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Enzymic mechanism of starch synthesis in ripening rice grains
ARCHIVES
OF
Enzymic
BIOCHEMISTRY
Mechanism
AND
of Starch
III. Mechanism TAKAO
MURATA,2
The International
113,
BIOPHYSICS
34-44
(1966)
Synthesis
in Ripening
of the Sucrose-Starch
TATSUO
SUGIYAMA,3 T. AKAZAWA3
Rice Research
Institute,
Received
May
Grains
MINAhIIIKAWA,3
AND
Conversion’
TAKAO
Los BaAos,
Rice
Laguna,
The Philippines
12, 1965
In a coupling system of sucrose-synthetase and starch-synthetase, both isolated from the ripening rice grains, the more efficient transfer of glucose-Cl4 from sucroseCl4 to starch occurred in the presence of ADP as compared to UDP, indicating the predominant role of ADPG in the process. However, UDP was found to inhibit profoundly the ADPG-mediated glucose-C I4 transfer to starch from sucrose-CL”, which was based on its specific inhibition of the ADPG-sucrose t,ransglucosylation reaction. The Km value of sucrose synt,hetase was determined to be 1.5 X 1OW M (ADP) and 1.1 X lo+ M (UDP), respectively. These findings may support a view that the sucrose breakdown proceeds through the reversal of UDPG-sucrose transglucosylation rather than directly by way of the ADPG-sucrose transglucosylation. By feeding the rice grains at the mid-milky stage with sucrose-W, incorporation of the radioactivit,y into the starch fraction was observed, although most of the radioactivity in the acidsoluble fraction was recovered as sucrose. It was noted also that a relatively high radioactivity was detected in glucose and fructose. The formation of a prominent radioactive compound(s) emerging around the fraction of glucose-6-phosphate was demonstrated in a Dowex-1 anion-exchange column chromatography; neither its chemical nature nor the role in the sucrose-starch conversion has been revealed. Both ADPG and UDPG became labeled to a much lesser extent than the above conpounds under the experimental conditions employed. Attempts to dist,inguish their predominant role in the biosynthetic conversion of sucrose to starch were msmcessful, although the specific radioactivity of ADPG was about twice as high as t,hat of UDPG. Based on t,he experimental results obtained, possible enzymic mechanisms underlying the sucrose-starch conversion in developing rice grains are discussed.
In a previous paper of this series (I), evidence was given for t’he dominant role of ADPG in sucrose-starch conversion in ripening rice grains. When coupled with either ADPG-sucrose transglucosylase (sucrose synthetase) or ADPG-pyrophosphorylase, bobh residing in the soluble fraction of the grain extract, ADPG-starch transglucosylase (starch synthetase) utilized both sucrose-Cl4 and glucose-l-phosphate-Cl4 as efficient glu-
cose donors in starch format,iou. On the other hand, the corresponding UDPG system appeared to operate much less efficiently in the process [see reference (2)]. The overall mechanism proposed therein was Eq. (I) :
1 Issued as I.R.R.I. Journal Series No. 48. 2 Present address: Tohoku National Agricultural Experiment Station, Morioka, Japan. 3 Address to which requests for reprints should be sent: Nagoya University, Anjo, Aichi, Japan.
The important role of ADPG in starch synthesis has been confirmed by other workers using different plant, materials (3, 4). The question remains, however, as to the role of
ADP
Sucrose Sucrose
synthetase
’ ADPG
(I) Starch
34
synthetase
L starch
ENZYMIC
MECHANISM
OF
STARCH
SYNTHESIS
UDPG in the carbohydrate metabolism in rice grains. Its participation in the sucrosesbarch conversion mechanism cannot be ignored completely, especially in view of the facts that: (i) t,he content of UDPG in ripening rice grains was much higher than that of ADPG (5, ci), and (ii) glucose transfer from UDPG to starch molecule occurred at a reasonable rate, although the rate was lower than that from ADPG (1). These two points have also been found to be true in some other plants, and Leloir (7) has presented a view that both the ADPG and UDPG pathways may operat’e concurrently in the starch synthesizing mechanism. On the other hand, De Fekete and Cardini (4) have postulat’ed that UDPG may have a primary role in the initial step of the sucrose-starch transformation, whereas &arch synthesis is exclusively catalyzed by the ADPG-starch transglucosylase. This concept has been based on the smaller Michaelis constant (Km) of the sucrose synthet,ase toward UDP as compared to ADP, and the specific inhibition of the ADPG-sucrose transglucosylase by UDP (8). Consequently the following mechanism, Eq. (II), has been presented by them: UDP
Sucrose
’
Sucrose-synthetase PPi
glucose-lphosphate
UDPGpyrophosphorylase
(II)
ATP
--
ADPG-pyrophosphorylase Starch
UDPG
synthetase
’
ADPG
’
starch
In the present experiment, we have atOempted to determine the crucial role of UDPG in the sucrose-starch conversion mechanism in ripening rice grains and have used both a cell-free enzyme system and sucrose-CWfeeding bechnique. Attention has been given in t’rying to visualize the regulatory mechanism underlying the interaction of two different nucleoside diphosphate sugars in starch synthesis. MllTERIALS Plants. Peta, used as in the
AND
METHODS
an indica type rice previous experiment
variety, (1, 2).
was The
IN
RIPENING
RICE
GRAINS
33
growing of the plants and the subsequent sampling of rice grains were essentially the same as described before. Preparation of enzymes. (a) ADPG-starch transglucosylase: Acetone powder starch granules were prepared by the same method as reported previously (1). (b) ADPG (UDPG).sucrose transglucosylase (sucrose synthetase) : A crude enzyme preparation obtained by (NHa)&Od precipitation of the grain extract showed a strong activity in sucrose synthesis (2)) and a partial purification of the enzyme was undertaken as follows. Freshly harvested rice grains (240 gm) were ground in a chilled mortar with 300 ml of 0.05 M phosphate buffer (pH 7.0) and strained through cheese cloth, and the filtrate was centrifuged at 25,000g for 15 minutes. To the supernatant solut,ion (about 150 ml) was added solid (NHd)#Od to 50% saturation, and the mixture was centrifuged again. The resulting precipitate was dissolved in 20 ml of water and dialyzed against 8 liters of water overnight in a cold room (2°C). To the dialyzate was added 0.06 volume of 1 ,V MnC12 , and the mixture was allowed to stand for 30 minutes and then centrifuged. The supernatant fraction was fractionated by adding (NH,)zSOd (30-507, saturation), and the precipit,ate was dissolved in G ml water after centrifugation. After dialysis against water for 1.5-2 hours, 4.0 ml of Ca-phosphate gel (about 20 mg per milliliter) was added to the clear solution and the material was allowed to stand for 30 minutes. To the supernatant fraction was added ?/4 volume of 0.02 M tris buffer (pH 7.5) and the resulting solution was applied to a column of DEAE-cellulose (1.1 X 9.0 cm), which was preequilibrated with 0.005 M tris buffer (pH 7.5). The protein was fractionated by 0.005 tris-NaCl as eluent, the concentration of the latter component increasing stepwise: 0, 0.05, 0.10, 0.15, 0.20, and 0.30 Jf, respectively. To 40 ml eluate as fractionated by 0.005 M tris- 0.15 llil NaCl (measured by t,he optical densit,y at 280 mp) was added solid (NH&SO4 to make a 50yc saturation, and the resulting precipitate was dissolved in a small quantity of water. Bfter dialysis against water for 2 hours, the final solution was used as a sucrosesynthetase preparation. This enzyme preparation was found to be devoid of ADPG (UDPG)-pyrophosphatase activity, and the specific activity (micromoles sucrose formed per 10 minutes per milligram protein-N) was about 10 times as high as that of the preparation obtained at the initial (NH,)&04 precipitation. (c) Crude soluble enzyme preparation for the sucrose-transformation experiment: This was prepared by essentially the same method as that for the hexokinase of rice grains (2). A precipitable fraction obtained by
2%
RIURATA
the 50-60’;; (NH,)zSOI saturation of the rice grain extract in 0.1 M tris buffer (pH 7.5) was used as the source of enzyme. Enzylne assay methods. (u) Sucrose-starch conversion: Glucose transfer from sucrose-W to the starch molecule was determined in the reaction mixture containing both granular starch synthetase and soluble sucrose synthetase. Modification of the reaction system is described in each case. (b) ADPG (UDPG)-sucrose transglLcosylase : The reaction system was designed so as to determine the rate of sucrose breakdown in t,he presence of either ADP or UDP. The formation of radioactive ADPG or UDPG from sucrose-C’” in the presence of various amount,s of either ADP or UDP was quantitatively assayed by means of paper chromatography. All the enzymic experiments u-ere duplicated aud average values are present,ed. (c) Transformation of sucrose by a crude enzyme system: Breakdown of sucrose-C11 by the reversal of sucrose-synthetase followed by the metabolism of the hexose unit, through the glycolgtic enzyme system was examined by the Dowes-1 ion-eschange column chromatographic separation of individual hexose phosphates and their radioassay. The method was essentially the same as that reported previously (2). Sucrose-C14 feeding experiment and Dowex-1 anion-exchange column chromatography. Panicles of rice plants at the mid-milky st,age were inserted for 30-90 minutes into a test tube cont,aining 2.0 ml of 0.01 M sucrose-C14 (2.44.1 X 10’ cpm). During the incubat,ion period almost all the sucrose solution was absorbed by the plants. The grains were immediately stripped off, combined with about) 30 gm of freshly harvested grains from intact plant,s, and ground with 0.6 S ITClOa in a chilled mortar. The extraction was repeated two more times in the same mauner. The total volume of the extract was usually about 200 ml. The insoluble residual material was washed thoroughly three times with Hz0 and two times more with acetone. A subsample of the dried residue was used for the radioassay of the starch fraction. The total perchloric acid extract, contaiuing free sugars, sugar phosphates, and nucleotides, was carefully neutralized with 10 N KOII to pH 6.0, aud t,he precipitated KCIOl was removed by centrifugation after placing in a deep freezer. The supernatant fraction was concentrated in z’aczlo at 30”-34°C and centrifuged again, the final volume being usually about 10 ml. It was then applied to a column of Dowex-1 anion-exchange resin (AG 1 X 8, chloride form, 200400 mesh, Calbiochem.) and chromatographed by the method reported previously (5, 6), which is a modification of the method used by Nakamura’s group (10, 11).
ET
AL.
Identification of stachyose. Rice grains (18.7 gm) fed wit,h sucrose-C” (3.5 X 10’ cpm) for 90 minutes were the starting material. Separation of sugars by ion-exchange column chromatography combined with paper chromatograp1l.v was used for the identificatiork of stachgose. fleagents. The sources of the rcagerlts were as follows : ATP, Sigma Chemical Company; ADP, National Biochemicals Corporation; AlJPG, UDP, UTP, UJIPG, sucrose-C’4, glucose-l-phosphate, glucose-&phosphate, and frlictose-(i-phosphate, California Corporation for Biochemical ltesearch. Both UDl’-glucose-C’” alrd ADP-glucose-C” were prepared by the sucrose-synthetase isolated from rice grains by the method reported previously (1). Stachvose was a crift of Dr. %. Nikllni nod Dr. R. S. Schallenberger. RESULTS
conversion in cell-free ensystem. E’igure 1 shows c*learly t,hat
Sucrose-starch xyuze
I
0
10 SUCROSE
20 S&THETA&-
30
40 ( pgPROTEIN-N)
50
FIG. 1. Glucose-Cl4 transfer from sucrose-Clf into st,arch in the coupling system of sucrosesynthetase [ADPG (UDPG)-sucrose transglucosylase] and starch synthetase [ADP(; (UDPG)starch transglucosylasel. Preparative method of the individual enzymes is described in the text. Standard reaction mixture contained (in pmoles) : t,ris buffer (pH 7.4), 4.0; EDTA, 0.2; NaF, 2.0; sucrose-W, 0.3 (10.1 X lo4 cpm); ADP (UDP), 0.25; starch granules, 6.0 mg; and varying quantities of sucrose synthetase in a total volume of 25bl. Incubation was at 37°C and the radioactivity measurement of the starch molecule was carried out as reported previously (1, 2).
ENZYblIC
MECIIANISM
OF
STARCH
SYNTHESIS
IN
RIPENIKG
RICE
GRAINS
37
understood from the following observations. That is, the reaction mixture as used for experiment shown in the figure, only omitting starch granules, was examined for t.he paper chromatographie deeecOion of the nucleotide sugars formed. It was found that ADP-glucase-Cl4 was scarcely detectable in t,he system containing both ADP and UDP (the ratio 1: 34 and 1: l), while a small amount of this compound was formed in a system containing the above two nucleotides at the ratio of 1:,$s5. In the former two cases, a marked formation of UDP-glucose-C14 was demonstrated, whereas the magnitude of its formation was much less in the lntt.er case. The inhibitory effect of UDP in the ADPGUDP sucrose transglucosylase was originally reported by Cardini and Recondo (8), who used wheat germ enzyme, and later in more 60 120 180 0 detail by the same workers using maize enTIME IN MINUTES zyme (4). FIG. 2. Interaction of UDP and ADP in the Km values of sucrose-synthetase to ADP and glucose-W transfer from sucrose-Cl* into starch UDP. In an attempt to determine whether in the coupling syst,em of sucrose-synthetase and there exists any preferential utilization by starch synthetase. Experimental conditions were the rice enzyme for ADP or UDP, Kv2 values the same as that used in experiment described in of the partia,lly purified sucrose-synthetase Fig. 1, except that various amounts of UDP as toward each of the two nucleot,ides were indicated in the figure were added. The content measured. As represented in Figs. 3a and 3b, of protein-N of sucrose synthetase was 25 pg. the Km value of the enzyme for ADP was glucose transferred readily from sucrose to determined to be 1.5 X 10e3 31, while that starch in the presence of ADP. The rate of for UDP was 1.1 X 1O-4 M. The Kv2 values reaction was much higher than that via the of the maize enzyme reported by the Argencorresponding UDPG system, in which ADP tine workers to each of ADP and UDP were was replaced by UDP. This result is basically 2.1 X 10e3 M and 6 X 10V5 M, respectively in agreement wit,h our previous finding (1). (4). Recently, Avigad (13) isolated a highly With ADP, the magnitude of the glucose purified UDPG-sucrose transglucosylase transfer from sucrose-C’* to the starch molefrom Jerusalem artichoke, and the Km to cule increased proportionally with increasing UDP was determined to be 3.2 X low4 M. amounts of sucrose synthet’ase; no such effect Thus, our above result may support the was observed in the utilization of sucrose-Cl4 view that sucrose breakdown proceeds via the UDPG system. Although these obthrough the reversal of UDPG-sucrose trans.servations may suggest the efficient coupling glucosylation rather than directly by way of of ADPG-sucrose transglucosylation with the ADPG-sucrose transglucosylation reacthe starch synthetase reaction in the over-all tion. process of sucrose-starch conversion, t>he On the other hand, the measurement of actual situation was formd to be more comthe apparent equilibrium constant of the plicated. Thus, as seen in Fig. 2, the addition sucrose synthetase of rice grains gave the of UDP to the reaction mixture of the ADPG following values in t,riplicate experiments; system markedly inhibited the glucose transfer from sucrose to starch. The fact that this (ADP) (sucrose) inhibition lies in the effect of UDP on the (ADPG) (fructose) ADPG-sucrose transglucosylase, but not on = 1.6 (1.1, 1.8, 1.9) the ADlY-starcah transglucosylase, can be
AD4+,yg5)
,:,,::
1
3x (UDP) (sucrose) K’ eg (UDPG)(fructose)
h’IUIiATA
1
ET
AL.
400 -
= 3.3 (3.0, 3.0,3.9). Although it is difficult to evaluate the physiological significance of such values, one can speculate about the specific role of ADPGsucrose transglucosylase in the sucrosestarch conversion, which is apparently a contradictory view with the above postulation. Avigad (13) report’ed the value of K’eq [(sucrose) (UDP)/(UDPG) (fructose)] = 1.4 to 1.8 (pH 7.6) by the enzyme isolated from Jerusalem artichoke, while the one reported by Cardini et al. (14) was 2.0. As an alternate approach, we turned to sucrose-CWfeeding experiments. A major purpose was to see whether there is a predominant incorporation of the glucose molecule from sucrose-Cl4 to either ADPG or UDPG. Sucrose transformation in intact rice grains. Feeding of rice panicles at. the milky stage with sucrose-C14 for 30-90 minutes showed a measurable incorporation of the radioactivity into starch molecules (about 052.0%) the sucrose-starch (Table I), indicating t,ransformat,ion t,o be a relatively rapid met’abolic process. This reflects a vigorous rate of &arch synthesis in rice grains at this specific developmental stage [cf. Fig. 1 of (a)]. Figure 4 shows a typical pattern of radioactivitv distribution in the individual fractions,“i.e., free sugars, sugar phosphates, and nucleot,ides. Sucrose was the major radioact,ive compound in the free sugar fractions (2.0 X lo6 cpm), making up more than 90% of the tot,al acid-soluble fraction (see column 5 of Table I). It, is notable, however, t,hat a measurable radioactivity was observed in both the glucose (5.1 X lo4 cpm) and fructose (4.0 X lo4 cpm) fractions. This was interesting because Mori and Nakamura (11) have reported that the careful pretreatment of plant extracts by Amberlite IR-120 and -4B resins, before applying to a Dowex-1 anion-exchange column, resulted in only about, 0.2 % hydrolysis of the sucrose. In the present chromatogram, the high percentage of radioactivity recovered in each of glucose and fruct,ose fractions, 2.4 and 2.0 %, respectively, per total cpm of acid-soluble
I 0
25
50 mM
75
100
125
ADP
-
2500,
02.5
0.5 mM
075
IO
125
UDP
FIG. 3. Determination of the Michaelis constant (Knz) of sucrose-synthetase toward ADP (a) and UDP (b). The reaction misture consisted of the following (in rmoles): tris buffer (pH 7.4), 4.0; EDTA, 0.2; NsF, 2.0; sucrose-(Y4, 1.0 (9.3 X lo* cpm) (ADPG-system) and 0.1 (5.5 X 104 cpm) (UDPG-system); various amounts of ADP or UDP; and sucrose synthetase in a total volume of 20 ~1. After incubation at 37°C for 10 minutes, the reaction was stopped by adding 30 ~1 of 80% ethanol, and a 30.~1 aliquot of the clear centrifugate was applied to a sheet of acid-washed Whatman No. 1 paper for paper chromatography. The solvent system used was ethanol-ammonium acetate (pH 3.8) of Paladini and Leloir (9). The areas corresponding to ADPG and UDPG were eluted with H,O and filled up to 0.4 ml, and their radioactivity was determined in a Tri-Carb liquid scintillation spectrometer. The contents of protein-N of sucrose synthetase were 1.76 pg for ADP system and 0.26 pg for UDP system.
ENZYMIC
MECHANISM
~JTILIZATION Experiment
Feeding time (min) Grains weight (pm) Total radioactivity Cl4 administered Total radioactivity (cpm) Tc Incorporation Total radioactivity soluble fraction 7. Incorporation Total radioactivity fraction (cpm) ~___
OF
OF
SUCROSE-W
:
in free
IN
SYNTHESIS TABLE
I
RICE
GRAINS
1
of sucrose(cpm) in starch
in (cpm)
STARCH
acid-
sugar
X 9.6 -
BY
RIPENING
FEEDING
2
30 11.8 2.4 X lo7
2.3
IN
lo6
4
107
60 4.4 2.4 X lo7
4.1
x
106
1.1
3.8
1.7 x
106
3.1
fraction, indicates a possible enzymic hydrolysis of the sucrose by invertase or some other enzymes in rice grains. Two small unknown radioactive sugar fractions were also detected, one emerging just after the sucrose fraction (I) and the other preceding the fructose fraction (II). Assuming each of these unknown sugar fractions to be raflinose and stachyose, respectively, we attempted their identification. However, by repeated chromatographic analyses of the radioactive free sugar fractions, obtained by the sucrose-C’4 feeding, the formation of the first fraction (I) was rather inconsistent. On the other hand, as represented in Fig. 5, paper chromatography and Dowex-I. ion-exchange column chromatography showed the second sugar fraction (II) to be stachyose. It is noteworthy that, the relative specific radioactivity (cpm/OD 490 rnp) of st,achyose (6.7 X 104) and the fraction I (8.9 X 104) was much higher than that, of sucrose and two hexoses, and their role in sucrose metabolism of rice merits further attention. The total radioactivity in the remainder of the acid-soluble compounds comprised a small fraction of the administered sucrose04. This fractJion was separated into sugar phosphates and nucleotides. As can be seen in Fig. 4, the intensity of the radioactivity in both glucose-l-phosphate and fructose-6phosphate was not very high; instead there was a broad and prominent radioactive compound(s) designated as “X,” emerging around the position of glucose-6-phosphate.
15.8 X lo6
2.9
x
12.9 x
106
5
GO 14.7 2.4 x
105
0.5 x 106
39
GRAINS
EXPERIMEXTS
3
60 17.8 2.4 x
3.2
RICE
10’
-
3.1
90 7.G 4.1 x 10’ 2.4
x
106
12.9 -
x
105
2.1
0.F x 106
2.09
5.1 x 10”
The measurement of radioactivitv in this fraction did not give a good co&idence curve with that of the calorimetric analysis of glucose-6-phosphate added as a carrier. Attempts to reveal the nature of this compound(s) have been unsuccessful. However, the following compounds have been excluded from the list of the possible shructures based on the ion-exchange column chromatography, i.e., sucrose phosphate, G-phosphogluconate, ribose&phosphate, 3-phosphoglyceraldehyde, and dihydroxyacetone phosphate. It is unknown whether this compound(s) occupies a key role in the sucrosestarch conversion or is merely formed as a by-product of the main path of sucrose transformation in rice grains. The bottom of Fig. 4, representing the radioactivity pattern in the nucleotide fraction, shows that’ ADI’G and CDPG became labelled to roughly t’he same extent within a 90-minute incubation. By t,he closer examination the specific radioactivity (cpm,/ /Imole) of the ADPG (570) was about t,wice as that, of UDPG (270), but it is nat,urally difficult from such figures to assess the predominant precursor role between these two nucleot,ide sugars in starch synthesis. Moreover, on the basis of the current cboncept, of the starch biosynthesis via, nucleotide sugar pat,hway,
the
magnitude
of
the
lshelling
in
t,hese compounds was much smaller than that in both free sugars and sugar phosphat,es, and even as compared with that in the starch fraction. It is desirable t)o shorten
I
I.1I-
0.00SMNaia,9+0,+
+
0.02YNq40,
0.03YNo2D,
0,
elucou suemu
1 I.0 i P 0 d
IO.0 8.0
6.0
% h
0.5
4.0
I g d IJ
2.0
c
SUGAR
PHOSPHATES
10.0
1.0
i B f Ii d
8.0
“0 *
4.0
Ii d 6
0.5
2.0
0
I
-z
IO
20
SO
40
0.4
iI d 6 0.2
0
50
200
loo FRACTION
NUYDCR
FIG. 40
4
%
6.0
ENZYMIC
MECHANISM
OF
l -
3.30
STARCH
1 M
SYNTHESIS
Na&,O,
------+
04 ELUATE
0.6
Cl8
THROUGH
RIPENING
0.02MNa2B44
Stachyose
a2
IN
RICE
GRAINS
41
4
i
1.0 COLUMN
1.2 (I
1.4
)
FIG. 5. Column chromatographic identification of stachyose. Construction of the Dowex-1 column was essentially t.he same as that shown in the upper part of Fig. 4. Sugar-containing fractions obtained in the first run of a Dowex-1 column were concentrated in ~lucuo, applied to a sheet of Whatman No. 1 filter paper as a streak, and subjected to a descending chromatography for 35 hours using n-butanolpyridine-water (80:80:40) as a developing solvent. The area containing stachyose was eluted with hot Hz0 three times, and an aliquot was again applied to a Dowex-1 column (borate form) after passing through Amberlite M B-3 resin. Sucrose (1.0 mg), stachyose (2.0 mg), and fructose (4.5 mg) were added as carriers. Both sugar analysis and radioactivity determination were carried out as before.
the time of sucrose-Cl4 feeding to distinguish the radioactivity incorporation into two nucleotides, but this type of pulse-experiment. could not be employed in t,he rice plant system. DISCUSSION
From the ready transfer of glucose from sucrose-Cl4 t’o the starch molecule through ADPG as demon&abed in cell-free enzyme system of ripening rice grains (1) and maize (4), there is now no doubt that ADPG is the best substrate, superior to UDPG, in the
starch synthetase reaction. However, from some experimental results presented in this report, we cannot entirely exclude the possible role of UDPG in the transformation of sucrose into starch. Information gained by an in vitro experiment may support a view that the UDPG-sucrose transglucosylase functions more readily in the initial step of sucrose breakdown rather than through the ADPG-system. This mechanism is supported by the specific inhibition of the ADPG-sucrose tjransglucosylase by UDP, in agreement with the observation of Cardini and Recondo
FIG. 4. Dowex-1 ion-exchange column chromatographic fraction of acid-soluble compounds isolated from the rice grains fed with sucrose-CLJ. Experimental details of the feeding and the subsequent treatment for extracting the acid-soluble compounds are described in the text. The first fraction containing free sugars was eluted from the column by H20, and was rechromatographed on a Dowex-1 column in a borate form as illustrated in the upper part. Thirty-ml fractions of the effluent were collected. Fractions containing sugar phosphates were eluted from the initial column by 0.005-0.01 N HCl. After concentration of the eluat,es in U~CIIO, about 10 mg each of glucose-l-phosphate, glucose-G-phosphate, and fructose-6-phosphate was added as carrier and was rechromatographed on a Dowex-1 column. Effluent fractions (40 ml) were collected and the sugar content was analyzed (middle part). Nucleotide fractions (10 ml) were directly collected from the initial column startsing from 0.01 IV HCl to 0.01 N NaCl as an eluent, and the absorbancy at 260 mp was measured (lower part). The identification of the individual compounds was based essentially on the method described previously (5, 6). Two ml each of the above effluent components was analyzed for radioactivity in a Packard Tri-Carb liquid scintillation spectrometer. Sugar analyses were carried out using a l.O-ml diquot after the method of Dubois et al. (12).
MUllATA
ET
AL.
1100 s :?OO 640 560
320 240 160 60
20
IO
II
i
30
40
m
IA
% ';; d 0
60
50
ml
J-
5
o
;: 0 p a 0
3
0.5-
HOUR
0.4-
G-l-P
0.3 -
0.2 -
240 160
0.1 60 ,\ 0
IO
20 FRACTION
30
40
50
60
NUMBER
FIG. G. Dowex-1 ion-exchange chromatographic analysis of hexose phosphates produced from sucroseCl4 by a crude rice grain enzyme. The composition of the reaction mixture (in rmoles) was: tris buffer, 14, 10 (1.4 X 105 cpm); ATP, 20; UDP, 12; NaF, 50; and 0.4 ml of (pH 7.4), 100; MgS04 , 30; sucrose-C the enzyme preparation (2.76 mg protein-N/ml) in a total volume of 1.6 ml. After incubation at 3O”C, the reaction was stopped by immersing t,he tube in a boiling water bath, and the clear supernatant fraction was subjected to a Dowex-1 ion-exchange column chromatography for the separation of the individual sugar phosphates. Construction of the Dowex-1 column was essentially the same as that shown in the middle part of Fig. 4. Eluents used were: I, 0.001 M NH40H; II, 0.025 !I4 NH&l, and 0.01 M Na2B40T ; III, 0.025 M NH&l, 0.025 M NH,OII, and 0.001 M NasBnO~ , and IV, 0.025 Jf NH&l, 0.025 M NH,OH, and 0.00001 M Na2B40~
ENZYMIC
MECHANISM
OF
STARCH
SYNTHESIS
(8). In this connection it is of value to point out the fact, t.hat the content of UDP in ripening rice grains was consistently much lower than that of ADP, whereas the UDPG content was about 3-5 times higher than ADPG (5, 6, 15). Alternately one can postulate the compartment.ation mechanism in grain cells enabling the hnrmonious participation of the two nucleot,ides in the sucrose-starch transformation. The results of feeding experiments (Fig. 4) may indicate a concurrent functioning of the ADPG and UDPG pathway in the sucrose-starch conversion in rice grains : Sucrose
.mP, UDP Surrose synthctase
(III)
’ starch
This is, in essence, a mechanism proposed by Leloir (7), in which he stated that the slower rate of the IYDPG-starch transglucosylation reaction as compared with t’hat of ADPGstarch transglucosylase might be well compensated for by the higher UDPG content in plant tissues. Another intriguing mechanism is the one based on Eq,. (IV) :
Sucrose
UDP
VDP -2
r uDPG
-iG-
ADPG i-
RIPENING
RICE
GRAINS
+ starch -I ADP
(IV)
So far, however, no report has been available in literat,ure supporting this energetically feasible mechanism in nature. Assuming the labelling pattern of both ADPG and UDPG as presented in Fig. 4 is based on the reaction IV, we sought in vain for a transglucosylat)ion reaction, UDP-glucose-Cl4 + + UDP, in rice ADP $ ADP-glucose-Cl4 grains, using Cl*-labelled nucleotide sugars. The possibility of the reaction mechanism (Eq. II) proposed by De Fekete and Cardini (4) cannot be excluded, but Dhe major objection to t,his mechanism lies in the fact that the glucose-l-phosphate formation from
43
UDPG by the pyrophosphorylase reaction is physiologically quite unfavorable. As has been emphasized by Kornberg (16), enzymic reactions releasing PPi are considered to be essentially irreversible due to the hydrolysis of PPi, thus making the over-all reaction a synthetic one. This concept is applicable t,o the sucrose-starch conversion mechanism in plants. Consequently the whole mechanism based on either one of I, III, and IV or their combinations, can be summed as Eq. (V): Sucrose
+
2ATP
+
acceptor
+ Hz0 -+ &arch
(G),
(G)n+z
+ 2ADP
Starch synthetase
f-----F 1
IN
07 + 2Pi
n’aturally this mechanism is energetically more efficient than the one involving the invertase reaction (Eq. VI), Sucrose! H,O
g1ucoseATP + __f i fructose 1 ATP
glucose-l-phosphate __f
WI)
ADPG 3 starch,
and plant cells are presumed to utilize the former mechanism to save ATP. The latter mechanism necessitates two additional ATP, which will make the reaction obviously uneconomical from the view of the energy conservation in living cells. Unexpectedly, however, the result’ of feeding experiment shown in Fig. 4 may indicate the possible format,ion of glucose and fructose by invertase reaction. This finding was interesting, because an independent experiment using a crude rice grain enzyme has given a picture indicating the sucrose metabolism through the glycolytic pathway (Fig. 6). The distribution of radioactivit,ies in the hexose phosphat,es fraction as revealed by irz vivo feeding experiment was irregular by comparison with results obtained by in vitro enzyme experiment. In particular, the prominent C14-incorporation into unknown compound(s) from sucrose-W may be a reflection of the more complicat,ed mechanism of the sucrose-starch conversion in intact rice grains. Certainly the more thorough analytical studies are necessary to disclose the picture of carbohydrat,e metabolism in rice.
44
XIURATA REFERENCES
1.
T., STGII’AMA, T., AND AKAZAWA, Hiochem. Biophys. 107, 92 (1964).
MURATA,
ilrch. 2. AKAZAWA,
T.,
Plant
3. FRYDM.AN, 242 (1963). FEKETE,
Awh.
6.
7. 8.
T.,
AND
MuRATA,
Phy.sioZ. 39, 371 (1964). K. B., Arch. Biochem. Biophys.
102,
M. A. R., .\ND CARDINI, C. E., Biochem. Biophys. 104, 173 (1964). MURATA, T., MINAMIKAWA, T., AND iluz.~w.&, T., Riochem. Biophys. Res. Commun. 12, 204 (1963). MURATA, T., MINAMIKAWA, T., AKAZ.I~A, T., AND SUGIYAMA, T., Arch. Biochem. Biophys. 106, 371 (1964). LELOIR, L. F., Biochem. J. 91, 1 (1964). CARDINI, C. E., AND RECONDO, E., Plant Cell Physiol. 3, 313 (1962).
4. DE
5.
T., MlN:\>fIKAWA,
T.,
ET
AL.
9. PAL.IDIKI, A. C., AKD LELOIR, L. F., Biochem. J. 51, 426 (1952). 10. 310~1, Ei., NAK.IX-RA, M., AND Fcsa~as~I, S., Bull. Agr. Chem. Sot. (Japan) 24, 3-M (1960). 11. Moar, Ii., .\XD N.IK.UVIURA, M, KuII. Agr. Chem. Sot. (Japan) 23, 389 (1959). 12. DTBOIS, M., GILLES, K. A., HAXILTOX, J. K., REBERS, P. A., AND SMITH, F., -l~l. Chew. 28, 350 (1956). 13. AT.IG.\D, G., J. Biol. Chem. 239, 3613 (1964). 14. GRDISI, C. E., LELOIR, L. F.,.IND CHIRIBOGA, J., J. Biol. Chem. 214, 149 (1955). 15. MURATA, T., S~GIYAMA, T., AND AK.J.z.\wA, T., Biochem. Biophys. Res. Commun. 18, 371 (1965). lti. KORNBERG, A., in “Horizons in Biochemistry” (&I. Kasha and B. Pullman, eds.), pp. 2512G4. Academic Press, New Turk (1962).
OF
Enzymic
BIOCHEMISTRY
Mechanism
AND
of Starch
III. Mechanism TAKAO
MURATA,2
The International
113,
BIOPHYSICS
34-44
(1966)
Synthesis
in Ripening
of the Sucrose-Starch
TATSUO
SUGIYAMA,3 T. AKAZAWA3
Rice Research
Institute,
Received
May
Grains
MINAhIIIKAWA,3
AND
Conversion’
TAKAO
Los BaAos,
Rice
Laguna,
The Philippines
12, 1965
In a coupling system of sucrose-synthetase and starch-synthetase, both isolated from the ripening rice grains, the more efficient transfer of glucose-Cl4 from sucroseCl4 to starch occurred in the presence of ADP as compared to UDP, indicating the predominant role of ADPG in the process. However, UDP was found to inhibit profoundly the ADPG-mediated glucose-C I4 transfer to starch from sucrose-CL”, which was based on its specific inhibition of the ADPG-sucrose t,ransglucosylation reaction. The Km value of sucrose synt,hetase was determined to be 1.5 X 1OW M (ADP) and 1.1 X lo+ M (UDP), respectively. These findings may support a view that the sucrose breakdown proceeds through the reversal of UDPG-sucrose transglucosylation rather than directly by way of the ADPG-sucrose transglucosylation. By feeding the rice grains at the mid-milky stage with sucrose-W, incorporation of the radioactivit,y into the starch fraction was observed, although most of the radioactivity in the acidsoluble fraction was recovered as sucrose. It was noted also that a relatively high radioactivity was detected in glucose and fructose. The formation of a prominent radioactive compound(s) emerging around the fraction of glucose-6-phosphate was demonstrated in a Dowex-1 anion-exchange column chromatography; neither its chemical nature nor the role in the sucrose-starch conversion has been revealed. Both ADPG and UDPG became labeled to a much lesser extent than the above conpounds under the experimental conditions employed. Attempts to dist,inguish their predominant role in the biosynthetic conversion of sucrose to starch were msmcessful, although the specific radioactivity of ADPG was about twice as high as t,hat of UDPG. Based on t,he experimental results obtained, possible enzymic mechanisms underlying the sucrose-starch conversion in developing rice grains are discussed.
In a previous paper of this series (I), evidence was given for t’he dominant role of ADPG in sucrose-starch conversion in ripening rice grains. When coupled with either ADPG-sucrose transglucosylase (sucrose synthetase) or ADPG-pyrophosphorylase, bobh residing in the soluble fraction of the grain extract, ADPG-starch transglucosylase (starch synthetase) utilized both sucrose-Cl4 and glucose-l-phosphate-Cl4 as efficient glu-
cose donors in starch format,iou. On the other hand, the corresponding UDPG system appeared to operate much less efficiently in the process [see reference (2)]. The overall mechanism proposed therein was Eq. (I) :
1 Issued as I.R.R.I. Journal Series No. 48. 2 Present address: Tohoku National Agricultural Experiment Station, Morioka, Japan. 3 Address to which requests for reprints should be sent: Nagoya University, Anjo, Aichi, Japan.
The important role of ADPG in starch synthesis has been confirmed by other workers using different plant, materials (3, 4). The question remains, however, as to the role of
ADP
Sucrose Sucrose
synthetase
’ ADPG
(I) Starch
34
synthetase
L starch
ENZYMIC
MECHANISM
OF
STARCH
SYNTHESIS
UDPG in the carbohydrate metabolism in rice grains. Its participation in the sucrosesbarch conversion mechanism cannot be ignored completely, especially in view of the facts that: (i) t,he content of UDPG in ripening rice grains was much higher than that of ADPG (5, ci), and (ii) glucose transfer from UDPG to starch molecule occurred at a reasonable rate, although the rate was lower than that from ADPG (1). These two points have also been found to be true in some other plants, and Leloir (7) has presented a view that both the ADPG and UDPG pathways may operat’e concurrently in the starch synthesizing mechanism. On the other hand, De Fekete and Cardini (4) have postulat’ed that UDPG may have a primary role in the initial step of the sucrose-starch transformation, whereas &arch synthesis is exclusively catalyzed by the ADPG-starch transglucosylase. This concept has been based on the smaller Michaelis constant (Km) of the sucrose synthet,ase toward UDP as compared to ADP, and the specific inhibition of the ADPG-sucrose transglucosylase by UDP (8). Consequently the following mechanism, Eq. (II), has been presented by them: UDP
Sucrose
’
Sucrose-synthetase PPi
glucose-lphosphate
UDPGpyrophosphorylase
(II)
ATP
--
ADPG-pyrophosphorylase Starch
UDPG
synthetase
’
ADPG
’
starch
In the present experiment, we have atOempted to determine the crucial role of UDPG in the sucrose-starch conversion mechanism in ripening rice grains and have used both a cell-free enzyme system and sucrose-CWfeeding bechnique. Attention has been given in t’rying to visualize the regulatory mechanism underlying the interaction of two different nucleoside diphosphate sugars in starch synthesis. MllTERIALS Plants. Peta, used as in the
AND
METHODS
an indica type rice previous experiment
variety, (1, 2).
was The
IN
RIPENING
RICE
GRAINS
33
growing of the plants and the subsequent sampling of rice grains were essentially the same as described before. Preparation of enzymes. (a) ADPG-starch transglucosylase: Acetone powder starch granules were prepared by the same method as reported previously (1). (b) ADPG (UDPG).sucrose transglucosylase (sucrose synthetase) : A crude enzyme preparation obtained by (NHa)&Od precipitation of the grain extract showed a strong activity in sucrose synthesis (2)) and a partial purification of the enzyme was undertaken as follows. Freshly harvested rice grains (240 gm) were ground in a chilled mortar with 300 ml of 0.05 M phosphate buffer (pH 7.0) and strained through cheese cloth, and the filtrate was centrifuged at 25,000g for 15 minutes. To the supernatant solut,ion (about 150 ml) was added solid (NHd)#Od to 50% saturation, and the mixture was centrifuged again. The resulting precipitate was dissolved in 20 ml of water and dialyzed against 8 liters of water overnight in a cold room (2°C). To the dialyzate was added 0.06 volume of 1 ,V MnC12 , and the mixture was allowed to stand for 30 minutes and then centrifuged. The supernatant fraction was fractionated by adding (NH,)zSOd (30-507, saturation), and the precipit,ate was dissolved in G ml water after centrifugation. After dialysis against water for 1.5-2 hours, 4.0 ml of Ca-phosphate gel (about 20 mg per milliliter) was added to the clear solution and the material was allowed to stand for 30 minutes. To the supernatant fraction was added ?/4 volume of 0.02 M tris buffer (pH 7.5) and the resulting solution was applied to a column of DEAE-cellulose (1.1 X 9.0 cm), which was preequilibrated with 0.005 M tris buffer (pH 7.5). The protein was fractionated by 0.005 tris-NaCl as eluent, the concentration of the latter component increasing stepwise: 0, 0.05, 0.10, 0.15, 0.20, and 0.30 Jf, respectively. To 40 ml eluate as fractionated by 0.005 M tris- 0.15 llil NaCl (measured by t,he optical densit,y at 280 mp) was added solid (NH&SO4 to make a 50yc saturation, and the resulting precipitate was dissolved in a small quantity of water. Bfter dialysis against water for 2 hours, the final solution was used as a sucrosesynthetase preparation. This enzyme preparation was found to be devoid of ADPG (UDPG)-pyrophosphatase activity, and the specific activity (micromoles sucrose formed per 10 minutes per milligram protein-N) was about 10 times as high as that of the preparation obtained at the initial (NH,)&04 precipitation. (c) Crude soluble enzyme preparation for the sucrose-transformation experiment: This was prepared by essentially the same method as that for the hexokinase of rice grains (2). A precipitable fraction obtained by
2%
RIURATA
the 50-60’;; (NH,)zSOI saturation of the rice grain extract in 0.1 M tris buffer (pH 7.5) was used as the source of enzyme. Enzylne assay methods. (u) Sucrose-starch conversion: Glucose transfer from sucrose-W to the starch molecule was determined in the reaction mixture containing both granular starch synthetase and soluble sucrose synthetase. Modification of the reaction system is described in each case. (b) ADPG (UDPG)-sucrose transglLcosylase : The reaction system was designed so as to determine the rate of sucrose breakdown in t,he presence of either ADP or UDP. The formation of radioactive ADPG or UDPG from sucrose-C’” in the presence of various amount,s of either ADP or UDP was quantitatively assayed by means of paper chromatography. All the enzymic experiments u-ere duplicated aud average values are present,ed. (c) Transformation of sucrose by a crude enzyme system: Breakdown of sucrose-C11 by the reversal of sucrose-synthetase followed by the metabolism of the hexose unit, through the glycolgtic enzyme system was examined by the Dowes-1 ion-eschange column chromatographic separation of individual hexose phosphates and their radioassay. The method was essentially the same as that reported previously (2). Sucrose-C14 feeding experiment and Dowex-1 anion-exchange column chromatography. Panicles of rice plants at the mid-milky st,age were inserted for 30-90 minutes into a test tube cont,aining 2.0 ml of 0.01 M sucrose-C14 (2.44.1 X 10’ cpm). During the incubat,ion period almost all the sucrose solution was absorbed by the plants. The grains were immediately stripped off, combined with about) 30 gm of freshly harvested grains from intact plant,s, and ground with 0.6 S ITClOa in a chilled mortar. The extraction was repeated two more times in the same mauner. The total volume of the extract was usually about 200 ml. The insoluble residual material was washed thoroughly three times with Hz0 and two times more with acetone. A subsample of the dried residue was used for the radioassay of the starch fraction. The total perchloric acid extract, contaiuing free sugars, sugar phosphates, and nucleotides, was carefully neutralized with 10 N KOII to pH 6.0, aud t,he precipitated KCIOl was removed by centrifugation after placing in a deep freezer. The supernatant fraction was concentrated in z’aczlo at 30”-34°C and centrifuged again, the final volume being usually about 10 ml. It was then applied to a column of Dowex-1 anion-exchange resin (AG 1 X 8, chloride form, 200400 mesh, Calbiochem.) and chromatographed by the method reported previously (5, 6), which is a modification of the method used by Nakamura’s group (10, 11).
ET
AL.
Identification of stachyose. Rice grains (18.7 gm) fed wit,h sucrose-C” (3.5 X 10’ cpm) for 90 minutes were the starting material. Separation of sugars by ion-exchange column chromatography combined with paper chromatograp1l.v was used for the identificatiork of stachgose. fleagents. The sources of the rcagerlts were as follows : ATP, Sigma Chemical Company; ADP, National Biochemicals Corporation; AlJPG, UDP, UTP, UJIPG, sucrose-C’4, glucose-l-phosphate, glucose-&phosphate, and frlictose-(i-phosphate, California Corporation for Biochemical ltesearch. Both UDl’-glucose-C’” alrd ADP-glucose-C” were prepared by the sucrose-synthetase isolated from rice grains by the method reported previously (1). Stachvose was a crift of Dr. %. Nikllni nod Dr. R. S. Schallenberger. RESULTS
conversion in cell-free ensystem. E’igure 1 shows c*learly t,hat
Sucrose-starch xyuze
I
0
10 SUCROSE
20 S&THETA&-
30
40 ( pgPROTEIN-N)
50
FIG. 1. Glucose-Cl4 transfer from sucrose-Clf into st,arch in the coupling system of sucrosesynthetase [ADPG (UDPG)-sucrose transglucosylase] and starch synthetase [ADP(; (UDPG)starch transglucosylasel. Preparative method of the individual enzymes is described in the text. Standard reaction mixture contained (in pmoles) : t,ris buffer (pH 7.4), 4.0; EDTA, 0.2; NaF, 2.0; sucrose-W, 0.3 (10.1 X lo4 cpm); ADP (UDP), 0.25; starch granules, 6.0 mg; and varying quantities of sucrose synthetase in a total volume of 25bl. Incubation was at 37°C and the radioactivity measurement of the starch molecule was carried out as reported previously (1, 2).
ENZYblIC
MECIIANISM
OF
STARCH
SYNTHESIS
IN
RIPENIKG
RICE
GRAINS
37
understood from the following observations. That is, the reaction mixture as used for experiment shown in the figure, only omitting starch granules, was examined for t.he paper chromatographie deeecOion of the nucleotide sugars formed. It was found that ADP-glucase-Cl4 was scarcely detectable in t,he system containing both ADP and UDP (the ratio 1: 34 and 1: l), while a small amount of this compound was formed in a system containing the above two nucleotides at the ratio of 1:,$s5. In the former two cases, a marked formation of UDP-glucose-C14 was demonstrated, whereas the magnitude of its formation was much less in the lntt.er case. The inhibitory effect of UDP in the ADPGUDP sucrose transglucosylase was originally reported by Cardini and Recondo (8), who used wheat germ enzyme, and later in more 60 120 180 0 detail by the same workers using maize enTIME IN MINUTES zyme (4). FIG. 2. Interaction of UDP and ADP in the Km values of sucrose-synthetase to ADP and glucose-W transfer from sucrose-Cl* into starch UDP. In an attempt to determine whether in the coupling syst,em of sucrose-synthetase and there exists any preferential utilization by starch synthetase. Experimental conditions were the rice enzyme for ADP or UDP, Kv2 values the same as that used in experiment described in of the partia,lly purified sucrose-synthetase Fig. 1, except that various amounts of UDP as toward each of the two nucleot,ides were indicated in the figure were added. The content measured. As represented in Figs. 3a and 3b, of protein-N of sucrose synthetase was 25 pg. the Km value of the enzyme for ADP was glucose transferred readily from sucrose to determined to be 1.5 X 10e3 31, while that starch in the presence of ADP. The rate of for UDP was 1.1 X 1O-4 M. The Kv2 values reaction was much higher than that via the of the maize enzyme reported by the Argencorresponding UDPG system, in which ADP tine workers to each of ADP and UDP were was replaced by UDP. This result is basically 2.1 X 10e3 M and 6 X 10V5 M, respectively in agreement wit,h our previous finding (1). (4). Recently, Avigad (13) isolated a highly With ADP, the magnitude of the glucose purified UDPG-sucrose transglucosylase transfer from sucrose-C’* to the starch molefrom Jerusalem artichoke, and the Km to cule increased proportionally with increasing UDP was determined to be 3.2 X low4 M. amounts of sucrose synthet’ase; no such effect Thus, our above result may support the was observed in the utilization of sucrose-Cl4 view that sucrose breakdown proceeds via the UDPG system. Although these obthrough the reversal of UDPG-sucrose trans.servations may suggest the efficient coupling glucosylation rather than directly by way of of ADPG-sucrose transglucosylation with the ADPG-sucrose transglucosylation reacthe starch synthetase reaction in the over-all tion. process of sucrose-starch conversion, t>he On the other hand, the measurement of actual situation was formd to be more comthe apparent equilibrium constant of the plicated. Thus, as seen in Fig. 2, the addition sucrose synthetase of rice grains gave the of UDP to the reaction mixture of the ADPG following values in t,riplicate experiments; system markedly inhibited the glucose transfer from sucrose to starch. The fact that this (ADP) (sucrose) inhibition lies in the effect of UDP on the (ADPG) (fructose) ADPG-sucrose transglucosylase, but not on = 1.6 (1.1, 1.8, 1.9) the ADlY-starcah transglucosylase, can be
AD4+,yg5)
,:,,::
1
3x (UDP) (sucrose) K’ eg (UDPG)(fructose)
h’IUIiATA
1
ET
AL.
400 -
= 3.3 (3.0, 3.0,3.9). Although it is difficult to evaluate the physiological significance of such values, one can speculate about the specific role of ADPGsucrose transglucosylase in the sucrosestarch conversion, which is apparently a contradictory view with the above postulation. Avigad (13) report’ed the value of K’eq [(sucrose) (UDP)/(UDPG) (fructose)] = 1.4 to 1.8 (pH 7.6) by the enzyme isolated from Jerusalem artichoke, while the one reported by Cardini et al. (14) was 2.0. As an alternate approach, we turned to sucrose-CWfeeding experiments. A major purpose was to see whether there is a predominant incorporation of the glucose molecule from sucrose-Cl4 to either ADPG or UDPG. Sucrose transformation in intact rice grains. Feeding of rice panicles at. the milky stage with sucrose-C14 for 30-90 minutes showed a measurable incorporation of the radioactivity into starch molecules (about 052.0%) the sucrose-starch (Table I), indicating t,ransformat,ion t,o be a relatively rapid met’abolic process. This reflects a vigorous rate of &arch synthesis in rice grains at this specific developmental stage [cf. Fig. 1 of (a)]. Figure 4 shows a typical pattern of radioactivitv distribution in the individual fractions,“i.e., free sugars, sugar phosphates, and nucleot,ides. Sucrose was the major radioact,ive compound in the free sugar fractions (2.0 X lo6 cpm), making up more than 90% of the tot,al acid-soluble fraction (see column 5 of Table I). It, is notable, however, t,hat a measurable radioactivity was observed in both the glucose (5.1 X lo4 cpm) and fructose (4.0 X lo4 cpm) fractions. This was interesting because Mori and Nakamura (11) have reported that the careful pretreatment of plant extracts by Amberlite IR-120 and -4B resins, before applying to a Dowex-1 anion-exchange column, resulted in only about, 0.2 % hydrolysis of the sucrose. In the present chromatogram, the high percentage of radioactivity recovered in each of glucose and fruct,ose fractions, 2.4 and 2.0 %, respectively, per total cpm of acid-soluble
I 0
25
50 mM
75
100
125
ADP
-
2500,
02.5
0.5 mM
075
IO
125
UDP
FIG. 3. Determination of the Michaelis constant (Knz) of sucrose-synthetase toward ADP (a) and UDP (b). The reaction misture consisted of the following (in rmoles): tris buffer (pH 7.4), 4.0; EDTA, 0.2; NsF, 2.0; sucrose-(Y4, 1.0 (9.3 X lo* cpm) (ADPG-system) and 0.1 (5.5 X 104 cpm) (UDPG-system); various amounts of ADP or UDP; and sucrose synthetase in a total volume of 20 ~1. After incubation at 37°C for 10 minutes, the reaction was stopped by adding 30 ~1 of 80% ethanol, and a 30.~1 aliquot of the clear centrifugate was applied to a sheet of acid-washed Whatman No. 1 paper for paper chromatography. The solvent system used was ethanol-ammonium acetate (pH 3.8) of Paladini and Leloir (9). The areas corresponding to ADPG and UDPG were eluted with H,O and filled up to 0.4 ml, and their radioactivity was determined in a Tri-Carb liquid scintillation spectrometer. The contents of protein-N of sucrose synthetase were 1.76 pg for ADP system and 0.26 pg for UDP system.
ENZYMIC
MECHANISM
~JTILIZATION Experiment
Feeding time (min) Grains weight (pm) Total radioactivity Cl4 administered Total radioactivity (cpm) Tc Incorporation Total radioactivity soluble fraction 7. Incorporation Total radioactivity fraction (cpm) ~___
OF
OF
SUCROSE-W
:
in free
IN
SYNTHESIS TABLE
I
RICE
GRAINS
1
of sucrose(cpm) in starch
in (cpm)
STARCH
acid-
sugar
X 9.6 -
BY
RIPENING
FEEDING
2
30 11.8 2.4 X lo7
2.3
IN
lo6
4
107
60 4.4 2.4 X lo7
4.1
x
106
1.1
3.8
1.7 x
106
3.1
fraction, indicates a possible enzymic hydrolysis of the sucrose by invertase or some other enzymes in rice grains. Two small unknown radioactive sugar fractions were also detected, one emerging just after the sucrose fraction (I) and the other preceding the fructose fraction (II). Assuming each of these unknown sugar fractions to be raflinose and stachyose, respectively, we attempted their identification. However, by repeated chromatographic analyses of the radioactive free sugar fractions, obtained by the sucrose-C’4 feeding, the formation of the first fraction (I) was rather inconsistent. On the other hand, as represented in Fig. 5, paper chromatography and Dowex-I. ion-exchange column chromatography showed the second sugar fraction (II) to be stachyose. It is noteworthy that, the relative specific radioactivity (cpm/OD 490 rnp) of st,achyose (6.7 X 104) and the fraction I (8.9 X 104) was much higher than that, of sucrose and two hexoses, and their role in sucrose metabolism of rice merits further attention. The total radioactivity in the remainder of the acid-soluble compounds comprised a small fraction of the administered sucrose04. This fractJion was separated into sugar phosphates and nucleotides. As can be seen in Fig. 4, the intensity of the radioactivity in both glucose-l-phosphate and fructose-6phosphate was not very high; instead there was a broad and prominent radioactive compound(s) designated as “X,” emerging around the position of glucose-6-phosphate.
15.8 X lo6
2.9
x
12.9 x
106
5
GO 14.7 2.4 x
105
0.5 x 106
39
GRAINS
EXPERIMEXTS
3
60 17.8 2.4 x
3.2
RICE
10’
-
3.1
90 7.G 4.1 x 10’ 2.4
x
106
12.9 -
x
105
2.1
0.F x 106
2.09
5.1 x 10”
The measurement of radioactivitv in this fraction did not give a good co&idence curve with that of the calorimetric analysis of glucose-6-phosphate added as a carrier. Attempts to reveal the nature of this compound(s) have been unsuccessful. However, the following compounds have been excluded from the list of the possible shructures based on the ion-exchange column chromatography, i.e., sucrose phosphate, G-phosphogluconate, ribose&phosphate, 3-phosphoglyceraldehyde, and dihydroxyacetone phosphate. It is unknown whether this compound(s) occupies a key role in the sucrosestarch conversion or is merely formed as a by-product of the main path of sucrose transformation in rice grains. The bottom of Fig. 4, representing the radioactivity pattern in the nucleotide fraction, shows that’ ADI’G and CDPG became labelled to roughly t’he same extent within a 90-minute incubation. By t,he closer examination the specific radioactivity (cpm,/ /Imole) of the ADPG (570) was about t,wice as that, of UDPG (270), but it is nat,urally difficult from such figures to assess the predominant precursor role between these two nucleot,ide sugars in starch synthesis. Moreover, on the basis of the current cboncept, of the starch biosynthesis via, nucleotide sugar pat,hway,
the
magnitude
of
the
lshelling
in
t,hese compounds was much smaller than that in both free sugars and sugar phosphat,es, and even as compared with that in the starch fraction. It is desirable t)o shorten
I
I.1I-
0.00SMNaia,9+0,+
+
0.02YNq40,
0.03YNo2D,
0,
elucou suemu
1 I.0 i P 0 d
IO.0 8.0
6.0
% h
0.5
4.0
I g d IJ
2.0
c
SUGAR
PHOSPHATES
10.0
1.0
i B f Ii d
8.0
“0 *
4.0
Ii d 6
0.5
2.0
0
I
-z
IO
20
SO
40
0.4
iI d 6 0.2
0
50
200
loo FRACTION
NUYDCR
FIG. 40
4
%
6.0
ENZYMIC
MECHANISM
OF
l -
3.30
STARCH
1 M
SYNTHESIS
Na&,O,
------+
04 ELUATE
0.6
Cl8
THROUGH
RIPENING
0.02MNa2B44
Stachyose
a2
IN
RICE
GRAINS
41
4
i
1.0 COLUMN
1.2 (I
1.4
)
FIG. 5. Column chromatographic identification of stachyose. Construction of the Dowex-1 column was essentially t.he same as that shown in the upper part of Fig. 4. Sugar-containing fractions obtained in the first run of a Dowex-1 column were concentrated in ~lucuo, applied to a sheet of Whatman No. 1 filter paper as a streak, and subjected to a descending chromatography for 35 hours using n-butanolpyridine-water (80:80:40) as a developing solvent. The area containing stachyose was eluted with hot Hz0 three times, and an aliquot was again applied to a Dowex-1 column (borate form) after passing through Amberlite M B-3 resin. Sucrose (1.0 mg), stachyose (2.0 mg), and fructose (4.5 mg) were added as carriers. Both sugar analysis and radioactivity determination were carried out as before.
the time of sucrose-Cl4 feeding to distinguish the radioactivity incorporation into two nucleotides, but this type of pulse-experiment. could not be employed in t,he rice plant system. DISCUSSION
From the ready transfer of glucose from sucrose-Cl4 t’o the starch molecule through ADPG as demon&abed in cell-free enzyme system of ripening rice grains (1) and maize (4), there is now no doubt that ADPG is the best substrate, superior to UDPG, in the
starch synthetase reaction. However, from some experimental results presented in this report, we cannot entirely exclude the possible role of UDPG in the transformation of sucrose into starch. Information gained by an in vitro experiment may support a view that the UDPG-sucrose transglucosylase functions more readily in the initial step of sucrose breakdown rather than through the ADPG-system. This mechanism is supported by the specific inhibition of the ADPG-sucrose tjransglucosylase by UDP, in agreement with the observation of Cardini and Recondo
FIG. 4. Dowex-1 ion-exchange column chromatographic fraction of acid-soluble compounds isolated from the rice grains fed with sucrose-CLJ. Experimental details of the feeding and the subsequent treatment for extracting the acid-soluble compounds are described in the text. The first fraction containing free sugars was eluted from the column by H20, and was rechromatographed on a Dowex-1 column in a borate form as illustrated in the upper part. Thirty-ml fractions of the effluent were collected. Fractions containing sugar phosphates were eluted from the initial column by 0.005-0.01 N HCl. After concentration of the eluat,es in U~CIIO, about 10 mg each of glucose-l-phosphate, glucose-G-phosphate, and fructose-6-phosphate was added as carrier and was rechromatographed on a Dowex-1 column. Effluent fractions (40 ml) were collected and the sugar content was analyzed (middle part). Nucleotide fractions (10 ml) were directly collected from the initial column startsing from 0.01 IV HCl to 0.01 N NaCl as an eluent, and the absorbancy at 260 mp was measured (lower part). The identification of the individual compounds was based essentially on the method described previously (5, 6). Two ml each of the above effluent components was analyzed for radioactivity in a Packard Tri-Carb liquid scintillation spectrometer. Sugar analyses were carried out using a l.O-ml diquot after the method of Dubois et al. (12).
MUllATA
ET
AL.
1100 s :?OO 640 560
320 240 160 60
20
IO
II
i
30
40
m
IA
% ';; d 0
60
50
ml
J-
5
o
;: 0 p a 0
3
0.5-
HOUR
0.4-
G-l-P
0.3 -
0.2 -
240 160
0.1 60 ,\ 0
IO
20 FRACTION
30
40
50
60
NUMBER
FIG. G. Dowex-1 ion-exchange chromatographic analysis of hexose phosphates produced from sucroseCl4 by a crude rice grain enzyme. The composition of the reaction mixture (in rmoles) was: tris buffer, 14, 10 (1.4 X 105 cpm); ATP, 20; UDP, 12; NaF, 50; and 0.4 ml of (pH 7.4), 100; MgS04 , 30; sucrose-C the enzyme preparation (2.76 mg protein-N/ml) in a total volume of 1.6 ml. After incubation at 3O”C, the reaction was stopped by immersing t,he tube in a boiling water bath, and the clear supernatant fraction was subjected to a Dowex-1 ion-exchange column chromatography for the separation of the individual sugar phosphates. Construction of the Dowex-1 column was essentially the same as that shown in the middle part of Fig. 4. Eluents used were: I, 0.001 M NH40H; II, 0.025 !I4 NH&l, and 0.01 M Na2B40T ; III, 0.025 M NH&l, 0.025 M NH,OII, and 0.001 M NasBnO~ , and IV, 0.025 Jf NH&l, 0.025 M NH,OH, and 0.00001 M Na2B40~
ENZYMIC
MECHANISM
OF
STARCH
SYNTHESIS
(8). In this connection it is of value to point out the fact, t.hat the content of UDP in ripening rice grains was consistently much lower than that of ADP, whereas the UDPG content was about 3-5 times higher than ADPG (5, 6, 15). Alternately one can postulate the compartment.ation mechanism in grain cells enabling the hnrmonious participation of the two nucleot,ides in the sucrose-starch transformation. The results of feeding experiments (Fig. 4) may indicate a concurrent functioning of the ADPG and UDPG pathway in the sucrose-starch conversion in rice grains : Sucrose
.mP, UDP Surrose synthctase
(III)
’ starch
This is, in essence, a mechanism proposed by Leloir (7), in which he stated that the slower rate of the IYDPG-starch transglucosylation reaction as compared with t’hat of ADPGstarch transglucosylase might be well compensated for by the higher UDPG content in plant tissues. Another intriguing mechanism is the one based on Eq,. (IV) :
Sucrose
UDP
VDP -2
r uDPG
-iG-
ADPG i-
RIPENING
RICE
GRAINS
+ starch -I ADP
(IV)
So far, however, no report has been available in literat,ure supporting this energetically feasible mechanism in nature. Assuming the labelling pattern of both ADPG and UDPG as presented in Fig. 4 is based on the reaction IV, we sought in vain for a transglucosylat)ion reaction, UDP-glucose-Cl4 + + UDP, in rice ADP $ ADP-glucose-Cl4 grains, using Cl*-labelled nucleotide sugars. The possibility of the reaction mechanism (Eq. II) proposed by De Fekete and Cardini (4) cannot be excluded, but Dhe major objection to t,his mechanism lies in the fact that the glucose-l-phosphate formation from
43
UDPG by the pyrophosphorylase reaction is physiologically quite unfavorable. As has been emphasized by Kornberg (16), enzymic reactions releasing PPi are considered to be essentially irreversible due to the hydrolysis of PPi, thus making the over-all reaction a synthetic one. This concept is applicable t,o the sucrose-starch conversion mechanism in plants. Consequently the whole mechanism based on either one of I, III, and IV or their combinations, can be summed as Eq. (V): Sucrose
+
2ATP
+
acceptor
+ Hz0 -+ &arch
(G),
(G)n+z
+ 2ADP
Starch synthetase
f-----F 1
IN
07 + 2Pi
n’aturally this mechanism is energetically more efficient than the one involving the invertase reaction (Eq. VI), Sucrose! H,O
g1ucoseATP + __f i fructose 1 ATP
glucose-l-phosphate __f
WI)
ADPG 3 starch,
and plant cells are presumed to utilize the former mechanism to save ATP. The latter mechanism necessitates two additional ATP, which will make the reaction obviously uneconomical from the view of the energy conservation in living cells. Unexpectedly, however, the result’ of feeding experiment shown in Fig. 4 may indicate the possible format,ion of glucose and fructose by invertase reaction. This finding was interesting, because an independent experiment using a crude rice grain enzyme has given a picture indicating the sucrose metabolism through the glycolytic pathway (Fig. 6). The distribution of radioactivit,ies in the hexose phosphat,es fraction as revealed by irz vivo feeding experiment was irregular by comparison with results obtained by in vitro enzyme experiment. In particular, the prominent C14-incorporation into unknown compound(s) from sucrose-W may be a reflection of the more complicat,ed mechanism of the sucrose-starch conversion in intact rice grains. Certainly the more thorough analytical studies are necessary to disclose the picture of carbohydrat,e metabolism in rice.
44
XIURATA REFERENCES
1.
T., STGII’AMA, T., AND AKAZAWA, Hiochem. Biophys. 107, 92 (1964).
MURATA,
ilrch. 2. AKAZAWA,
T.,
Plant
3. FRYDM.AN, 242 (1963). FEKETE,
Awh.
6.
7. 8.
T.,
AND
MuRATA,
Phy.sioZ. 39, 371 (1964). K. B., Arch. Biochem. Biophys.
102,
M. A. R., .\ND CARDINI, C. E., Biochem. Biophys. 104, 173 (1964). MURATA, T., MINAMIKAWA, T., AND iluz.~w.&, T., Riochem. Biophys. Res. Commun. 12, 204 (1963). MURATA, T., MINAMIKAWA, T., AKAZ.I~A, T., AND SUGIYAMA, T., Arch. Biochem. Biophys. 106, 371 (1964). LELOIR, L. F., Biochem. J. 91, 1 (1964). CARDINI, C. E., AND RECONDO, E., Plant Cell Physiol. 3, 313 (1962).
4. DE
5.
T., MlN:\>fIKAWA,
T.,
ET
AL.
9. PAL.IDIKI, A. C., AKD LELOIR, L. F., Biochem. J. 51, 426 (1952). 10. 310~1, Ei., NAK.IX-RA, M., AND Fcsa~as~I, S., Bull. Agr. Chem. Sot. (Japan) 24, 3-M (1960). 11. Moar, Ii., .\XD N.IK.UVIURA, M, KuII. Agr. Chem. Sot. (Japan) 23, 389 (1959). 12. DTBOIS, M., GILLES, K. A., HAXILTOX, J. K., REBERS, P. A., AND SMITH, F., -l~l. Chew. 28, 350 (1956). 13. AT.IG.\D, G., J. Biol. Chem. 239, 3613 (1964). 14. GRDISI, C. E., LELOIR, L. F.,.IND CHIRIBOGA, J., J. Biol. Chem. 214, 149 (1955). 15. MURATA, T., S~GIYAMA, T., AND AK.J.z.\wA, T., Biochem. Biophys. Res. Commun. 18, 371 (1965). lti. KORNBERG, A., in “Horizons in Biochemistry” (&I. Kasha and B. Pullman, eds.), pp. 2512G4. Academic Press, New Turk (1962).