate membrane using a semi-dry
blotting apparatus (Bio-Rad, California, U.S.A.) based on the
modified procedure of Towbin et al. (1979). Rabbit antibodies
raised against mung bean SS were used (Arai et al. 1992).
Results
Appearance of SS isoforms in Japanese pear fruit—
The chromatography of the extract from young fruit on
DEAE-Sephacel showed one large peak of SS activity
(Fig. 1A), while that from mature fruit showed two peaks
of SS activity (Fig. IB). The former peak was eluted at the
same KC1 concentration as that of the SS extracted from
young fruit. The latter peak, which was eluted at a high
KC1 concentration was mainly involved in the SS activity in
mature fruit. Thus, these SSs are isoforms (or isozymes)
with different electrical charges; and named SS I and SS II
which are mainly involved in young and mature fruit, respectively.
Purification of SS I and SS II— SS I and SS II were
purified to specific activities of 3.37 and 4.26 (units (mg pro
tein)" 1 ), respectively, according to the procedures described in Table 1. The recovery of SS II (0.91%) was lower
than SS I (10.4%) and the Phenyl Sepharose purification
step was omitted for SS II. The peak of activity in SS I or
SS II appeared in the same fraction and corresponded to
each protein peak on the gel filtration (data not shown).
The final preparation of purification steps contained one
major polypeptide by silver staining after SDS-PAGE
(Fig. 2). Each major polypeptide after SDS-PAGE was
revealed clearly by Western blot using the polyclonal antibodies raised against the mung bean SS protein (Fig. 2).
Each molecular weight of both SS I and SS II polypeptide
was estimated to be 83,000.
Determination of molecular weight of native SSs—
The molecular weight of native proteins of each isoform
was determined to be 360,000 by Superose-6 gel filtration
Sucrose synthase isoforms from Japanese pear Fruit
410
1.0
(A)
-, 500
(B)
(kDa)
(kDa)
10
15
20
25
l.Or—
TT
L
ssn-
•l
/
\X
~ 0.4c
10
15
Act
42-
-42
20
M
M
-I 0
5
-66
SSI
'1\ J 1-
/\
5 0.2-
66-
-1 500
/
0.8-
fraction)
p
'
7
;' i
-116
J o
30
Fraction number
(B)
116-
25
30
Fraction number
Fig. 1 Elution profile of sucrose synthase (SS) from DEAESephacel chromatography. SSs from young (A) and mature (B)
fruits were eluted with the elution medium of a linear gradient of
0 to 500 mM KC1 in buffer A. Each fraction of 3 ml was collected.
The activity was determined by assay C (sucrose cleavage).
Fig. 2 SDS-PAGE and western blot of SSs. SDS-PAGE was
carried out in 7.5% (W/V) polyacrylamide gels and proteins were
stained by silver. Immunoblot after SDS-PAGE of SS was done
with the rabbit antibodies raised against mung bean SS. (A): lane
1; silver staining of SS I; lane 2, immunoblot of SS I. M, marker.
(B): lane 1; immunoblot of SS II, lane 2; silver staining of SS II.
M, marker.
Table 1 Purification of sucrose synthase from Japanese pear fruit.
(A)
Fraction
Crude extract
40% ammonium sulfate
DEAE Sephacel
Sepharose CL-6B
Hydoroxyapatite
Phenyl-Sepharose
Total activity
(unit)
Total protein
(mg protein)
Specific activity
(unit (mg protein)"1)
Yield
3.56
3.55
1.52
0.75
0.69
0.37
596
126
25.2
3.67
1.22
0.11
0.006
0.028
0.060
0.20
0.56
3.37
100
99.7
42.7
20.9
19.5
10.4
Total activity
(unit)
Total protein
(mg protein)
Specific activity
(unit (mg protein) 1 )
Yield
4.76
2.92
1.71
0.30
0.11
0.043
313
51.9
13.5
0.51
0.03
0.01
0.015
0.056
0.13
0.60
3.67
4.26
100
61.6
36.2
6.30
2.33
0.91
(B)
Fraction
Crude extract
40% ammonium sulfate
DEAE Sephacel
Sepharose CL-6B
Hydoroxyapatite
Surperose 6
(A): SS I, (B): SS II. Activity was determined by assay C (sucrose cleavage).
Sucrose synthase isoforms from Japanese pear Fruit
Thyrogloburin
•a
12
13
14
SS I ( O ) and SS II ( • ) -
15
16
Elution volume (ml)
Fig. 3 Molecular weight of SS I and SS II estimated by Superose
6 gel filtration. Thyrogloblin, 669,000; /?-amylase, 200,000; bovine
serum albumin, 66,000 and blue dextran, 6,000,000 were used as
the standard protein samples.
(Fig. 3). The major polypeptides of SS I and SS II after
SDS-PAGE were estimated to be 83,000 each as shown in
Fig. 2. Therefore, SS I and SS II from Japanese pear fruit
seem to be a tetramer.
Optimum pH—The sucrose synthesis activities of both
411
SS I and SS II fell sharply at a lower pH, while high sucrose
cleavage activities appeared in a relatively broad pH range
(data not shown). The optimum pH of SS I for sucrose
synthesis and sucrose cleavage was 8.5 and 7.5, respectively, and that of SS II was 8.0 and 7.0, respectively.
Kinetic parameters—Sucrose synthesis and cleavage
reactions of these enzymes nearly conformed to MichaelisMenten kinetics (data not shown). Kinetic parameters by
Lineweaver-Burk plot are shown in Table 2. For SS I, the
Km value for UDP-glucose was 0.05 mM, which was almost
the same as the Km value for UDP (0.069 mM), when they
were used as the variable substrate (Table 2). The Km values
for fructose and sucrose as the variable substrates were
18.6 and 37.6 mM, respectively. For SS II, it had a higher
affinity for UDP-glucose (Km = 0.03) than UDP (Km =
0.41), although the Km value of the former did not vary
greatly from that of SS I. The Km value for fructose (9.77
mM) in SS II was smaller than that in SS I (18.6 mM),
while the Km value for sucrose was equal to that in SS I.
Km values of SS I for ADP-glucose and ADP as the first
substrate were 0.17 and 0.11, respectively, and that of SS II
were 0.78 and 1.94 mM, respectively. Comparing the Km
Table 2 Kinetic parameters of SS I and SS II from Japanese pear fruit
(A)
Variable substrate
Fixed substrate
Sucrose synthesis
Fructose
UDP-glucose
ADP-glucose
Sucrose cleavage
Sucrose
UDP
ADP
'max
(mM)
(unit (mg protein) ! )
UDP-glucose
Fructose
Fructose
18.6 ±2.6
0.05 ±0.003
0.17 ±0.01
3.42±0.58
3.00±0.48
1.95+0.39
UDP
Sucrose
Sucrose
37.6 ±6.79
0.069±0.01
0.11 ±0.013
3.56±0.16
3.59±0.10
2.85±0.18
(mM)
(unit (mg protein) l)
9.77 ±1.7
0.03 ±0.001
0.78 ±0.096
2.86±0.04
2.50±0.08
1.58 + 0.33
36.0 ±3.90
0.41 ±0.14
1.94 ±0.037
2.40 ±0.04
2.28 ±0.004
0.82±0.07
(B)
Variable substrate
Fixed substrate
Sucrose synthesis
Fructose
UDP-glucose
ADP-glucose
UDP-glucose
Fructose
Fructose
Sucrose cleavage
Sucrose
UDP
ADP
UDP
Sucrose
Sucrose
'max
(A): SS I, (B): SS II. Km and Fmax were calculated using Lineweaver—Burk plots.
Activity for sucrose synthesis and cleavage were determined by assay B (sucrose synthesis) and assay
C (sucrose cleavage), respectively. Values are mean of two determinations ±SE.
Sucrose synthase isoforms from Japanese pear Fruit
412
values of the first substrates examined, SS II showed higher
affinity for sucrose synthesis than SS I. The Kmax values for
sucrose cleavage were almost the same as those for sucrose
synthesis in SS I or SS II, when UDP/UDP-glucose and
sucrose/fructose were used as the substrates. However, the
Pmax values for ADP and ADP-glucose substrates were
lower than those of UDP and UDP-glucose substrates
in both SS I and SS II (Table 2).
Effect of divalent cations—For sucrose synthesis reaction, both SS I and SS II activities were stimulated by
Mg 2+ , Mn 2+ and Ca 2+ , especially by Mg 2+ (Table 3). On
the other hand, for sucrose cleavage reaction, both activities were weakly inhibited by these cations. Cu 2+ , Zn 2+ and
Hg 2+ strongly inhibited the reactions in both directions.
Table 3 Effects of divalent cations on sucrose synthase
activity
(A)
Discussion
Concentration (mM)
1.0
0.1
5.0
Sucrose synthesis
Mg
Mn
Ca
Cu
Zn
Hg
100
99
101
62
58
36
123
121
117
19
23
21
139
95
91
2
N.D.
N.D.
Sucrose cleavage
Mg
Mn
Ca
Cu
Zn
Hg
101
101
102
104
35
38
93
83
87
6
12
N.D.
68
70
87
N.D.
12
N.D.
(B)
Concentration (mM)
0.1
1.0
5.0
Sucrose synthesis
Mg
Mn
Ca
Cu
Zn
Hg
107
118
97
47
11
52
160
113
115
16
N.D.
N.D.
158
126
117
N.D.
N.D.
N.D.
Sucrose cleavage
Mg
Mn
Ca
Cu
Zn
Hg
81
72
83
8
35
N.D.
85
84
87
N.D.
7
N.D.
58
66
66
N.D.
N.D.
N.D.
(A): SS I, (B): SS II, Activity for sucrose synthesis and cleavage
were determined by assay A (sucrose synthesis) and assay C (sucrose cleavage), respectively. Relative activity in % as compared
to that in the absence of metal ions. N.D.; not determined.
Japanese pear fruit involves two isoforms of SS, one
of which is SS I occurring mainly in young fruit and the
other is SS II in mature fruit. They have a different ionic
charge, as in cucumber (Gross and Pharr 1982), since
they were eluted from DEAE-Sephacel chromatography at
different KC1 concentrations (Fig. 1). SS I and SS II proteins had the same molecular size on SDS-PAGE and gel
filtration, but differed in their electrical charge. Thus, SS I
and SS II did not result from the modification of each SS
by protease action during its purification. The specific activities of SS I and SS II corresponded to 3.37 and 4.26
(units (mg protein)"1), respectively (Table 1), and were
similar to those of sweet potato root and rice grain (Murata
1971, Nomura and Akazawa 1973).
Chourey et al. (1986) proposed that SS in maize is
composed of SSI and SS2 subunits, and involves five forms
of two homotetramers and three heterotetramers. Five
forms of SS were also detected in barley endosperm (Guerin and Carbonero 1997). However, SS 1 and SS II in this
study revealed only one peptide band on SDS-PAGE, with
a molecular weight estimated to be about 83,000, respectively, and each molecular weight of the native protein was
determined to be 360,000 by gel filtration (Fig. 3). Therefore, SS I and SS II from Japanese pear fruit seemed to be
mainly a tetramer similar to those in soybean nodules
(Morell and Copeland 1985) and rice grains (Nomura and
Akazawa 1973).
The Km values for UDP and UDP-glucose of SS I were
different from those of SS II (Table 2). These data of SS
isoforms were different from that reported previously
(Suzuki et al. 1996). Because of the use of purified SS
isoforms, our data may be more accurate. Our findings
show that SS I had an equal affinity for UDP-glucose and
UDP, which is similarly observed in SS from tapioca tuber
(Shukla and Sanwal 1971). SS I activity mainly appeared
in young Japanese pear fruit, which accumulated much
starch and much cell wall polysaccharides but less sucrose
(Yamaki et al. 1979). Thus, the prominent role of SS I
seems to be to provide the substrate (UDP- and ADP-glucose) for the syntheses of starch and cell wall polysaccharides by cleavage of sucrose. In many plant materials, SS
isozymes have the same property, that is higher activity to
cleavage sucrose than to synthesize sucrose (Murata 1971,
Nomura and Akazawa 1973, Morell and Copeland 1985,
Sucrose synthase isoforms from Japanese pear Fruit
Avigar 1964). SS I may have the same function as that of
SS in reported previously. On the other hand, SS II had a
higher affinity for UDP-glucose than for UDP, which
means that it is likely to have predominant activity for sucrose synthesis, although the direction of SS reaction depends on the balance of UDP/UDP-glucose in vivo. SS in
peach fruit was suggested to be responsible for sucrose
synthesis in vivo since it had a higher affinity for UDPglucose than UDP which consequently favored the accumulation of sucrose with fruit ripening (Moriguchi and
Yamaki 1988). This affinity was also observed in SS II of
the present study. From these results, SS I and SS II, which
have a peculiar characteristic, may have different roles for
sucrose utilization or accumulation in young and mature
Japanese pear fruit, respectively.
The activity of SS I was stimulated by Mg 2+ , Mn 2+
and Ca 2+ as SSs in cucumber, tomato fruit and soybean
(Gross and Pharr 1982, Morell and Copeland 1985,
Schaffer and Petreikov 1997). SS II isozyme which has a
function different from SS I, also showed the same inclination for Mg 2+ , Mn 2+ and Ca 2+ . Recently, phosphorylation of SS was found to stimulate the sucrose cleavage
reaction by increasing the affinity for sucrose and UDP
(Huber et al. 1996, Nakai et al. 1998). Moreover, Winter et
al. (1997) proposed that phosphorylation controlled the
cellular localization of SS protein rather than stimulation
of SS activity. Further studies about the phosphorylation
of SS I and SS II might make clearer the details of their
physiological roles.
We are grateful to Dr. H. Mori, Graduate School of
Bioagricultural Sciences, Nagoya University for supplying polyclonal antibodies against mung bean sucrose synthase. We also
thank Dr. Y. Kanayama, Faculty of Agriculture, Tohoku University, and Dr. K. Shiratake, Graduate School of Bioagricultural
Sciences, Nagoya University, for their advices. This research was
supported in part by a grants-in-aid (No. 09306002) from the
Ministry of Education, Science, Sports and Culture of Japan.
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(Received June 11, 1999; Accepted January 13, 2000)