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Enzymic mechanism of starch synthesis in sweet potato roots
ARCHIVES
OF
BIOCHEMISTRY
Enzymic
AND
BIOPHYSICS
Mechanism
126, 873-879 (1968)
of Starch
I. Requirement
Synthesis
of Potassium
in Sweet
Potato
Roots’
Ions for Starch Synthetase
T. MURATA Department
of Physiology
and Genetics, National
Institute
of Agricultural
Sciences, Kitamoto,
Saitama, Japan AND
T. AKAZAWA Biochemical
Regulation
Research Institute School of Agriculture, Nagoya, Japan
Nagoya
University
Chikusa,
Received May 9, 1968; accepted June 17, 1968 The effect of K+ on the starch synthesis catalyzed by the granules-bound ADPGZand UDPG-starch transglucosylase (starch synthetase) prepared from sweet potato roots, rice grains, white potato roots, and taro roots was examined. The magnitude of the stimulation was most marked by the sweet potato root ADPG-starch transglucosylase and was least by the taro and rice enzyme. From the results of kinetic experiment using sweet potato root enzyme, it was found that the addition of 0.1 M Kf increases the enzyme activity about 7-fold, and KA value was determined to be 1.3 x lo-* M. The level of K+ giving themaximum starchsynthetase activity was found to be about 0.05-0.1 M, roughly equivalent to the cellular concentration of K+ in the sweet potato roots. However, both K, (ADPG) and Ki (ADP) values were not affected by the addition of K+ to the reaction system. Pretreatment of the sweet potato enzyme with .K+ caused an appreciable protective effect against the heat-inactivation of enzyme (SO’ for 30 min), while the nearly complete loss of the enzyme activity occurred without Kf-treatment. Some physiological implications have been made concerning the dynamic role of K+ in regulating the starch synthesizing reaction in the sweet potat, roots. Many
workers
have
attempted
to eluci-
date the mechanism of the essentiality of K+ ions for the plant growth at the enzymic level. Several enzyme reactions have been shown to require K+ (1). A particularly notable advancement in this field has been made by Evans and his associates (2), showing the maintainance of the conformational structure of pyruvic kinase molecule in association with K+ as well as Rb+ and NH,+. 1 This research was supported in part by a research grant of Agricultural Research Services of U.S.D.A. under the P.L. 480 Program (FG-Ja 126). 2 Abbreviations used are : ADPG, adenosine diphosphate glucose; UDPG, uridine diphosphate glucose. 873
Some classical works have led to circumstantial evidence indicating the close connection between K+ and starch synthesis in plant cells (2, 3). As yet no single enzyme involved in the polysaccharide synthesis has been shown explicitly to require K+ (cf. 1). Basic enzymic mechanism of the starch synthesizing reaction in plant cells has been established (4), and attention of many recent workers appears to be focused on the regulatory
mechanism
of the polysaccharide
bio-
synthesis (5). Akatsuka and Nelson (6) found the organ-specific nature of starchsynthetase of maize, and reported that the magnitude of the stimulation of the starch synthesizing reaction by K+ addition is
874
MURATA
AND AKAZAWA
markedly different between embryonic and endospermic enzymes. They demonstrated also that some kinetic properties are distinguishable between two enzymes, but the crucial role of K+ in the starch synthesis reaction has not been fully studied by them. It has long been known that the application of K+-fertilizer is effective for the growth of sweet potato roots (7-9). During the course of our study on the regulatory mechanism of starch-synthesis, we have found that the granules-bound starch synthetase of sweet potato roots prepared by the thorough washing wit,h glass-pot distilled Hz0 showed the very low enzyme activity, while the addition of K+ greatly promoted the enzyme reaction. This paper deals with the detailed experimental results of the stimulation of the sweet potato starch synthetase by K+, and some inferences have been drawn regarding the physiological role of K+ in plant metabolism. MATERIALS
AND METHODS
Enzyme preparation. Granules-bound starch synthetase was prepared from appropriate plant tissues based on essentially the same way as that reported previously (10). For preparing the granular enzyme from root or tuber tissues, chilled 0.25% @-mercaptoethanol was used throughout. In order to wash thoroughly the starch granulesbound enzyme demineralized Hz0 was further subjected to the glass pot-distillation. Water washed enzyme samples were further washed with chilled acetone (-15”), and stored in vacua over P& in a cold room without much loss of the enzyme activity for several weeks. Enzyme assay method. Optimum conditions for the enzyme assay were found in a preliminary experiment to be at pH 8.5 (glycine buffer) and at 34”. Composition of the following reaction mixture (in rmoles) was selected as a standard system; glycine-NaOH buffer (pH 8.5), 5; EDTA, 0.3; ADPG-Cr4, 0.154 (13,420 dpm) or UDPG-Ci4, 0.144 (26,400 dpm) ; and either 2 mg (ADPG system) or 4 mg (UDPG system) of starch granules in a total volume of 16 al. Various cations were added to this reaction mixture to examine their effect on the enzyme reaction. At the termination of the reaction for 60 min, the Cl*-glucose incorporation into starch molecule was determined following the method as reported previously (11, 12). Radioactivity measurement was carried out using a Packard liquid scintillation spectrometer. All the assays were duplicated and average values are
40
0
30
60
90
INCUBATION
0 TIME
I
UDPG
30
60
SCI
(MINUTES)
FIG. 1. Stimulative effect of K+ on the activity of ADPG- and UDPG-starch transglucosylase of sweet potato roots. The standard reaction mixture described in text was used.
presented. Changes in protocols of reaction mixture are described in each experimental system. RESULTS
Experimental results presented in Fig. 1 show a marked stimulative effect of Kf on the activity of starch synthetase associated with the sweet potato starch granules; ADPG and UDPG being used as the glucose donor. It can be seen that up to 90 minutes of the incubation, the C14-glucose incorporation into the starch molecules proceeded nearly in a linear fashion in both systems. At the saturation level, the percentage utilization of glucose was about 35% for ADPG- and about 22% for UDPG-system, respectively. From the kinetic analyses of K+ concentration vs. the rate of the starch synthetase reaction (Fig. 2), the following values were obtained : KA (K+) = 1.33 X 10e2 M, and V msx= 27.1 mpmoles C14-glucose incorporated into starch/hr/mg starch granules. The flame photometric analysis has shown the presence of a minute amount of K+ attached to the starch granules3 Thus, assuming the activity seen without addition of K+ is due to K+ endogenous to the granules, we have used the values of AV for the double reciprocal analysis, where AV is the increase in enzyme activity due to addition of K+ (inset of the figure). There is naturally a possibility that the activity reflects either a combination of cations in the starch granules s Y. Tanaka and T. Akazawa, unpublished.
K+ AND STARCH
or simply enzyme activity in the absence of any cation (cf. Fig. 4). The level of K+ giving the maximum reaction velocity was about 0.05-0.1 M, which was roughly equivalent to the cellular concentration of K+ in the sweet potato root tissues. Experimental results concerning the effect of univalent cations on the activity of starch synthetase of sweet potato roots are presented in Table I. They clearly show that the most pronounced stimulative effect is
FIG. 2. Stimulative effect of various concentrations of K+ on the activity of ADPG-starch transglucosylase of sweet potato roots. The standard reaction mixture, except for various amounts of KC1 as shown in the figure, described in text was used. Incubation was at 34” for 60 min.
TABLE
I
EFFECT OF UNIVALENT CATIONS ON ADPGSTARCH TRANSGLUCOSYLASE ACTIVITY OF SWEET POTATO ROOTS~ Saks (0.1 aa)
None KC1 K&J04 (0.05 M) KI LiCl NaCl NazSOc (0.05 M) RbCl CsCl NH&l
“C-Glucose
6.7 50.1 49.7 42.3 19.0 11.3 12.0 47.0 32.3 30.7
100 748 742 631 284 169 179 702 482 459
0 The standard reaction mixture described in text, except for different types of cations (0.1 M or 0.05 M), was employed. Incubation was at 34’ for 60 min.
875
SYNTHETASE TABLE
II
EFFECT OF DIVALIZNT CATIONS ON UDPG-STARCH TRANSGLUCOSYLASE ACTIVITY OF SWEET POTATO ROOTS “C-Glucose
Salts
Concn. (as) K+ (0.1 n)
Relahve
incorporated activity into starch (%I (mpmoles)
Experiment I None CaClz 0.005 0.05 MgClz 0.005 0.05 MnClz 0.005 0.05 CUSOI 0.005 0.05 NiC12 0.005 0.05 CoClz 0.005 0.05
-
3.6 6.5 1.4 10.6 2.0 11.9 0.7 2.7 0 7.0 0.4 7.9 0.3
100 180 39 294 56 330 19 70 0 194 11 219 8
Experiment II None CaClt 0.005 0.05 MgClz 0.005 0.05 MnClz 0.005 0.05 cuso4 0.005 0.05 NiClz 0.005 0.05 CoClz 0.005 0.05
+ + + f + + + + + + f -I+
25.2 27.6 10.7 32.3 13.6 31.7 6.3 2.2 0 15.4 0.4 22.7 3.0
100 109 42 128 54 126 25 9 0 61 2 90 12
0 The standard reaction mixture as described in text, except for different types of cations, was employed. EDTA was omitted from the assay mixture. Incubation was at 34” for 60 min.
given by K+, while Rb+, Csf, and NH,+ exhibited also a marked stimulation. However, neither Na+ nor Li+ as well as anions exhibit a great stimulation on the enzyme reaction. We then examined to see whether or not there exists any interaction between K+ and various other divalent metal ions. In this particular experiment, UDPG was used as the glucose donor. As shown in Table II, in the presence of K+, Ca++ and Mg++ exhibited a slight stimulative effect on the starch synthesizing reaction at their low concentration (5 X 10e3 M), while at
876
MURATA
AND
AKAZAWA
TABLE III 0.05 M they were inhibitory. Even at 5 X EFFECT OF K+ ON UDPG-STARCH TRANSGLUCOSYL1O-3 M, Cu++, Ni++, and Co++ were inhibiASE ACTIVITY OF DIFFERENT PLANT ORIGINS” tory. In the absence of K+, all the cations except Cu++ showed an appreciable stimula“C-Glucose ReConcenti;eFcpo;lative tive effect at the low concentration (5 X Expt. Source of enzyme ration of activity no. starch K+ (~1 1O-3 M), but was inhibitory at 0.05 M. (%) bnlLmoles) The effect of CaClz and MgCL at their various concentrations on the enzyme reac3.5 100 1. Sweet potato 0 28.0 800 tion is summarized in Fig. 3, showing the 0.01 20.2 577 0.1 marked stimulation of both salts at 5 X 10m3 2. White 1
I
3. Rice
4. Taro
pot,ato
0 0.01 0.1
12.4 18.9 13.9
100 148 109
0 0.01 0.1 0 0.01 0.1
15.4 19.6 18.5 0.7 0.6 0.6
100 127 120 -
a The standard reaction mixture as described in text, except for the addition of K+, was employed. Incubation was at 34” for 60 min.
=uoL--. SALTS
(N?M)
FIG. 3. Effect of C&12 and MgC& on the activity of UDPG-starch transglucosylase of sweet potato roots. The standard reaction mixture, except for various amounts of either CaClz or MgClz as shown in the figure, was used. Incubation was at 34’ for 60 min. 1
I’
uo,
0
and the inhibitory effect of 5 X 1O-2 M CaCL Results are reminiscent of the curtailment of the sweet potato root growth by the surplus application of the Ca++-fertilizer (9). We next examined the stimulative effect of K+ on the starch synthetase reaction catalyzed by the granules-bound enzyme of white potato, taro, and rice (Fig. 4). It will be noted that the level of K+ giving the maximum effect was around 0.02 M for white potato enzyme. In both rice and taro enzyme, on the other hand, the optimum Kf level was at 0.024.05 M and higher K+ concentrations caused a somewhat inhibitory effect. However, the increase of C14-glucose incorporation in white potato starts from a higher initial enzyme activity than that in rice. Accordingly, the percentage increase is much the same between white potato and rice; white potato increases from 32 to about 60 mpmoles (nearly 100 % increase) and rice from 14 to 28 mpmoles (100 % increase). The Ii+-eff ect was least in the taro enzyme. Results presented in Table III show the effect of K+ (0.01 M and 0.1 M) on the starch synthetase activity of different plant origins, using UDPG as the substrate. In every case, except tar0 enzyme, essentially a trend similar to that observed M
012345
.I!.
002 cm4+006 008 0.1 ” a5 K (Ml
FIG. 4. Stimulative effect of various concentrations of K+ on the activity of ADPG-starch transglucosylase of white potato roots, taro roots, and rice grains. The standard reaction mixture, except for various amounts of KC1 as shown in the figure, was used. Incubation was at 34’ for 60 min. For the method of enzyme preparation see text.
K+ AND
STARCH
by the ADPG-starch transglucosylation reaction was obtained. Regardless of the addition of K+ to the reaction mixture, apparent failure to detect the enzyme activity in the granules-bound taro root enzyme is particularly noteworthy. There has not been reported any granules-bound starch synthetase of root or tuber origin, requiring ADPG specifically as the glucose donor (13, 14). We thus examined the substrate specificity of the taro enzyme in some detail. Results shown in Fig. 5 clearly demonstrate the complete inertness of UDPG in the starch synthetase prepared from the taro root tissues. We have previously reported the kinetic analysis of the ADP-inhibition on the starch synthetase of rice grains, in a particular relation to the protective effect of ADP against the monoiodoacetate inhibition of the enzyme reaction (11). It was thought to be of value to examine the possible effect of K+ on the ADP-inhibition of the sweet potato enzyme. But results of Fig. 6 show that K+ does not affect either the apparent affinity of the enzyme toward ADPG or the inhibition by ADP, K, (ADPG), 3.4 X 10M3M, and Ki (ADP), 1.9 X IOh M being essentially the same, regardless of the addition of K+ to the assay mixture. The Vmax value was determined to be 6.8 mpmoles C14-glucose incorporated into starch/hr/mg starch granules in the absence of K+ and 51.3 mpmoles/hr/mg starch granules in the presence of I(+, respectively. Akatsuka and Nelson (6) reported the
FIG. 5. Effect of K+ on the activity of ADPGand UDPGstarch transglucosylase of taro roots. The standard reaction mixture was used for examining the effect of K+ on the enzyme activity.
SYNTHETASE
‘4 FIG. 6. Double reciprocal plot of the K+-effect on the ADP-inhibition of ADPG-starch transglucosylase of sweet potato roots. The standard reaction mixture, except for various amounts of ADPG as shown in the figure, was used. To determine the effect of each of K+ and ADP, each of 0.1 M KC1 and 9.93 X NYM ADP was added to the reaction mixture in a total volume of 22 ~1. Incubation was at 34” for 30 min.
FIG. 7. Protective effect of K+ against the heatinactivation of ADPG-starch transglucosylase of sweet potato roots. To 2 mg sweet potato starch granules were added, (a) 5 wales glycine buffer (pH 8.5), (b) plus 0.3 rmole EDTA, (c) plus 1.6 pmoles KCl, and (d) plus EDTA and KCl. Total volume was 16 ~1 in every case. The whole mixture was then incubated at either 0 or 66” for 30 min. Afterwards, activity of ADPG-starch transglucosylase was determined by adding either 0.154 pmole of ADPG (unshaded bars) or ADPG plus 1.6 pmoles of KC1 (shaded bars). In order to unify the condition of the enzyme reaction, EDTA (0.3 pmole) was added to the reaction mixture (a) and (c). Incubation was at 34” for 60 min.
MURATA
AND AKAZAWA
k
(M)
FIG. 8. Protective
effect of various concentrations of K+ against the heat-inactivation of ADPG-starch transglucosylase of sweet potato roots. Basic procedures of treating starch granules were the same as those shown in experiments of Fig. 7, except 0.3 rmole of EDTA (+EDTA) or various amounts of KC1 as shown in the figure, were added to the incubation mixture (30 minutes, 0 or 60’). Then, the standard assay system as explained in text was used by adding 0.154 rmole of ADPG for determining the enzyme activity. EDTA was omitted from the reaction mixture except (+EDTA) system. Incubation was at 34” for 60 min.
stabilizing effect of K+ on the maize embryo starch synthetase as reflected in the protection against the heat-inactivation of the enzyme by the K+-addition. Our results shown in Fig. 7 also demonstrate that the pretreatment of the sweet potato enzyme with K+ markedly protected the enzyme against the subsequent heat-treatment at 60” for 30 min. Experimental results portrayed in Fig. 8 show that the protective effect of K+ against the heat-inactivation was detectable only at above 0.025 M, much higher than the level causing the stimulative effect on the enzyme reaction (cf. Fig. 2). It thus appears that the mode of action of K+ in stimulating the starch synthesizing reaction is distinguishable from that of the enzyme protection against heat-inactivation. DISCUSSION
Explicit in the experimental results reported in this paper is the indispensable requirement of K+ for the starch synthesizing reaction catalyzed by the granules-bound enzyme of sweet potato roots. Less markedly,
a similar stimulative effect was observed by the granular enzyme prepared from white potato, taro and rice. Although there is no evidence that K+ content fluctuates in the cell due to any metabolic activity, it is tempting to speculate about the rate of the starch synthesizing reaction as being held in check by the availability of K+ in plant cells. Some plants, e.g., sweet potato, white potato and sugar beet, are often called “Kalipflanzen” because of their high rate of K+absorption. From the decrease in starch content in these plants under the K+-deficient conditions, a possible involvement of K+ in the polysaccharide-synthesizing system has been postulated (1, 3). It has long been known that the root growth (starch accumulation) of sweet potato is most effectively stimulated by the K+-fertilizer, compared with some other plants. Thus our present experimental results at the enzyme level report for the first time the novel stimulative effect of K+ on the st,arch synthesis in plant cells related to the phenomena occurring in viva. The development of cereal grains, e.g., rice, and root growth of taro are known to be less markedly affected by the K+-application. It merits description about the effect of K+ on starch synthetase of white potato. Cellular levels of Kf in white potato tuber are as high as in sweet potato, though K+-fertilizer is not effective compared with the latter plant. In fact results shown in Fig. 3 indicate that the stimulative effect of K+ on the activity of the granules-bound starch synthetase of white potato starts to level off at the relatively low concentration, about 0.02 M. It is indeed an intriguing subject for future investigations to elucidate the nature of the differential effect of Kf on the different enzymes from the standpoint of the structural organization of the polysaccharideenzyme complex. The uniqueness of K+-stimulation of the granules-bound sweet potato starch synthetase is in marked contrast to the reported K+-effect on the starch synthetase of Chlorella (15) as well as on the bacterial a(1 -+ 4) glucansynthetases (16, 17). In the case of the latter three enzymes, a marked decline of the enzyme activity occurred by
K+ AND STARCH
withdrawing K+ together with G-SH and bovine serum albumin from the reaction mixture. Furthermore, our experimental results have shown that the activity of both ADPG- and UDPG-starch transglucosylase was stimulated by K+, whereas Nelson and Akatsuka (6) reported that the K+-addition was inhibitory to UDPG-starch transglucosylase of maize seed. Our present experimental result does not provide a direct clue as to the essentiality of K+ in starch synthetase at the molecular level. Although the heat-inactivation of the granular enzyme was appreciably protected by the pretreatment of the enzyme with K+, some kinetic constants, K, (ADPG) and Ki (ADP), were not affected by the addition of K+. One of the interpretations would be that K+ causes stronger binding of the starch granules to the enzyme molecule, and in this way stabilizes the enzyme. The mode of activation of starch synthetase by K+ appears to be quite different from that of some other K+-requiring enzymes (1, 2), and the more thorough investigation is needed to substantiate the point. ACKNOWLEDGMENTS The authors wish to record their most sincere thanks to Dr. David F. Houston of Western Regional Research Laboratory of U.S.D.A. Agricultural Research Services, Albany, California, for his counsel and support of this research. They are also much indebted to Miss S. Fukuchi for her competent technical assistance.
879
SYNTHETASE REFERENCES
1. EVANS, H. J., AND SORGER, G. J., Ann. Rev. PIant Physiol. 17,47 (1966). 2. SORGER, G. J., FORD, R. E., AND EVANS, H. J., Proc. Natl. Acad. Sci. 64,1614 (1965).
3. LUNDEGARDH, H., “Klima und Boden in ihrer Wirkung auf das Pjlanzenleben” p. 361. Japanese Edition. Iwanami Pub. Co. Ltd. Tokyo (1964). 4. AKAZAWA, T., Plant Biochemistry (J. Bonner and J. E. Varner eds.), p. 258. Academic Press, New York (1965). 5. GHOSH, H. P., AND PREISS, J., J. Biol. Chem. Ml,4491
(1966).
6. ARATSUKA, T., AND NELSON, 0. E., J. Biot. Chem. 241, 2280 (1966).
7. SUGAWARA, T., J. Sci. Soil Manure Japan
12, 154 (1938). 8. TOGARI, Y., Nogyo oyobi Engei (in Japanese) 23, 299 (1948). 9. KAMATANI, E., Nogyo oyobi Engei (in
Japanese) 20,373 (1945). 10. AKAZAWA, T., MINUIIKAWA, T., AND MURATA, T., Plant Physiol. 39, 371 (1964). 11. MURATA, T., SUGIYAMA, T., MINAMIKAWA, T., AND AKAZAWA, T., Arch. Biochem. Biophys. 113, 34 (1966).
12. MTJRATA, T., NAKAYAMA, AND AKAZAWA, T., Arch.
N., TANAKA, Y., B&hem. Biophys.
123,97 (1968). 13. TANAKA, Y., AND AKAZAWA, T., Plant Cell Physiol. 9, 445 (1968). 14. TANAKA, Y., AND AKAZAWA, T., submitted to Arch. Biochem. Biophys. (1968). 15. PREISS, J., AND GREENBERG, E., Arch. Biothem. Biophys. 118,702 (1967). 16. GREENBERG, E., AND PREISS, J., J. Biol. Chem. 940, 2341 (1965). 17. PREISS, J., AND GREENBERG, E., Biochemistry
4,232s (1965).
OF
BIOCHEMISTRY
Enzymic
AND
BIOPHYSICS
Mechanism
126, 873-879 (1968)
of Starch
I. Requirement
Synthesis
of Potassium
in Sweet
Potato
Roots’
Ions for Starch Synthetase
T. MURATA Department
of Physiology
and Genetics, National
Institute
of Agricultural
Sciences, Kitamoto,
Saitama, Japan AND
T. AKAZAWA Biochemical
Regulation
Research Institute School of Agriculture, Nagoya, Japan
Nagoya
University
Chikusa,
Received May 9, 1968; accepted June 17, 1968 The effect of K+ on the starch synthesis catalyzed by the granules-bound ADPGZand UDPG-starch transglucosylase (starch synthetase) prepared from sweet potato roots, rice grains, white potato roots, and taro roots was examined. The magnitude of the stimulation was most marked by the sweet potato root ADPG-starch transglucosylase and was least by the taro and rice enzyme. From the results of kinetic experiment using sweet potato root enzyme, it was found that the addition of 0.1 M Kf increases the enzyme activity about 7-fold, and KA value was determined to be 1.3 x lo-* M. The level of K+ giving themaximum starchsynthetase activity was found to be about 0.05-0.1 M, roughly equivalent to the cellular concentration of K+ in the sweet potato roots. However, both K, (ADPG) and Ki (ADP) values were not affected by the addition of K+ to the reaction system. Pretreatment of the sweet potato enzyme with .K+ caused an appreciable protective effect against the heat-inactivation of enzyme (SO’ for 30 min), while the nearly complete loss of the enzyme activity occurred without Kf-treatment. Some physiological implications have been made concerning the dynamic role of K+ in regulating the starch synthesizing reaction in the sweet potat, roots. Many
workers
have
attempted
to eluci-
date the mechanism of the essentiality of K+ ions for the plant growth at the enzymic level. Several enzyme reactions have been shown to require K+ (1). A particularly notable advancement in this field has been made by Evans and his associates (2), showing the maintainance of the conformational structure of pyruvic kinase molecule in association with K+ as well as Rb+ and NH,+. 1 This research was supported in part by a research grant of Agricultural Research Services of U.S.D.A. under the P.L. 480 Program (FG-Ja 126). 2 Abbreviations used are : ADPG, adenosine diphosphate glucose; UDPG, uridine diphosphate glucose. 873
Some classical works have led to circumstantial evidence indicating the close connection between K+ and starch synthesis in plant cells (2, 3). As yet no single enzyme involved in the polysaccharide synthesis has been shown explicitly to require K+ (cf. 1). Basic enzymic mechanism of the starch synthesizing reaction in plant cells has been established (4), and attention of many recent workers appears to be focused on the regulatory
mechanism
of the polysaccharide
bio-
synthesis (5). Akatsuka and Nelson (6) found the organ-specific nature of starchsynthetase of maize, and reported that the magnitude of the stimulation of the starch synthesizing reaction by K+ addition is
874
MURATA
AND AKAZAWA
markedly different between embryonic and endospermic enzymes. They demonstrated also that some kinetic properties are distinguishable between two enzymes, but the crucial role of K+ in the starch synthesis reaction has not been fully studied by them. It has long been known that the application of K+-fertilizer is effective for the growth of sweet potato roots (7-9). During the course of our study on the regulatory mechanism of starch-synthesis, we have found that the granules-bound starch synthetase of sweet potato roots prepared by the thorough washing wit,h glass-pot distilled Hz0 showed the very low enzyme activity, while the addition of K+ greatly promoted the enzyme reaction. This paper deals with the detailed experimental results of the stimulation of the sweet potato starch synthetase by K+, and some inferences have been drawn regarding the physiological role of K+ in plant metabolism. MATERIALS
AND METHODS
Enzyme preparation. Granules-bound starch synthetase was prepared from appropriate plant tissues based on essentially the same way as that reported previously (10). For preparing the granular enzyme from root or tuber tissues, chilled 0.25% @-mercaptoethanol was used throughout. In order to wash thoroughly the starch granulesbound enzyme demineralized Hz0 was further subjected to the glass pot-distillation. Water washed enzyme samples were further washed with chilled acetone (-15”), and stored in vacua over P& in a cold room without much loss of the enzyme activity for several weeks. Enzyme assay method. Optimum conditions for the enzyme assay were found in a preliminary experiment to be at pH 8.5 (glycine buffer) and at 34”. Composition of the following reaction mixture (in rmoles) was selected as a standard system; glycine-NaOH buffer (pH 8.5), 5; EDTA, 0.3; ADPG-Cr4, 0.154 (13,420 dpm) or UDPG-Ci4, 0.144 (26,400 dpm) ; and either 2 mg (ADPG system) or 4 mg (UDPG system) of starch granules in a total volume of 16 al. Various cations were added to this reaction mixture to examine their effect on the enzyme reaction. At the termination of the reaction for 60 min, the Cl*-glucose incorporation into starch molecule was determined following the method as reported previously (11, 12). Radioactivity measurement was carried out using a Packard liquid scintillation spectrometer. All the assays were duplicated and average values are
40
0
30
60
90
INCUBATION
0 TIME
I
UDPG
30
60
SCI
(MINUTES)
FIG. 1. Stimulative effect of K+ on the activity of ADPG- and UDPG-starch transglucosylase of sweet potato roots. The standard reaction mixture described in text was used.
presented. Changes in protocols of reaction mixture are described in each experimental system. RESULTS
Experimental results presented in Fig. 1 show a marked stimulative effect of Kf on the activity of starch synthetase associated with the sweet potato starch granules; ADPG and UDPG being used as the glucose donor. It can be seen that up to 90 minutes of the incubation, the C14-glucose incorporation into the starch molecules proceeded nearly in a linear fashion in both systems. At the saturation level, the percentage utilization of glucose was about 35% for ADPG- and about 22% for UDPG-system, respectively. From the kinetic analyses of K+ concentration vs. the rate of the starch synthetase reaction (Fig. 2), the following values were obtained : KA (K+) = 1.33 X 10e2 M, and V msx= 27.1 mpmoles C14-glucose incorporated into starch/hr/mg starch granules. The flame photometric analysis has shown the presence of a minute amount of K+ attached to the starch granules3 Thus, assuming the activity seen without addition of K+ is due to K+ endogenous to the granules, we have used the values of AV for the double reciprocal analysis, where AV is the increase in enzyme activity due to addition of K+ (inset of the figure). There is naturally a possibility that the activity reflects either a combination of cations in the starch granules s Y. Tanaka and T. Akazawa, unpublished.
K+ AND STARCH
or simply enzyme activity in the absence of any cation (cf. Fig. 4). The level of K+ giving the maximum reaction velocity was about 0.05-0.1 M, which was roughly equivalent to the cellular concentration of K+ in the sweet potato root tissues. Experimental results concerning the effect of univalent cations on the activity of starch synthetase of sweet potato roots are presented in Table I. They clearly show that the most pronounced stimulative effect is
FIG. 2. Stimulative effect of various concentrations of K+ on the activity of ADPG-starch transglucosylase of sweet potato roots. The standard reaction mixture, except for various amounts of KC1 as shown in the figure, described in text was used. Incubation was at 34” for 60 min.
TABLE
I
EFFECT OF UNIVALENT CATIONS ON ADPGSTARCH TRANSGLUCOSYLASE ACTIVITY OF SWEET POTATO ROOTS~ Saks (0.1 aa)
None KC1 K&J04 (0.05 M) KI LiCl NaCl NazSOc (0.05 M) RbCl CsCl NH&l
“C-Glucose
6.7 50.1 49.7 42.3 19.0 11.3 12.0 47.0 32.3 30.7
100 748 742 631 284 169 179 702 482 459
0 The standard reaction mixture described in text, except for different types of cations (0.1 M or 0.05 M), was employed. Incubation was at 34’ for 60 min.
875
SYNTHETASE TABLE
II
EFFECT OF DIVALIZNT CATIONS ON UDPG-STARCH TRANSGLUCOSYLASE ACTIVITY OF SWEET POTATO ROOTS “C-Glucose
Salts
Concn. (as) K+ (0.1 n)
Relahve
incorporated activity into starch (%I (mpmoles)
Experiment I None CaClz 0.005 0.05 MgClz 0.005 0.05 MnClz 0.005 0.05 CUSOI 0.005 0.05 NiC12 0.005 0.05 CoClz 0.005 0.05
-
3.6 6.5 1.4 10.6 2.0 11.9 0.7 2.7 0 7.0 0.4 7.9 0.3
100 180 39 294 56 330 19 70 0 194 11 219 8
Experiment II None CaClt 0.005 0.05 MgClz 0.005 0.05 MnClz 0.005 0.05 cuso4 0.005 0.05 NiClz 0.005 0.05 CoClz 0.005 0.05
+ + + f + + + + + + f -I+
25.2 27.6 10.7 32.3 13.6 31.7 6.3 2.2 0 15.4 0.4 22.7 3.0
100 109 42 128 54 126 25 9 0 61 2 90 12
0 The standard reaction mixture as described in text, except for different types of cations, was employed. EDTA was omitted from the assay mixture. Incubation was at 34” for 60 min.
given by K+, while Rb+, Csf, and NH,+ exhibited also a marked stimulation. However, neither Na+ nor Li+ as well as anions exhibit a great stimulation on the enzyme reaction. We then examined to see whether or not there exists any interaction between K+ and various other divalent metal ions. In this particular experiment, UDPG was used as the glucose donor. As shown in Table II, in the presence of K+, Ca++ and Mg++ exhibited a slight stimulative effect on the starch synthesizing reaction at their low concentration (5 X 10e3 M), while at
876
MURATA
AND
AKAZAWA
TABLE III 0.05 M they were inhibitory. Even at 5 X EFFECT OF K+ ON UDPG-STARCH TRANSGLUCOSYL1O-3 M, Cu++, Ni++, and Co++ were inhibiASE ACTIVITY OF DIFFERENT PLANT ORIGINS” tory. In the absence of K+, all the cations except Cu++ showed an appreciable stimula“C-Glucose ReConcenti;eFcpo;lative tive effect at the low concentration (5 X Expt. Source of enzyme ration of activity no. starch K+ (~1 1O-3 M), but was inhibitory at 0.05 M. (%) bnlLmoles) The effect of CaClz and MgCL at their various concentrations on the enzyme reac3.5 100 1. Sweet potato 0 28.0 800 tion is summarized in Fig. 3, showing the 0.01 20.2 577 0.1 marked stimulation of both salts at 5 X 10m3 2. White 1
I
3. Rice
4. Taro
pot,ato
0 0.01 0.1
12.4 18.9 13.9
100 148 109
0 0.01 0.1 0 0.01 0.1
15.4 19.6 18.5 0.7 0.6 0.6
100 127 120 -
a The standard reaction mixture as described in text, except for the addition of K+, was employed. Incubation was at 34” for 60 min.
=uoL--. SALTS
(N?M)
FIG. 3. Effect of C&12 and MgC& on the activity of UDPG-starch transglucosylase of sweet potato roots. The standard reaction mixture, except for various amounts of either CaClz or MgClz as shown in the figure, was used. Incubation was at 34’ for 60 min. 1
I’
uo,
0
and the inhibitory effect of 5 X 1O-2 M CaCL Results are reminiscent of the curtailment of the sweet potato root growth by the surplus application of the Ca++-fertilizer (9). We next examined the stimulative effect of K+ on the starch synthetase reaction catalyzed by the granules-bound enzyme of white potato, taro, and rice (Fig. 4). It will be noted that the level of K+ giving the maximum effect was around 0.02 M for white potato enzyme. In both rice and taro enzyme, on the other hand, the optimum Kf level was at 0.024.05 M and higher K+ concentrations caused a somewhat inhibitory effect. However, the increase of C14-glucose incorporation in white potato starts from a higher initial enzyme activity than that in rice. Accordingly, the percentage increase is much the same between white potato and rice; white potato increases from 32 to about 60 mpmoles (nearly 100 % increase) and rice from 14 to 28 mpmoles (100 % increase). The Ii+-eff ect was least in the taro enzyme. Results presented in Table III show the effect of K+ (0.01 M and 0.1 M) on the starch synthetase activity of different plant origins, using UDPG as the substrate. In every case, except tar0 enzyme, essentially a trend similar to that observed M
012345
.I!.
002 cm4+006 008 0.1 ” a5 K (Ml
FIG. 4. Stimulative effect of various concentrations of K+ on the activity of ADPG-starch transglucosylase of white potato roots, taro roots, and rice grains. The standard reaction mixture, except for various amounts of KC1 as shown in the figure, was used. Incubation was at 34’ for 60 min. For the method of enzyme preparation see text.
K+ AND
STARCH
by the ADPG-starch transglucosylation reaction was obtained. Regardless of the addition of K+ to the reaction mixture, apparent failure to detect the enzyme activity in the granules-bound taro root enzyme is particularly noteworthy. There has not been reported any granules-bound starch synthetase of root or tuber origin, requiring ADPG specifically as the glucose donor (13, 14). We thus examined the substrate specificity of the taro enzyme in some detail. Results shown in Fig. 5 clearly demonstrate the complete inertness of UDPG in the starch synthetase prepared from the taro root tissues. We have previously reported the kinetic analysis of the ADP-inhibition on the starch synthetase of rice grains, in a particular relation to the protective effect of ADP against the monoiodoacetate inhibition of the enzyme reaction (11). It was thought to be of value to examine the possible effect of K+ on the ADP-inhibition of the sweet potato enzyme. But results of Fig. 6 show that K+ does not affect either the apparent affinity of the enzyme toward ADPG or the inhibition by ADP, K, (ADPG), 3.4 X 10M3M, and Ki (ADP), 1.9 X IOh M being essentially the same, regardless of the addition of K+ to the assay mixture. The Vmax value was determined to be 6.8 mpmoles C14-glucose incorporated into starch/hr/mg starch granules in the absence of K+ and 51.3 mpmoles/hr/mg starch granules in the presence of I(+, respectively. Akatsuka and Nelson (6) reported the
FIG. 5. Effect of K+ on the activity of ADPGand UDPGstarch transglucosylase of taro roots. The standard reaction mixture was used for examining the effect of K+ on the enzyme activity.
SYNTHETASE
‘4 FIG. 6. Double reciprocal plot of the K+-effect on the ADP-inhibition of ADPG-starch transglucosylase of sweet potato roots. The standard reaction mixture, except for various amounts of ADPG as shown in the figure, was used. To determine the effect of each of K+ and ADP, each of 0.1 M KC1 and 9.93 X NYM ADP was added to the reaction mixture in a total volume of 22 ~1. Incubation was at 34” for 30 min.
FIG. 7. Protective effect of K+ against the heatinactivation of ADPG-starch transglucosylase of sweet potato roots. To 2 mg sweet potato starch granules were added, (a) 5 wales glycine buffer (pH 8.5), (b) plus 0.3 rmole EDTA, (c) plus 1.6 pmoles KCl, and (d) plus EDTA and KCl. Total volume was 16 ~1 in every case. The whole mixture was then incubated at either 0 or 66” for 30 min. Afterwards, activity of ADPG-starch transglucosylase was determined by adding either 0.154 pmole of ADPG (unshaded bars) or ADPG plus 1.6 pmoles of KC1 (shaded bars). In order to unify the condition of the enzyme reaction, EDTA (0.3 pmole) was added to the reaction mixture (a) and (c). Incubation was at 34” for 60 min.
MURATA
AND AKAZAWA
k
(M)
FIG. 8. Protective
effect of various concentrations of K+ against the heat-inactivation of ADPG-starch transglucosylase of sweet potato roots. Basic procedures of treating starch granules were the same as those shown in experiments of Fig. 7, except 0.3 rmole of EDTA (+EDTA) or various amounts of KC1 as shown in the figure, were added to the incubation mixture (30 minutes, 0 or 60’). Then, the standard assay system as explained in text was used by adding 0.154 rmole of ADPG for determining the enzyme activity. EDTA was omitted from the reaction mixture except (+EDTA) system. Incubation was at 34” for 60 min.
stabilizing effect of K+ on the maize embryo starch synthetase as reflected in the protection against the heat-inactivation of the enzyme by the K+-addition. Our results shown in Fig. 7 also demonstrate that the pretreatment of the sweet potato enzyme with K+ markedly protected the enzyme against the subsequent heat-treatment at 60” for 30 min. Experimental results portrayed in Fig. 8 show that the protective effect of K+ against the heat-inactivation was detectable only at above 0.025 M, much higher than the level causing the stimulative effect on the enzyme reaction (cf. Fig. 2). It thus appears that the mode of action of K+ in stimulating the starch synthesizing reaction is distinguishable from that of the enzyme protection against heat-inactivation. DISCUSSION
Explicit in the experimental results reported in this paper is the indispensable requirement of K+ for the starch synthesizing reaction catalyzed by the granules-bound enzyme of sweet potato roots. Less markedly,
a similar stimulative effect was observed by the granular enzyme prepared from white potato, taro and rice. Although there is no evidence that K+ content fluctuates in the cell due to any metabolic activity, it is tempting to speculate about the rate of the starch synthesizing reaction as being held in check by the availability of K+ in plant cells. Some plants, e.g., sweet potato, white potato and sugar beet, are often called “Kalipflanzen” because of their high rate of K+absorption. From the decrease in starch content in these plants under the K+-deficient conditions, a possible involvement of K+ in the polysaccharide-synthesizing system has been postulated (1, 3). It has long been known that the root growth (starch accumulation) of sweet potato is most effectively stimulated by the K+-fertilizer, compared with some other plants. Thus our present experimental results at the enzyme level report for the first time the novel stimulative effect of K+ on the st,arch synthesis in plant cells related to the phenomena occurring in viva. The development of cereal grains, e.g., rice, and root growth of taro are known to be less markedly affected by the K+-application. It merits description about the effect of K+ on starch synthetase of white potato. Cellular levels of Kf in white potato tuber are as high as in sweet potato, though K+-fertilizer is not effective compared with the latter plant. In fact results shown in Fig. 3 indicate that the stimulative effect of K+ on the activity of the granules-bound starch synthetase of white potato starts to level off at the relatively low concentration, about 0.02 M. It is indeed an intriguing subject for future investigations to elucidate the nature of the differential effect of Kf on the different enzymes from the standpoint of the structural organization of the polysaccharideenzyme complex. The uniqueness of K+-stimulation of the granules-bound sweet potato starch synthetase is in marked contrast to the reported K+-effect on the starch synthetase of Chlorella (15) as well as on the bacterial a(1 -+ 4) glucansynthetases (16, 17). In the case of the latter three enzymes, a marked decline of the enzyme activity occurred by
K+ AND STARCH
withdrawing K+ together with G-SH and bovine serum albumin from the reaction mixture. Furthermore, our experimental results have shown that the activity of both ADPG- and UDPG-starch transglucosylase was stimulated by K+, whereas Nelson and Akatsuka (6) reported that the K+-addition was inhibitory to UDPG-starch transglucosylase of maize seed. Our present experimental result does not provide a direct clue as to the essentiality of K+ in starch synthetase at the molecular level. Although the heat-inactivation of the granular enzyme was appreciably protected by the pretreatment of the enzyme with K+, some kinetic constants, K, (ADPG) and Ki (ADP), were not affected by the addition of K+. One of the interpretations would be that K+ causes stronger binding of the starch granules to the enzyme molecule, and in this way stabilizes the enzyme. The mode of activation of starch synthetase by K+ appears to be quite different from that of some other K+-requiring enzymes (1, 2), and the more thorough investigation is needed to substantiate the point. ACKNOWLEDGMENTS The authors wish to record their most sincere thanks to Dr. David F. Houston of Western Regional Research Laboratory of U.S.D.A. Agricultural Research Services, Albany, California, for his counsel and support of this research. They are also much indebted to Miss S. Fukuchi for her competent technical assistance.
879
SYNTHETASE REFERENCES
1. EVANS, H. J., AND SORGER, G. J., Ann. Rev. PIant Physiol. 17,47 (1966). 2. SORGER, G. J., FORD, R. E., AND EVANS, H. J., Proc. Natl. Acad. Sci. 64,1614 (1965).
3. LUNDEGARDH, H., “Klima und Boden in ihrer Wirkung auf das Pjlanzenleben” p. 361. Japanese Edition. Iwanami Pub. Co. Ltd. Tokyo (1964). 4. AKAZAWA, T., Plant Biochemistry (J. Bonner and J. E. Varner eds.), p. 258. Academic Press, New York (1965). 5. GHOSH, H. P., AND PREISS, J., J. Biol. Chem. Ml,4491
(1966).
6. ARATSUKA, T., AND NELSON, 0. E., J. Biot. Chem. 241, 2280 (1966).
7. SUGAWARA, T., J. Sci. Soil Manure Japan
12, 154 (1938). 8. TOGARI, Y., Nogyo oyobi Engei (in Japanese) 23, 299 (1948). 9. KAMATANI, E., Nogyo oyobi Engei (in
Japanese) 20,373 (1945). 10. AKAZAWA, T., MINUIIKAWA, T., AND MURATA, T., Plant Physiol. 39, 371 (1964). 11. MURATA, T., SUGIYAMA, T., MINAMIKAWA, T., AND AKAZAWA, T., Arch. Biochem. Biophys. 113, 34 (1966).
12. MTJRATA, T., NAKAYAMA, AND AKAZAWA, T., Arch.
N., TANAKA, Y., B&hem. Biophys.
123,97 (1968). 13. TANAKA, Y., AND AKAZAWA, T., Plant Cell Physiol. 9, 445 (1968). 14. TANAKA, Y., AND AKAZAWA, T., submitted to Arch. Biochem. Biophys. (1968). 15. PREISS, J., AND GREENBERG, E., Arch. Biothem. Biophys. 118,702 (1967). 16. GREENBERG, E., AND PREISS, J., J. Biol. Chem. 940, 2341 (1965). 17. PREISS, J., AND GREENBERG, E., Biochemistry
4,232s (1965).