Enzymic Mechanism Starch Synthesis Ripening …...Enzymic Mechanism of Starch Synthesis in Ripening Rice Grains1 2 T. Akazawa, T. Minamikawa, and T. Murata TheInternational Rice Research

Enzymic Mechanism of Starch Synthesis in Ripening Rice Grains 1 2T. Akazawa, T. Minamikawa, and T. Murata

The International Rice Research Institute, Los Banos, Laguna, The Philippines 3

It has long been believed that phosphorylase isinvolved in the synthesis of starch, which constitutes70 to 80% of the mature rice grain. Aimi and hisassociates (1, 2, 3) found that during the ripeningprocess a parallel relationship exists between starchsynthesis and phosphorylase activity in rice grains.But we can not exclude the possible existence ofmechanisms other than phosphorylase reaction forstarch formation. The high ratio of inorganic phos-phate to sugar phosphate in plants, which is unfavor-able to starch formation (1, 10, 31), raises doubtsabout the role of phosphorylase in polysaccharidesynthesis.

An important development in the field of poly-saccharide biochemistry has been the recent dis-covery of starch synthetase by Leloir's group (8, 14),which proposed the following enzymic reaction:UDPG + acceptor (G)n ->

UDP + starch (G)n+1. I

Many workers have accepted this view (23, 27) andin contrast, have relegated the role of phosphorylaseto the breakdown of polysaccharides (8, 36).

In some plants, including rice, sucrose is synthe-sized in the leaves and stems, and is translocated tothe grains where it eventually is transformed intostarch (12, 25, 26, 35). In assigning starch synthe-tase to its role in starch synthesis, the connection be-tween this enzymic mechanism and sucrose metabolismshould be recognized. Enzymic evidence pertainingto sucrose-starch interconversion has not been re-

ported, but since sucrose synthesis is catalysed bysucrose synthetase (6, 22), the possible existence ofa link between the 2 reactions involving UDPG isworth exploration. The present investigation is pri-marily concerned with the study of starch synthetasein ripening rice grains, and secondly, with experi-ments on sucrose synthetase as a possible clue to themechanisms of sucrose-starch interconversion.

Materials and MethodsGrowing and Sampling of Rice Plants. Peta, an

1 Received July 8, 1963.2 Submitted by the International Rice Research Insti-

tute as IRRI Journal Series No. 13.3 Mailing Address: The International Rice Research

Institute, Manila Hotel, Manila, The Philippines.Abbreviations: ADPG, adenosine disphosphate glu-

cose; UTP, uridine-5'-triphosphate; UDP, uridine-5'-diphosphate; UDPG, uridine diphosphate glucose.

371

indica variety, and Chianung 242, a japonica variety,were used. Plants were grown in soil in a greenhouse. Peta and Chianung 242 flowered 4 monthsand 3 months, respectively, after planting. Paniclesat the same developmental stage were taken from dif-ferent pots. The grains were stripped off the pani-cles, mixed thoroughly, and a subsample removed foranalysis of sugar components in the grains. For theenzymic study, panicles with grains in the midmilkstage (usually 10-15 days after pollination) werechosen.

Sugar Analyses. A certain amount of fresh wholerice grains including seedcoat was first heated at 900for 30 minutes, then dried at 600 for 4 hours and thedry weight determined. The dried grains wereground with a Wiley mill (40 mesh). Usually, a3-g sample of the powdered grain was continuallyextracted with 95% ethanol, and the residual matter(starch) was hydrolyzed with 2% HCl for glucoseanalysis. The ethanol extract was concentrated in aflash evaporator, treated with lead acetate, and finallydeionized with a small amount of Amberlite IR-120and IR-4B ion-exchange resins. It was then madeup to a 20-ml volume, and aliquots were analyzed forthe total sugar content (9) and for reducing sugars(33). The amount of nonreducing sugar was cal-culated from the difference of the above 2 values. Forfurther column chromatographic analyses of sugarcomponents, the method of Mori and Nakamura (18)was employed.

Enzymic StudiesStarch Synthetase. This enzyme was prepared

using 15 g of the immature rice grains (14). Theaverage total nitrogen content was 7 to 8 Lg per milli-gram of starch for the 2 varieties. Total and proteinnitrogen were determined by nesslerization. Starchgranules prepared in this manner were kept in a deepfreeze (-20°) without much loss of activity over a2 month period. The enzyme assay was a modifiedmethod of Leloir et al. (14); a standard incubationmixture contained 6,moles of glycine buffer (pH8.4), 0.2 ,umole of EDTA, 0.3 ,.umole of UDPG, 0.4iLmole of glucose-6-P (when added) in a total volumeof 17 1.l and with stated amounts of starch granules.The mixture was then incubated at 370 for varyingperiods of time. For longer incubation periods, thetest tubes were stoppered to prevent evaporation.After the incubation, the UDP formed was spectro-photometrically assayed (520 mg) by the pyruvic

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PLANT PHYSIOLOGY

phosphiokinase system which is a miiodification of the-metlio(I of Leloir aIndI Goldenmherg (15).

For the isotope experinments, UDP-glucose-C14was diluted twofold with a 6 x 10-2 NI UDPG solu-tion, andl 0.18 to 0.24 /mnole was added to each testtube. After the incubation, the transfer of glucose-Cl" to starch was determined according to the methodof Leloir et al. (14). The reaction system for theglucose-C'4 transfer from sucrose-C14 to starch con-taine(l: 4 ,umoles of glycine buffer (pH 8.4), 0.1mniole of EDTA. 0.3 ,umole of MgSO4, 0.36 /Amole ofUDP. 5 ,umoles of sucrose-C14 (2.28 X 105 cpm),5 ,ul of sucrose svnthetase (fraction II, see below),an(I 6 iing of starch granules in a total volume of 17 ,ul.Chatnges in the comiiposition of this reaction mixtureare indlicatedi in the table. Incubation was for 3hour.,. To inisure comlplete solubilization of the sample,a lheate(l starchi solutioin was further hydrolyzed with(lilute HCI. 10 nml of scinitillator (28) was a(lded to0.1 mul aliquot- of tthe sami)le containe(l in vials andra(lioactivity was (leterllline(l with a Packard Tri-Carbliqui(l scitntillation spectrometer.

Sucrose Sn thletase. In preparing the enzyme(14) usuallv 15 of rice graiins wvere ground with 40ml of 0.05 NM potassium phosphate buffer (pH 7.0).The extract squeezed througlh cheese cloth wvas spundown at 15,500 rpml for 15 nminutes to obtain the super-natanit (fractioni I). A fraction p)recipitable by 0.2to 0.6 saturation of (NH,)2S04 was dissolved in10 nil of dlistilled w-ater and (lialyze(l against water forseveral hour; in a cold room. It wx-as then centri-fuged to get a clear supernatant fluid (fraction II)whiclh was stable for about a week in the refrigerator(1'). A standard reaction mixture contained 100/Amoles of Tris buffer (pH 7.4), 3 munoles of MIgSO4,5 ,unmoles of fructose, 0.02 to 0.1 nml of an appropriateenzyme preparation, and a final adlditioni of 1.2 ,umolesof UDPG in a total volume of 0.6 nml after 10 minutespreincubation at 370. Sucrose formlationi was dleter-mined by the resorcinol metho(d of Roe (30).

UDPG-pvrophosphorvlasc anid UDP-ph-ospho-kiiiase. Fractioni IT obtained above was used toexanine the activity of these 2 enzyme systems.Instea(1 of determining UDPG formlation, the reac-tiotn was couple(l to the sucrose synthesizing enzymeand sucrose formiiation x-as (letermlinedl. The assaymixtture was idenitical to that of the sucrose svnthetasesystemi, except that TDPG Nvas substituted by 1 ,umoleof UTP and 2 junioles of glucose-i-P for the pyro-phosphorylase and 2 ,nuoles of UDP and 5 umoles ofATP for the kinase svstems.

Herokincasc. The preparation of the enzyme wasbased oni the miietlhod of Saltmiian (32): 70 g of ricegrains were groundl in a mortar wxith 50 ml of 0.1 MATris buffer (pH 7.5), the extract squeezed through1cheese cloth,, ani(I spun (lowvn at 9,200 rpmii for 5 min-utes. The superniataint fluidl was again centrifugedat 15,500 rpmi for 20 iminutes to get a crudle extract,fraction A. The latter fraction was treated with 1 Nacetic acid to adjust the pH to 5.5 anid the precipitatewa, reniiove(l by cenltrifugationi. The pH of the su-

pernatant fluid was adjusted to 7.5 wvith 1 N NaOH(fraction B). Fraction B was treated with solid(NH,) 2SO4 and the fraction precipitable at 0.5saturation (fraction C) and 0.5 to 0.6 saturation(fraction D) were separated by centrifugation. Eachwas (lissolved in 0.1 At Tris buffer (pH 7.5). Beforethe enzymic assay, each fraction wvas dialyzecd againstwater for 4 to 6 hours. The reaction mlixture con-tained 100 ,umoles of Tris buffer (pH 7.5), 10 u.molesof MgSO,, 5 ,umoles of glucose or fructose, 10 unmolesof ATP, 50 u.tmoles of NaF and 0.2 nml of the enyzmepreparationi in a total volume of 1.0 ml. The samiipleswvere incubated for 30 minutes at 300, and 1.0 ml eachof 0.3 N Ba (OH). and 5% ZnSO, were ad(led toremove the esterified sugar. The resi(lual freehexoses were (letermiiined by an appropriate miiethod(30, 33).

.Oxidativ' P/iosph orliation. The particulate frac-tion was prepared fromii 50 g of rice grains, using themethodl for obtaining mitochondria from sweet po-tato roots (4). The final precipitate was susp)endedin 2.5 ml of the me(liumii. The assay miixture con-

tainedl 12.5 ,umoles of potassium phosphate, 20 Lmiolesof a-ketoglutarate, 2.5 ,amoles of ATP, 1 FLmnole ofDPN, 0.5 mg of thiamiiine pyrophosplhate, 10 1uLmioles ofMgCl, 20 gmoles of glucose, 100 units of crystallinehexokilnase, and 0.5 ml of the particulate suspensionin a final volunme of 1.5 ml. Incubation was at 300for 45 minutes and Pi disappearaince was measuredby Nakamura's method (21).

Ion -exchanige Chroinatographic Scparatioii ofSutgar Phosphates. The following reaction imixtureswere employed: hexokinase systemn-100 ummoles ofTris buffer (pH 7.4), 30 ,umoles of MIgSO,, 30 ,unmolesof fructose-C'4 (2.24 X 106 cpml), 60 ,umoles ofATP, 50 ,umoles of NaF, and 0.4 ml of enzymepreparation (fraction D as described above), in atotal volume of 1.8 ml; and sucrose utilizationi sys-tem-l100 Mulmoles of Tris buffer (pH 7.4), 30 ,unolesof MrgSO,, 30 ,umoles of sucrose -C'4 (1.37 X 107cpmll), 20 ytmioles of ATP, 12 Mmoles of UDP, 50,umoles of NaF, andl 0.4 ml of the enzyme preparationin a total volumiie of 1.6 ml. At the en(d of incubation,the entire reaction mlixture was heated in a boilingwrater bath for 4 minutes, and later (leionized by adld-ing a small amount of Amberlite ion exchange resinis,IR-120 and IR-4B, and centrifugedl. The clearcentrifugate was subjected to ion exchanige columiinchromatography for sugar phosplhates, using Dowex-1resin [AG 1 -X8 (200-400 mesh, chlori(le form-l )Calbiochemii.] originally developed by Khym-n anl(ICohn (13) andl described by Mlori et al. (19). Abouti mg each of glucose-i-P and glucose-6-P were ad(ledas carriers. An authentic sample of fructose-6-Pwas not available for use as a carrier. This sugarphosphate, therefore, was identified from its elutionfrom the column, as quoted in the literature. Fortymililiters of effluent was collectecl andl an aliquot wasanalyzed for sugar content bv the miietho(d of Duboiset al. (9) andcl radioactivity. The miietlho(d of Roe

372



AKAZAWA ET AL.-STARCH SYNTHESIS IN RICE GRAINS

0

E

E

Days after Flowering

20-

._

0

CP 10

E

5

Sucrose

= = _ Glucose

Fructose-A._

0 6 9 12 15 Is 21

Days after Flowering

FIG. 1 (upper). Change of carbohydrate content inrice grain (variety Peta) during ripening. Contents ofstarch and sugars are plotted as mg per 1 and 100 grains,respectively.

FIG. 2 (lower). Changes of the individual sugar com-

ponents in rice grain (variety Peta) during ripening.

(30) was used for analyses of the fractions containingfructose-6-P.

Reagents. The following reagents were pur-

chased from the California Corporation for Bio-chemical Research: UTP, UDP, UDPG, glucose-1-P,glucose-6-P, phosphoenolpyruvic acid, a-ketoglutaricacid, pyruvic phosphokinase, thiamine pyrophosphate,and sucrose-C'4. ATP was purchased from Nutri-tional Biochemical Corporation. UDP-glucose-C14and hexokinase were gifts of Dr. Elizabeth F. Neufeldof the University of California, and Sigma ChemicalCompany, respectively.

ResultsCarbohydrate Analysis. Figure 1 illustrates the

changes in starch and sugar contents of the grainduring ripening. A sharp increase of both reducingand nonreducing sugars occurred immediately afterthe onset of flowering, reaching a maximum at about9 days. Thereafter, a steady decrease occurred to-ward the full maturation period of the seeds. Starchsynthesis at first increased in parallel with the low-molecular weight sugars, continued steadily even after

120

100

80

60

40

20

0

o

0 2 4 6 8

mg Starch Granules

FIG. 3. Relationship between amount of starch gran-

ules and UDPG-starch transglucosylase activity. Starchgranules were prepared from Peta, and the standard reac-tion mixture contained 0.4 ,umole of glucose-6-P. Incuba-tion time was 90 minutes.

the decline of the latter, and the accumulation reacheda maximum level at about 30 days. Sucrose was themain component of the nonreducing sugars, while glu-cose and fructose were the main reducing sugars, as

borne out by chromatography data from ion-exchangecolumns (fig 2). The changes in the 3 sugars duringripening follow the patterns obtained from those ofthe nonreducing and reducing sugars in figure 1.In fact, the amount of the disaccharide and monosac-

charides agree closely with the total amounts of non-

reducing and reducing sugars respectively. Hence,no significant amounts of other sugars appeared tobe present in the grain.

Starch Synthetase. The average starch synthe-tase activity of the starch granules, expressed as

mp moles of UDP formed/mg enzyme hr, was 10 forPeta and 5 for Chianung 242. A roughly propor-

tional increase in starch synthesis resulted from in-creasing amounts of starch granules (fig 3). Heat-ing of the starch granules in the dry state at 100° for10 minutes did not cause much enzyme inactivation.When the granules were heated in glycine buffer forthe same length of time, a complete loss of activityresulted.

Nearly all of the glucose portion of the UDPGmolecule is transferred to the starch acceptor mole-cules (fig 4). The apparent equilibrium constantK'eq = (UDP)/(UDPG) was calculated to bemore than 10. Thus, the reaction favors polysac-charide synthesis.

Iodoacetamide caused about 70% inhibition of thestarch enzyme under the stated conditions (table I),

cfa

CPO0)

373

15~



PLANT PHYSIOLOGY

Table 1Effect of Iodoacetamide on UDPG-starch

Transglucosylase3.5 mg. of starch granules prepared from Peta were

added to the standard reaction mixture.

Iodoacetamide Incubation UDP formedconcentration min m,umoles

... 60 54.42.5 X 10-3M 60 17.2

... 90 86.42.5 X 10-3M 90 23.5

indicating the presence of an SH-functional group.Under the same reaction conditions, this inhibitor didnot affect the pyruvic phosphokinase system for UDPdetermination. Glucose-6-P did not stimulatemarkedly the starch synthetase system, although thissugar phosphate was reported to be essential in theglycogen synthetase system (15, 16).

The results of the isotope experiments eliminatedthe possibility of UDP formation by simple hydrolyticcleavage of the UDPG molecule catalyzed by thestarch granules. Vigorous incorporation of glu-cose-C14 into the pre-existing starch molecules didoccur, and this was taken as conclusive evidence ofenzymic transglucosylation. The possible contamina-tion by oligosaccharides formed by the transfer ofglucose-C14 also was eliminated since the starchsamples had been washed thoroughly.

200E

0

150 //0

o 100E

so.E

50

60 120 180 240 300 360 420 480

Time in Minutes

FIG. 4. Reaction course of starch synthetase. Starchgranules (6 mg.) (see legend in fig 3) were added to thestandard reaction mixture containing 0.24 umole ofUDPG. Arrow indicates the maximum level of the re-

action.

To determine whether glucose was incorporatedinto both amylose and amylopectin after incubationwith UDP-glucose-C14, the starch granules were

fractionated into these 2 components following themethod of Leloir et al. (14), and the radioactivitywas determined in each fraction. Both amylose andamylopectin incorporated glucose-C14 after 2 to 10hours incubation, and the specific radioactivity in theamylose fraction was consistently higher than thatin the amylopectin fraction (table Ha).

Table IIGlucose Transfer from UDP-glucose-C14 (a) and Sucrose-C14 (b) to Amylose and Amilylopectin

4.0 mg of starch granules prepared from Chianung 242 were added to each standard reaction mixture. Each ofUDP-glucose-C14 and sucrose-C14 added were 0.24 Amole (3.31 x 104 cpm) and 7 ,umoles (6.84 X 105 cpm) respec-tively.

(a)

Incubation Starch Total C14 Glucose Specifichr fractions cpm content activity

,umoles cpm/,xmole

Amylose 3,530 5.9 5982

Amylopectin 6,400 18.7 342

Amylose 9,370 6.8 1,3785


Amylose 12,800 5.9 2,16910


(b)

Incubation Starch Total C14 Glucose Specifichr fractions cpm content activity

,umoles cpm/,mole

Amylose 4,990 7.0 7312


Amylose 5,660 6.5 8715


374




Table IIIIsolation of Sucrose Synthetase from Rice Grains

The enzyme was prepared from Chianung 242. One enzyme unit is defined as the amount of enzyme synthesizing 1,umole of sucrose in 10 minutes under the given conditions.

SpecificEnzyme Volume Activity Total amt. Protein-N activityfraction ml units/ml units mg/ml units/mg

protein-N

I 42.0 10.3 432.6 0.17 60.0II 11.8 18.4 217.1 0.15 122.5

Sucrose Synthetase and Sucrose-starch Intercon-version. A constant supply of UDPG must somehowbe provided for the starch synthesis to proceed viastarch synthetase. As a first step towards solvingthis problem, and considering the large amounts ofsucrose present in ripening rice seeds (fig 1,2), thesystem involving sucrose synthetase was investigatedas a possible source of UDPG. The crude extractas well as the partially purified preparation (frac-tions I, 11) exhibited strong enzyme activities (tableIII). In a complete reaction system, fraction I en-

zyme produced 0.52 ,umole and 0.84 umole of sucrose

after 10 minutes and 30 minutes incubation, respec-tively; negligible amounts of sucrose were formedwhen either UDPG or fructose was omitted or whenboiled enzyme was used. The apparent equilibriumconstant of the reaction [K'eq = (UDP) (sucrose)/(UDPG) (fructose)] was calculated to be about 2.0,an average value from 3 experiments. Thus, underthe present experimental conditions, the equilibriumis in favor of sucrose synthesis instead of UDPGformation. However, the reversal of sucrose synthe-tase may be energetically possible when coupled withstarch synthetase (I), with UDPG as an inter-mediate and sucrose serving as the glucose donor(II).Sucrose + acceptor (G),

fructose + starch (G)n+1. II

The results shown in table IV indicate that in thepresence of sucrose synthetase, a roughly proportionalincrease of glucose transfer from sucrose-C14 to

starch was observed with increasing amounts ofstarch granules (experiment 1-4). The increase inthe rate of glucose-C14 transfer as a function of UDPconcentration is shown also (experiment 5-7).There was very little incorporation of glucose-C14without UDP, whereas a marked stimulating effect ofUDP on the glucose transfer was noted. Thus, theexperimental results tend to verify the reversal ofsucrose synthesis as a generator of UDPG necessary

for starch formation. The percentage utilization ofsucrose-C14 as a glucose donor was rather low ineach case, however. As will be seen in the table(experiment 8-9), ATP was found to inhibit ratherthan stimulate the glucose-C14 transfer from sucrose.

Provided sucrose functions truly as a glucose do-nor in starch formation, the pattern of C14 distribu-tion of each fraction of amylose and amylopectinshould be identical to that from UDP-glucose-C14.The results of the radioactivity determinations in the2 fractions are shown in table IIb. The pattern issimilar to the case of glucose transfer from UDP-glucose-C'4.

Another possibility for the synthesis of UDPGmay be by the UDPG pyrophosphorylase reaction(24, 34), and the coupling of this system with UDPphosphokinase to get the over-all reaction III.

UDP + ATP + glucose-i-P :±

UDPG + ADP + PPi. IIIInstead of measuring UDPG formation, both thesereactions were tested for by coupling each -one withsucrose synthetase, using fraction II enzyme. A

Table IVGlucose Transfer from Sucrose-C14 to Starch under Various Conditions

Experiments 1 through 9 were carried out simultaneously and in duplicate; experiment 3 served as the standard sys-tem, in which the reaction mixture described in the text were employed. Sucrose-C14 added was 2.28 x 105 cpm.Starch granules were prepared from Peta.

Experiment Starch granules UDP ATP Sucrose- C14 in starch Incorporationmg m,moles 'Umoles synthetase cpm %1 2 360 - + 1,070 0.472 4 " - + 1,290 0.573 6 " - + 2.440 1.074 8 " - + 3,060 1.34

5 6 + 980 0.436 ,, 240 + 1,720 0.757 " 720 - + 4,280 1.88

8 6 360 0.2 + 2,080 0.919 " 1.0 + 1,390 0.61

375



Table VIsolation of Hexokinase from Rice Grain1s

The enzyme was prepared from Chianung 242. One enzyme unit is defined as the amount of enzyme svnthesizingI,umole of hexose phosphate in 30 minutes under the given condition.

Specifc activityunits/mg protein-N

glucose

10.0611.443.97

15.15

fructose

6.198.473.30

13.90

Ratiofructose/glucose

0.620.740.830.91

measurable amiiount of sucrose is formed in the com-

plete system (fig 5A, 5B). Omission of either one

of the reaction components resulted in little sucrose

formation. Nakamura (22) ma(le similar observa-tions, using partially purifiecl sucrose synthesizingenzyme from beans.

Hexokinase and Oxidative Phiosph orylation. Todetermine the fate of free glucose and fructose, thephosphorylation of these sugars was examined. Ofthe various preparations, the (NH4)2SO4 precipit-able fraction at 0.5 to 0.6 saturation (fraction D) ex-

hibited a strong hexokinase activity to both sugars

(table V). The ratios of the activities of the 2 sugars(fructose/glucose) were somewhat consistent in sub-sequent purification steps, but it is not certain whetherthere is a fructose-specific kinase in rice grains.Fructokinase was isolated from peas (17).

The energy required for the hexokinase reactionmay possibly be generated from the particulate com-

ponents (mitochondria) of the grain cells throughoxidative phosphorylation. The particulate fractionisolated from rice grain was tested for the presence

of the latter system, and P/O ratios of 0.85 an(d 1.32were observed in 2 separate experiments. However,both oxidative and phosphorylative activities ob-taine(I were rather weak, as compared to other plantmitochondria.

It was of interest to verify the enzymic transforma-tion of fructose to glucose-i-P, which may establishthe over-all process of sucrose-starch conversion as

reaction IV, because the fructose moiety of reactionII will eventually be converted to UDPG by reactionIII.Sucrose + 2 ATP + 2 acceptor (G)n

2 starch (G),,+ + 2 ADP + PPi. IV

Froml- our basic knowledge of the carbohydrate me-

tabolism, it is highly predictable that the glycolyticpathway exists in rice seeds, and in fact, Dowex-1ion-exchange column chromatographic separation ofthe reaction products of the fructose-C14 phosphoryla-tion system described above showed the formation of

fructose-6-P, glucose-6-P, and glucose-i-P. Simi-larly, formation of such hexose phosphates was sub-stantiated by incubating sucrose-C14 in the presenceof UDP and ATP, supporting the presumption of thepresence of glycolytic enzymes tranisforming the fruc-tose moiety dlerived froml sucrose to glucose-i-P.

0)

E

0

(0

0

E

2. 0K. UDP

B ATP + GIP + fructose * ADP + sucrose +PPI1. 0

0. 5

/- UDP7,- ATP

0 30 60 120 I80

.oA UTP + GIP + fructose ^ UDP + sucrose + PPi

0.5 [

- UTP

/_ G P

0 30 60 120 180 240

Time in Minutes

FIG. 5. (A) Coupling of UDPG-pyrophosphorylaseNxith sucrose synthetase. A twofold dilution (0.5 ml) offraction II described in table III was used as the enzyme.

Open circles are for the complete system. GIP repre-

sents glucose-i-P.(B) Coupling of UDPG-pyrophosplhorylase and LUDP-

kinase with sucrose synthetase. A twofold dilution (0.1ml) of fraction II described in table III was used as theenzyme. Other experimental conditions were same as in(A). Open circles are for the complete system. In (A)and (B) arrows indicate the maximunm level of the reac-

tion.

DiscussionThe net content of starch per grain is muclh lhigher-

than that of sucrose and the other 2 hexoses (fig 1, 2).This over-all trend indicates sucrose utilization and itsaccumulation in grains during the enzymic transfor-mation to starch. Assuming sucrose to be a directprecursor in starch formation, a possible enzymicmechanism governing the over-all interconversionwould be reaction II. This enzymic linking was

proposed by De Fekete et al. (8), and our experi-mental results (table IV) tend to verify that such a

mechanism operates in ripening rice grains. Thequestion. will be raised, however, whether stcrose

Activityunits/mlEnzyme

fraction

ABC

D

glucose

S.456.755.60

10.30

fructose

5.205.004.659.45

Protein-Nmg/ml

0.840.591.410.68 . .376 PLANT PHYSIOLOGY




Enzymatic Mechanism of Starch Synthesis

Pi

4 ~~ppi Acceptor (G)n

UDPG

GIP Starch

i UTP (G)nl |

ADP |ructose

UDP ATP

ATP jSucrose

ADP

G6P - F6P

FIG. 6. Proposed enzymic mechanism of starch bio-synthesis. F6P, G6P, and GlP represent fructose-6-P,glucose-6-P, and glucose-1-P, respectively.

forms UDPG by the direct transglucosylation in sucha reaction system. Distribu,tion of sucrase isubiquitous in the plant kingdom, and the enzyme maybe engaged in the metabolic breakdown or utilizationof sucrose, but the activity was found to be weak inthe rice grain extract. Also if the entire processinvolves the mechanism:sucrose -* glucose + fructose

hexose phosphate UDPG -> starchATP should enhance the glucose transfer from sucroseto starch, because of the requirement of hexose phos-phorylation. However, ATP was in fact found to beinhibitory rather than stimulative (c.f. experiment8,9 table IV). By the isotope experiments it wasfound that UDPG was synthesized from sucrose onlyin the presence of UDP (Paper submitted to Arch.Biochem. Biophys.), substantiating the mechanismwritten as reaction II. Thus, by taking into accountthe experimental evidences in hand, the consequentover-all reaction mechanism of sucrose-starch con-version will be reaction IV and may be diagram-matically illustrated as figure 6. The validity of thisview requires more study. For example, Recondoand Leloir (29) and Frydman (11) found that ADPGis much more active than UDPG as a glucose donorof starch synthetase, while UDPG is the dominantsubstrate for the sucrose synthesis (7). We havealso isolated ADPG from ripening rice grains inde-pendently and its more efficient utilization in starchformation was confirmed (20). The inefficient utili-zation of sucrose-C14 in starch synthesis in vitro maybe a reflection of the more important role of theADPG pathway in the system, and further work is

needed to elucidate the interconnection of this newpathway with the presently investigated UDPG sys-tem. Another reason for the poor utilization of su-crose-C14 in starch synthesis in vitro may result fronmdifficulties inherent in reconstituting such a system.As has been noted by Leloir et al. (14) the structuralrelation between the enzyme and polysaccharide inthe starch synthetase reaction system is similar to theconditions existing in whole grains. The colloidalcondition of the reaction system was not maintained,however, when the 2 enzymes were combined. Thisresulted in a heterogeneous mixture consisting mainlyof the liquid phase. Therefore, the over-all mech-anism of sucrose-starch conversion in vivo also maybe more efficient than the results obtained in a testtube.

Leloir's group (14,16) established that the mole-cular structure of polysaccharides synthesized by bothglycogen and starch synthetase is the a- (1-4) glu-cosidic type, thus the enzyme is often called amylosesynthetase (36). Nelson and Rines (23) recentlyreported, however, the deficiencv of starch synthetasein waxy corn and proposed a possible regulation mech-anism in the biosynthesis of amylose and amylo-pectin. Although we have not examined this viewwith waxy and nonwaxy rice grains, the results intable II show a measurable incorporation of glu-cose-C14 from UDPG to both the amylose and amylo-pectin fractions. These results are not easily ex-plained by the observation of Nelson and Rines.

The important role of phosphorylase in starchsynthesis cannot be neglected as has been discussedby Badenhuizen (5) and Porter (26), and more workis needed to clarify its role in the starch formation inthe developing rice grains.

SummaryStarch granules catalyzing starch synthesis were

prepared from ripening rice grains. Starch synthesiswas confirmed by both the enzymic assay for uridinediphosphate liberated during the transglucosylasereaction and glucose-Cl4 transfer to starch moleculesfrom uridine diphosphate glucose-C14. Glucose-C'4was incorporated into both the amylose and amylo-pectin fractions of the starch molecules.A measurable amount of glucose-C14 was trans-

ferred from sucrose-C14 to starch in the presence ofstarch granules, sucrose synthetase and uridine diphos-phate, indicating a possible operation in vivo of thereversal of sucrose synthesis to provide uridine di-phosphate glucose. Glucose-C'4 was incorporatedfrom sucrose into both the amylose and amylopectinfractions of the starch molecules in much the sameratio as that obtained from uridine (liphosphateglucose-C'4.A crude enzyme preparation catalyzing sucrose

synthesis was also found to have uridine diphosphateglucose pyrophosphorylase and uridine diphosphatephosphokinase activities, indicating the existence inrice grains of a mechanism for uridine diphosphateglucose formation from glucose-l-phosphate. The

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crude rice grain extract also exhibited strong hexo-kinase activity. Fructose-6-phosphate, glucose-6-phosplhate, and glucose-i-phosphate were formed whensucrose utilization was combined with the hexokinasereaction.

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Enzymic Mechanism Starch Synthesis Ripening …...Enzymic Mechanism of Starch Synthesis in Ripening Rice Grains1 2 T. Akazawa, T. Minamikawa, and T. Murata TheInternational Rice Research