Enzymic mechanism of starch synthesis in ripening rice grains

ARCHIVES

OF

Enzymic

BIOCHEMISTRY

AND

BIOPHYSICS

Mechanism

VII. Purification

166, 644-652

of Starch

Synthesis

and Enzymic

T. NOMURA

(1973)

Properties AND

in Ripening of Sucrose

Rice Grains

Synthetase’

T. AKAZAWA

Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya, Japan Received

October

6, 1972

Sucrose synthetase (UDP-glucose:n-fructose-2-glucosyltransferase, EC 2.4.1.13) from ripening rice seeds was purified by ammonium sulfate fractionation and column chromatography of microgranular DEAE-cellulose (DE-32) and Neusilin (MgO. Al~Os.2SiOz). An enzyme preparation obtained was homogeneous as examined by polyacrylamide gel electrophoresis. The enzyme, having a molecular weight, 4.0 X 105, consists of 4 identical subunits, each having a molecular weight, 1.0 X 106. Examination of reaction kinetics of both sucrose synthesis and cleavage catalyzed by sucrose synthetase revealed that the rate of synthesis follows a Michaelis-Ment’en equation having the followmg parameters: K,(fructose)unr+lucose, 6.9 mM; K,(fructose)AD&~lucose, 40 mM; K,(UDP-glucose), 5.3 mM; and K,(ADP-glucose), 3.8 mM. The cleavage reaction yielded the following values: K,(UDP), 0.8 mu; K,(ADP), 3.3 mM; and K,(SUCrOSe)vDp, 290 mM. In the latter reaction the rate deviat,ed from the Michaelis equation when ADP was used as the glucose acceptor, the n value being 1.6 by the Hill plot analysis and &.~(sucrose)AnP, 400 mM. At high concentration of ADP the cleavage reaction was inhibited, while the synthesis reaction was inhibited with high concentrations of fructose.

Since the classical study of Cardini, Leloir and Chiriboga (l), considerable research has been conducted on sucrose synthetase which catalyzes reversibly the formation and cleavage of sucrose according to Eq. (1) (a-12). UDP-(or

ADP-)glucose ti sucrose

+ fructose + UDP

(or ADP).

(1)

At one time this was considered to be the dominant pathway for sucrose synthesis in plants. However, recent studies indicate that Eqs. (2) and (3) mediated by sucrose B-phosphate synthetase and sucrose 6-phosphate phosphatase express the principal pathway of sucrose synthesis in higher plants. UDP-glucose

+ fructose

6-P

--+ sucrose

6-P + UDP,

sucrose 6-P + Hz0 + sucrose

+ Pi.

(2)

MATERIALS

(3)

AND

METHODS

Plant Material

Of interest is that glucose m0iet.y of ADP-

Ripening rice seeds, harvested in Sept., 1971, were used throughout this experiment. They were

1 Supported in part by a research grant from the Ministry of Education of Japan (786007). 644 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

glucose resulting from sucrose cleavage [Eq. (l)] was found to be transferred to a (l-4) glucan in ripening rice seeds (15, 16). In an earlier experiment (16) using a mixture of sucrose synthetase and granule-bound starch synt’hetase, the overall reaction was inhibited by UDP which occurs in relatively high concentrations in rice seeds. The present paper examined this interaction between ADP- and UDP-glucose and describes some structural and kinetic properties of sucrose synthet’ase and a method for its purification. During the preparation of this article, Delmer (17) reported on the purification of sucrose synthetase from Phase&m aureus and gave some of its st’ructural characters which are similar to these report’ed herein.

SUCROSE

SYNTHETASE

stored at -20°C until analyzed. Seeds collected at this stage and stored at this temperature lost very little enzyme activity (18).

Enzyme PuriJication ltice grains (approx 300 g) were immersed in liquid N2 and ground finely in a cold mortar. The powder was suspended in 750 ml of 50 mM Trisacetate buffer (pH 7.0) and t,he resulting suspensionpassed through 3 layers of gauze. Unless ot,herwise stated, the buffer solution at pH 7.0 used throughout this study contained 0.1 mM EDTA, 5 IIIM o-mercaptoethanol and 50 mM potassium acetate. Solid (NHn)tSOa was added to the filtrate to 0.G saturation and the contents were centrifuged (lO,OOOg, 15 min). The pellet was resuspended in the homogenizing buffer and dialyzed against 5 mM Tris-acetate buffer (pH 7.0). After centrifuging the dialysate, the precipitate was discarded and the supernatant was transferred to a column of microgranular DEAE-cellulose (DE-32) (3.6 X 36 cm), preequilibrated with 5 mM Trisacetate buffer (pH 7.0), and the enzyme was eluted by a linear gradient of potassium acetate from 0.05 to 0.3 M in 5 mM Tris-acetate buffer (pH 7.0) (Fig. 1). The enzymically active fractions (tube Nos. 281-325, 670 ml) were charged into a column of Keusilin (MgO.Al203.2Si02) (2.7 X 20 cm), preequilibrated with 5 mM Tris-acetate buffer (pH 7.0). The enzyme was then eluted with 0.2 M potassium phosphate buffer (pH 7.0), and the eluate was diluted 2-fold with 5 mM Tris-acetate buffer (pH 7.0). The diluted enzyme sample was applied again to another column of Neusilin (1.4 X 23 cm), preequilibrated with the diluting buffer. The column was first washed with 0.1 M potassium phosphate buffer (pH 7.0), and the enzyme was eluted by a linear gradient of potassium phosphate buffer (pH 7.0) from 0.1 to 0.4 M (Fig. 2). The enzymically active fractions (tube Nos. 52-88, 200 ml) were pooled, concent,rated in a collodion membrane bag and then dialyzed against 5 mM Trisacetate buffer (pH 7.0) cont’aining 0.1 mM EDTA, 3 mM dithiothreitol and 50 mM potassium acetate. The final volume of t,he dialysate obtained was 5 ml. The sample was stored in a cold room (4°C) and was appropriately diluted by 5 rn>f Tris-acet,ate buffer (pH 7.0) before use.

Enzyme Assay The enzyme activities were determined in each of sucrose synthesis and cleavage reaction as follows. a. Sucrose synthesis. The standard reaction mixture contained: Tris-acetate buffer (pH KO), 143 mM; UDP-glucose, 32 mM or ADP-glucose, 45 mM; fructose, 10 mM with UDP-glucose system, or 70 mM with ADP-glucose system; and diluted enzyme

FROM

RICE

SEEDS

645

solution (ca. 2.0 fig protein) in a total volume of 35 ~1. Reaction was carried out at 37°C for 15 min, and stopped by heating the reaction mixture in a boiling water bath for 1 min. On adding 0.5 ml of 0.1 N NaOH, the mixture was heated in a boiling water bath for 10 min to degrade the unreacted fructose molecule. The amount of sucrose formed was determined by measuring the absorbancy at 490 nm (19). b. Sucrose cleavage. The reaction mixture contained: Tris-acetate buffer (pH 6.0), 100 mM; sucrose, 600 IIIM; UDP, 11 mM or ADP, 4.7 mM; and diluted enzyme solution (0.95-3.8 rg protein), in a total volume of 50 ~1. The reaction was carried out at 37°C for 15 min, and stopped by heating the mixture in a boiling water bath for 1 min. The amount of fructose formed was analyzed according to the method of Nelson-Somogyi with slight, modification by measuring the absorbancy at 660 nm (14). c. Optimum pH for sucrose sytcthetase. For measuring the pa-activity relationship, BrittonRobinson’s (H8POrCH&OOH-HsB08-NaOH) wide range buffer (20) was used. The assay mixture contained: (A) buffer solution, 125 mM; UDPglucose, 32 rnM; fructose, 70 mM; and diluted enzyme solution (ca. 2.0 rg protein): or (B) buffer solution, 100 mM; sucrose, 400 mM; UDP, 11 rn%f; and diluted enzyme solution (0.95 pg) in a total volume of 50 ~1. The reaction was carried out at 37°C for 15 min.

Polyacrylamide

Gel Disc ElectTophoresis

Polyacrylamide gel electrophoresis (7.5yQ cross linkage) at pH 8.9 was carried out at the constant current of 4 ma/tube at 4”C, following the method of Ornstein and Davis (21). To locate the zones of enzyme activities finely sliced polyacrylamide gel fractions (1 mm thick) were immersed in 100 pl of 50 mM Tris-acetate buffer (pH 7.0) overnight at 4°C and 20 ~1 aliquot of the diffusate was applied to the standard assay mixture of sucrose synthetase reaction (UDP-glucose + fruct,ose) and incubated at 37°C for 1 hr (cf. Fig. 3). To estimat,e the molecular weight of sucrose synthetase gel electrophoresis at different gel concentrations was employed as recommended by Hedrick and Smith (22). The following marker proteins were used to make t,he calibration curve, moleclllar weight vs slope : chymotrypsinogen, 2.5 X 104; hexokinase, 9.6 X 104; bovine serum albumin (monomer), 6.7 X 10”; bovine serum albumin (dimer), 1.34 X 105; aldolase, 1.47 X 105; bovine serum albumin (trimer), 2.01 X 105; catalase, 2.4 X 105; and ferritin (apoferritin), 4.5 X 105. Apoferritin and ferritin have the same molecular size as tested by gel electrophoresis (23, 24). The inclusion of Fe3+ by the protein changes its molecular weight but not its molecular size and its

646

NOMURA

AND

net charge. Therefore, the molecular weight of apoferritin (4.5 X 105) was used (22). For the molecular weight determination of the monomeric subunit of the enzyme, sodium dodecyl sulfatepolyacrylamide gel electrophoresis was carried out after the method of Weber and Osborn (25). Standard marker proteins of known molecular weight used were as follows: ovalbumin (monomer), 4.5 X 104; bovine serum albumin (monomer), 4.5 X 104; ovalbumin (dimer), 9.0 X 104; and bovine serum albumin (dimer), 1.34 X 105.

Estimation

AKAZAWA tion following the method reported by Martin and Ames (27). The purified preparation of sucrose synthetase (350-400 fig) was layered on top of 17 ml of glycerol gradient solution (5-200/L, w/v) dissolved in 0.05 M Tris-acetat,e buffer (pH 7.0). Centrifugation was carried out for 15 hr at a rotor speed of 23,000 rpm in an SW 25-3 rotor of Spinco Model L-2 preparative ultracentrifuge (10%). After centrifugation, 0.5-ml fractions were collected and absorbancy at 280 nm as well as sucrose synthetase activities using the standard assay mixture (UDP-glucose + frllctose) were measured. The internal marker proteins used were spinach ribulose-1,5-diphosphate carboxylase (100 rg), s20,w = 18.6 (28, 29) and beef liver catalase (100 fig), s*o,,~ = 11.3 (30). In both cases only uv absorbancy at 280 nm was measured.

of Molecular Weight by Sephadez G-200 Gel Filtration

The molecular weight of sucrose synthetase was also estimated using a column (2.7 X 92 cm) of Sephadex G-200 according to the method of Andrews (26). The standard protein samples of known molecular weight used were: ovalbumin, 4.5 X 104; bovine serum albumin, 6.7 X 104; aldolase, 1.47 X 105; catalase, 2.4 X 105; ferritin, 5.4 X 105; and blue dextran, 2 X 106. Enzyme proteins were eluted with 5 msl potassium phosphate buffer (pH 7.0) containing 0.1 M potassium acetate, 0.1 mM EDTA and 5 mM P-mercaptoethanol. Each 3-ml fraction collected was tested for enzyme activity in the standard assay system described above for sucrose synthesis and uv absorbancy measurement at 280 nm.

Determination

of Protein Content

Protein content was determined by the colorimetric method of Lowry et al. (31) using bovine serum albumin dissolved in 0.1 N NaOH as a standard.

Reayen ts and Chemicals All nucleotides were purchased from Boehringer Mannheim Co. Ltd. UDP and ADP were purified by paper chromatography using a solvent system of 95% ethyl alcohol and 1.0 M ammonium acetate (5:2, v/v) (pH 7.0), to eliminate contaminants of mono- and triphosphate esters of each nucleotide and other unknown impurities. Microgranular DEAE-cellulose (DE-32) was a product of Whatman Co. Ltd. Neasilin (MgO.Al203.2SiOJ was

Glycerol Density Gradient Cen,trifugation The s20,w of sucrose synthetase was determined by means of glycerol density gradient centrifuga-

DE-32

50

loo

150 FRACTION

x0

250 NUMBER

300

350

400

( 15ml )

FIG. 1. Microgranular DEAE-cellulose (DE-32) ion exchange column chromat,ography of rice seed sucrose synthetase. Experimental details are described in the t)ext. Enzyme assay conditions are the same as the standard assay conditions (UDP-glucose + fructose) except for the reaction time 10 min.

SUCROSE SYNTHETASE FROM RICE SEEDS purchased

from

Fuji

Kagaku Kogyo Co. Ltd.,

Toyama, Japan. RESULTS Enzyme PuriJcation. ,1fter several attempts at purifying sucrose synthetase from rice seeds, e.g., Sephadex I

.f

s 1 T

Neusilin

647

G-200 gel filtration and column chromat,ogrsphy on DEAE-cellulose, DEAE-Sephadex, and hydroxyapatite, the most satisfactory procedure finally adopted was the use of DE-32 and Neusilin (MgO .A1203. 2 SiOZ) as chromatographic adsorbents. Typical experimental results are presented in Figs. 1 and 2, showing the effective separation of the enzyme protein. The final enzyme preparation obtained was judged to be homogeneous from the coincidence of its catalyt,ic activities and its electrophoretic behavior (Fig. 3A). An 11-fold purification at the final step was obtained (Table I). Molecular Weight

20

40

1-M

FRACTION %IMZ

( 5 nlpp

FIG. 2. Neusilin (MgO. A1203.2Si02) adsorption column chromatography (2nd) of rice seed sucrose synthetase. Experimental details are described in the t,ext. The same reaction mixtrue as that described in Fig. 1 was used.

t marker( BFB)

By using polyacrylamide gel electrophoresis of the enzyme and :I series of marker proteins at different gel concentrations, the molecular weight of the rice seed sucrose synthrtasc was determined to be 4.1 X lo5 (Fig. 4). This value agrees well with 4.4 X 10” obtained by an analytical column of Sephadex G-200 (Fig. 5). With the sodium dodecyl sulfato-polyacrylamide gel electrophorcsis, tlw cnzymct molecule W:IS shown to

25 20 15 IO Fmction Number

5

c

1 start t *y.

t

!

(A) pH8.9

(f-3)SDS

FIN. 3. Klectrophoretograms of the purified sucrose synthetase from rice seeds. (A) Polyacrylamide gel disc electrophoresis at pH 8.9 after Ornstein and Davis (21). (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis after Weber and Osborn (25). Other experimental det,ails are described in the text. Approximately 29 fig prot,ein samples were assayed and the zone of the sucrose synthetase activities corresponding to the protein band in (A) located in a parallel experiment using the sliced gel diffusate as explained in the text.

648

NOMURA

AND

AKAZAWA

TABLE PCIUFUXTI~N

OF SUCROSE

I

SYNTHETASE

FROM

RIPXNING

RICE

SI<:F:DS~

Fraction

Total protein b-d

Total activity (rmoles sucrose formed/ min)

Specific activity &moles sucrose formed/ min/mg protein)

Crude extract Ammonium sulfate DE-32

1460 775 138

730 543 304

0.5 0.7 2.2

1 1.4 4.4

100 74.4 41.6

152 64

3.6 5.7

7.2 11.4

20.8 8.8

Neusilin

(1st) (2nd)

42.2 11.2

Activity recovery (%I

Purification

= The assay mixture contained Tris-acetate buffer (pH 8.0), 143 mM; UDP-glucose, 32 mM; fructose, 10 mM; and enzyme sample 20 ~1 in a total volume of 35 ~1. The mixture was incubated for 10 min at 37°C. Other experimental details are described in the text.

25

Fblyauylamide Gel Electrvphaesir (H&rick

& Smith) I

Gd ,n, L

20

10 Molecular

40

Weight

( x 17% I

FIG. 4. Determination of molecular weight of rice seed sucrose synthetase by the polyacrylamide gel electrophoresis at different gel concentrations. Experimental details of the method by Hedrick and Smith (22) are described in the text. Relative mobility (R,) vs gel concentration was plotted with each protein sample to determine from which the calibration curve, slope vs molecular weight was derived to estimate the molecular weight of sucrose synthetase.

consist of 4 identical subunits (Fig. 3B), each having a molecular weight of 1.0 X lo5 compared to several marker proteins. The &,I,~ determined by the glycerol gradient centrifugation in reference to spinach leaf ribulose1,5-diphosphate carboxylase (18.6s) was 13.3s (Fig. 6). The same value was obtained when crystalline catalase (11.3s) was used as an internal marker. Optimum pH The enzyme activity-pH relationship in the sucrose synthesis and the cleavage reac-

tions in Fig. 7 reveals a broad pH optimum between 7.0-9.5 in the former, and a sharp optimum at pH 6.0 in the latter reaction. The same results were obtained by replacing each of ADP-glucose and ADP with TJDPglucose and UDP, respectively. Reaction Kinetics a. Sucrose synthesis. The reaction rate of sucrose synthesis was measured as a function of fructose concentration in the presence of UDP-glucose (32 mM) or ADP-glucose (45 mM). From the double reciprocal plot of the

SUCROSE

\

RICE

(A)Sucmw

Sephadex G-200

odbumin

FROM

649

SEEDS

1

1

I

400

SYNTHETASE

synthgit :I25 ‘E g P -loo-

(8) Swmre cleam~e -

.f 5 1 75 z

5

I50

, 4.5

* 50

55

6.0

65

cog(M.W.1

FIG. 5. Determination of molecular weight of rice seed sucrose synthetase by the analytical column of Sephadex G-200. Experimental details of t,he method of Andrews (26) are described in the text. Exclusion volume of sucrose synthetase was measured by determining the enzymic activities in the standard assay system (UDP-glucose + fructose).

clycrol (A)

Suck

Gmdiit

Gntrifvgwcm

synth,t&

6

I

(5-20X, w/v)

7 8 PH

9

IO

FIG. 7. Activity-pH relationships of sucrose synthesis (UDP-glucose + fructose) (A) and sucrose cleavage (UDP + sucrose) (3) reactions catalyzed by rice seed sucrose synthetase. Details of the reaction mixture and assay method in each reaction system are described in the text. TABLE

II

KINETIC PARAMETERS OF RICE SEED SUCROSE SYNTHETASE IN SUCROSE SYNTHESIS REACTIONS T First substrate Second substrate K, 17 (pmoles sucrose (concn fixed) (concn varied) wf) formed/ (maa) min/mg protein)

’

0.12

03

5

UDP-glucose ADP-glucose

Fructose Fructose

Fructose

UDP-glucose (32) ADP-glucose (45)

Fructose

/

(10) (70)

5.3 3.8

6.3 13.3

6.9

7.1

40.0

15.4

I

5 The assay conditions are described in the text. Data obtained were applied to calculate the kinetic values by the double reciprocal plots.

Tube

Number

FIG. 6. Glycerol density gradient centrifugat,ion of rice seed sucrose synthetase. Experimental procedures basically following the method of Martin and Ames (27) are described in the text. The sedimentation of sucrose synthetase was monitored by measuring both uv absorbancy at 280 nm and enzyme activities in the standard assay system (UDP-glucose + fructose).

data., K,(fruct.ose) UDP-prucose and K,(fructose)Anp-piuoosevalues were calculated to be 6.9 and 40 mM, respectively. The substrate saturation curves for UDP-glucose and ADP-glucose gave the following data; K,(UDP-glucose)fructose-lOml\l, 5.3 mM and Kn(ADP-glucose)fructose-70mM, 3.S mM. The data are summarized in Table II. b. Sucrose cleavage. Examination of substrate concentrations on the sucrose cleavage reaction revealed that the rate curves of fructose formed as a function of UDP and ADP at the fixed concentration of sucrose (600

NOMURA

2

I

,

4

14

AND

16

FIG. 8. The saturation curves of UDP or ADP in the sucrose cleavage reaction. The concentration of sucrose was 660 mM in each system. Data obtained were applied to the double reciprocal plot t’o obtain V values. Then values were used for the Hill plot analysis (inset).

AKAZAWA

respectively. The molecular weight of the monomeric subunit was determined to be 1.0 X lo5 by sodium dodecyl sulfatc-polyacrylamide gel electrophoresis. We, thus, conclude that the rice seed sucrose synthetase has a tetrameric oligomeric organization. This finding agrees with that of Delmer (17) who recently reported that sucrose synthetase from Phaseolus aureus has a molecular weight of 3.75 X 105, and consists of 4 identical monomeric subunits each having a molecular weight of 9.4 x 104. The kinetic properties of the purified rice seed sucrose synthetase merit description. The enzyme was shown to behave differently

Sucrose

cleovoge

(B )

10 -

mM) were hyperbolic for which K,(UDP) and (ADP) were 0.8 and 3.3 mM, respectively (Fig. 8). However, ADP above 5 mM was inhibitory. The saturation curve for sucrose was, likewise, hyperbolic in the presence of UDP as the second substrate (11 mM), K,(sucrose) being 290 mM. On the other hand, it deviated from the Michaelis-Menten equation in the presence of ADP (4.7 mM), n(sucrose) and SO.s(sucrose) being 1.6 and 400 mM, respect’ively, from the Hill plot analysis (Fig. 9). DISCUSSION

The specific activity of the purified preparation of sucrose synthetase from ripening rice seeds was 5-7 pmoles sucrose formed/ min/mg protein with UDP-glucose + fructose under standard assay conditions. This activity is higher than that reported for wheat germ (l), sugar beet (14), potato tuber (6), t’apioca tuber (ll), andsweet potato root enzyme (12). The molecular weight of rice seed sucrose synthetase was estimated to be approximately 4 X 105. The value approximates those reported for the enzyme obtained from the sweet potato (5.4 X 105) by Murata (12) and potato (2.9 X 105) by Pressey (6),

100

200 Sucrose

300

400

( mM)

FIG. 9. The saturation curves of sucrose in the sucrose cleavage reaction. The concentrations of UDP and ADP were 11 and 4.7 mM, respectively. Data obtained were applied to the double reciprocal plot to obtain V values from intercept of the ordinate by extrapolation. Values were then applied to the Hill plot analysis (inset). In the reaction system, sucrose + ADP, the plots follow equation, l/v = (Km/V) X (l/s)l.B + l/V (V = 5 fimoles/min/mg protein).

SUCROSE

SYNTHETASE

in t,hc sucrose synthesis and t,he cleavage reactions as manifested by their different optimum pH values and substrat’e saturation curves. In the sucrose synthesis react,ion hyperbolic saturation curvrs were obtaincd \vith UDP- and ADP-glucose as this finding agrees with wports of most other workers (l-7, l&12), although the Km values for UDP-glucose was greater than that for ADl’-glucose in our study. This finding is not ncccssarily in agreement with the generally acwptcd notion conrcrning the toward pwferencc of sucrose synthetaw UDP-glucosr (L2,5, 1%). But Grimes, Jones and Albcrsheim (7) reported that sucrose synthetase from Phaseolus aweus seedlings c>xhibits the lurgwt V values with respect, to ADP-glucow among other nucleosidc diphosphate glucoses tested. That fructose has an inhibitory effect at high conccntration in the UDP-glucose system concurs with the findings of Cardini, Leloir and Chiriboga (1) and Avigad (4). Hinet,ic values and their implicat,ions IF ported by different workers on the sucrose clcavagc reaction, which may have a physiologically important role, vary. Avigad (4) and Shukla and Sanwal (11) using Jcrusalcm artichoke and tapioca t’ubers, rrsprctively, report,cd the Michaelian saturation curves for substrates, whcrcas Murata (10, 12) obtained sigmoidal saturation curves for all the substrate molecules examined in the reaction. Dclmer (17) reported that sucrose synthetase from Phaseolus aweus llas K, values of 17 rn>I (in the presence of UDP) and 0.19 rnM for sucrose and UDP, respectively. She proposed that’ the formation of UDP-glucose was the major metabolic role played by the enzyme. In the cleavage rcaction catalyzed by rice seed sucrose synthctasc, UDP was found to bc a more efficient, glucose acceptor than ADP. Howver, it should be point,ed out that K, value for sucrose of the rice seed sucrose synthctase is cxccedingly lurgc. Some workers (6, 12) also reported large K, values of the clnzyme for sucrose, and it is implied that sucrose s>.nth(+lso plays :I role of forming UDPglucose in the dtwtloping riw seed endosperm where sucrose content is high. Thus it apprlars that while tlro physical proprrtiw

FROM

RICE

651

SEEDS

of sucrose synthetasw from Phase&s aureus seedlings (17) and ripening rice seeds are similar, tht>y arc readily distinguishable enzymically. REFERENCE:S 1.

2. 3. 1.

5. G.

7. 8. 9. 10. 11.

12’ 13. l-1.

15. 16.

15. 18. 19. 20. 21.

22.

23. 24.

C. E., LI.:LOIH, 1,. F., AND CHIllII%OG.\, J. (1955) J. Viol. Chem. 214,149-156. &RDINI, C. E., END I~ECONDO, E. (19(i2) Plmt Cell Physiol. 3,313-318. 1)~ FEKETE, M. A. I:., AND C.\RDINI, C. I<. (1963) Arch. Rioch,em. Hiophys. 104,173-184. AVIG.\D, G. (19&I) J. Hiol. Chem. 239, 31i133618. SL.\~NIK, I<., FHYI)M.\N, I<. B., AND C.UCUINI, C. E. (1968) Plan/ Ph!lsiol. 43,lO(i3-1068. PILI:SSEY, I<. (1969) Plaut Physiol. 44, 759%7G4. (~IZIYI~X, W. J., JONKS, B. K., AND ALW:KSFII~:IM, P. (1970) J. Riol. Chem. 246, 188-197. ~)I~;LMER, 1). P., .\ND ALIH~IWII~XM, P. (1970) Plats1 Physiol. 46, 78%78(i. ~~UILLTA, T. (1971) .\.ippoj! .Vogei k’ccguh-zc Kc&hi 46, 441-448. MuI~.\T.\, T. (1971) Agr. Hid. Chem. 36, 297299. C>WINI,

8HUKLA,

I<.

?;.,

.\SI)

s.\NL\..\L,

(;.

(;.

(1971)

Arch. Biochem. Hiophgs. 142,303-309. MI:R.IT.\, T. (1971) Agr. Niol. Chem. 36, 14-211448. ~;LoIR, L. F., .%ND C.\KDINI, c. 15. (19%) J. Biol. Chem. 214.157-165. AVIG.\D, G., .\NDMILNER, V. (1966) i(LMethods in Enzymology (S. P. Colowick and N. 0. Kaplan, eds.) Vol. VIII, 341-315, Academic Press, New York. i%RIT.\, T., SU‘iIy.\M.\, T., .&ND A~iaza\v.\, T. (1964) Arch. Biochem. Biophys. 10’7, 92-101. MLII~~T~~, T., SUGIY~M.\, T., MIN.\MIK.\KY, T., .\NI) AIL~z.\L~.\, T. (1966) Arch. Riochem. Iliophys. 113, 34-44. ~)ELMIGR, I>. P. (1972) J. Hiol. Chem. 247, 38223828. MUKIT.\, T., Aktaz~w.\, T., .\ND FUKUCHI, S. (1968) Plan1 Physiol. 43, 1899-1905. ICors, J. Ii. (1934) J. Hiol. Chem. 107,15-22. BRITTON, II. T. S., .YND R~IHNSON, R. A. (1931) J. Chew Sot. 458-473, 115G14G2. ORNSTKIN, IA., .\ND I).\vIs, B. J. (19ti2) Disc Electrophoresis. Reprinted bp Distillation Products Industries, Rochester, N. Y. HEDRICK, J. L., .1x1) SMITH, A. J. (1968) Arch. J?iochem. Hiophys. 126, 155-164. SURAN, A. A., .XND T.utvm, H. (1965) Arch. Biochem. Biophys. 111, 399306. HIRRISON, P. WI., .YND (;RI:GOIiy, 1). W. (1965) -1. Mol. Bid. 14, C“(iXi29.

652

NOMURA

AND

25. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 44Of-4412. 26. ANDREWS, P. (1964) Biochem. J. 91, 222-233. 27. MARTIN, R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. 28. THORN, P. W. (1965) Biochemistry 4,90&918. 29. SUGIYAMA, T., NAK~Y.~M.~, N., AND AKAU~A,

AKAZAWA T. (1968) Arch. Biochem. Biophys. 126, 737745. 30. SOBER, H. A. (1968) Handbook of Biochemistry, C-11, Chem. Rubber Co., Cleveland. 31. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.