Seasonal Changes of Activities of Asparagine Synthetase and Asparaginase in Relation to Asparagine Content in Sugarcane

( 45) Nippon Shokuhin Kogyo Gakkaishi Vol. 34, No. 10, 673～679 (1987) 〔Article〕 673

Toshiyuki MATSUI* and Hirotoshi KITAGAWA* *D epartment of Agroindustrial Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07

We studied on the seasonal changes of asparagine, aspartic acid, glutamine and glutamic acid contents and activities of asparagine and glutamine synthetases and asparaginase and glutaminase in stalks and roots of two varieties of sugarcanes, 'Chikusha' and 'N:CO 310'. The highest activity of asparagine synthetase was observed in the basal part of stalks in August, whereas that of asparaginase was observed in September. Asparagine synthetase showed the highest activity at root in June and/or July. In 'N:CO', asparagine content in the basal part of stalk continued to decrease until October, whereas that in the upper part of stalk increased in September to November. In 'Chikusha', asparagine showed the highest content in the stalk in July, and its content increased in the middle and top parts of the stalk in September to November. The best harvest time of 'Chikusha' sugarcane is considered to be in November on the basis of the highest weight and asparagine contents in stalk.

The quality of the "Wasanbon" sugar great- Materials and Methods ly depends on the contents of several compo- nents in sugarcane such as amino acids, reduc- Materials ing sugar, aroma substances and sucrose. The Sugarcanes germinated for one month in amino acids cause very important coloring green house were transferred to the field at the matters formed by so-called amino-carbonyl end of April. Twenty kg of compost, 15kg of reaction. In a previous paper1), we reported rice bran and 10kg of complex fertilizer, "Nissan 525" per 10mm2 already on seasonal changes of the enzyme were given to the field activities in relation to sucrose. at the beginning of April, and 5kg of the com- Asparagine was shown to be a key compound plex fertilizer was given as the additional one of aroma and browning substances2) formed by to the field at the beginning of June. Three amino-carbonyl reaction. In the harvest time sugarcanes of each variety were harvested at of both varieties, asparagine, aspartic acid, random on the 25th of each month. glutamine and glutamic acid contents are on Methods average around 46, 13, 5.6 and 6% of total free Enzyme extraction: Each stalk and each amino acid contents , respectively3). The root were ground in a cooled motar and pestle primary object of this study is to determine the with twice volumes of 0.1M tris-HCl buffer accumulation of asparagine , glutamine, as- (pH 7.0) containing 4mM L-cysteine. The re- partate and glutamate in stalks on the basis of sulting homogenate was centrifuged at 9000 the activities of asparagine and glutamine syn- rpm for 15min. The supernatant was filled up thetase during its growth. to 10ml with 0.01M tris-HCl buffer (pH 7.0) and the mixture was used as the crude enzyme after passing through Sephadex G-25. All pro-

cedures were carried out at 4℃. 674 日本食品工業学会誌第34巻第10号 1987年10月 ( 46)

Enzyme assay: In order to remove the droxamine6). Soluble protein was determined

disturbing substances during the assay, 2ml of according to the method of LOWRY et al.7)

the enzyme solution was applied to Sephadex using bovine serum albumin as the standard.

G-25 fine column (1.5i.d.×10cm), which had The activity of asparagine synthetase was

been equilibrated with 0.01M tris-HCl buffer represented as μmol of aspartyl hydroxamate

(pH 7.0). The column was eluted with the formed per min per mg protein, whereas that

same buffer at flow rate of 24ml per h. Each of glutamine synthetase was indicated as μ

2ml fraction was collected and was monitored mol of glutamyl hydroxamate formed per min

by absorbance at 280nm for protein and the per mg protein.

enzyme activities were assayed as described as Determination of asparagine, aspartic acid,

below. The fraction with the highest protein glutamine and glutamic acid: The sample (10

content (tube No. 6) was used for enzyme assay. g) was treated in a POTTER's homogenizer with

The asparaginase and glutaminase assays were 20-fold volumes of 80% ethyl alcohol. The

carried out at 37℃ for 1h according to the mixture was heated at about 80℃ for 30min

procedures described by DUNLOP et al.4) with under a reflux condenser. The alcoholic ex-

some modification as described here. For the tract was evaporated under reduced pressure

standard asparaginase assay method, the for- and it was made up to 25ml with water. An

mation of aspartic acid was determined spec- aliquot (5ml) of the solution was charged on

trophotometrically by measuring the decrease Amberlite IR-120 B (H+ form) column (1.8i.d.

in NADH at 340nm and pH 7.5. After 3min ×5cm) and the free amino acid absorbed on

preliminary incubation, the assay mixture con- the resin was eluted from the column. The

tained (in mmol) K3PO4, pH 7.4 (200), α-keto- eluate was evaporated and then the syrupy

glutarate (3), asparagine (4), NADH (disodium liquid was made up to 10ml with 0.5N lithium salt, 0.05), 4 units of glutamate dehydrogenase buffer (pH 2.2). Quantitative analysis of free

and 0.1ml crude enzyme in a total volume of amino acid in sugarcane samples were per-

2.0ml. formed with an automatic high pressure amino

For the glutaminase assay, asparagine (4m acid analyzer (Hitachi 835). mol) was substituted for glutamine (4mmol). Results and Discussion The both enzyme activities were represented as

μ mol of aspartic acid or glutamic acid decom- Seasonal changes of asparaginase, aspra- posed per min per mg of protein. The asparagine synthetase, glutaminase, and gluta- gine and glutamine synthetase assays were per- mine synthetase formed at 30℃ for 10min according to the Asparaginase and asparagine synthetase:

procedures by RAVEL, et al.5) The standard as- Asparaginase, hydrolytic enzyme catalyzes the say medium for asparagine synthetase consisted hydrolysis of amide and produces NH3. It is of 10μmol aspartic acid, 50μmol of tris-HCl distributed widely in plant, but the physiologi- (pH 7.0), 6μmol manganese chloride, 5μmol cal role of this enzyme is not clear8). On the ADP, 5μmol hydroxamate, 0.1ml of crude other hand, asparagine synthetase normally enzyme in a total volume of 0.5ml. transfers the amide nitrogen from glutamine to In the case of glutamine synthetase assay, aspartic acid9). Due to detoxification of am- asparagine was substituted for 10μmol of glu- monia in tissues of higher plants, asparagine tamine. The reaction mixture was incubated synthetase can react with ammonium directly at 30℃ for 10min, and the reaction was under large exogenous source of ammonia10). stopped by the addition of 1.5ml of 10% ferric The activities of the asparaginase and aspara- chloride reagent. After centrifugation, the O. gine synthetase from three equally divided D. was measured at 540nm and the amount of parts from the stalk are shown in Figs. 1 (N: aspartyl hydroxamate formed was calculated CO) and 2 (Chikusha), and those from roots from standard curve of synthetic aspartyl hy- are shown in Fig. 3 (N:CO and Chikusha). ( 47) MATSUI and KITAGAWA: Seasonal Changes of Activities of Asparagine Synthetase 675

Fig. 1 Seasonal changes of the activities of asparagine synthetase and asparaginase in stalks of 'N:CO310' Fig. 3 Seasonal changes of the activities of asparagine synthetase and asparaginase in roots of both varieties

In both varieties, the activity of asparagine synthetase increased in the basal stalk until August and then decreased. It increased in upper part of the stalk in August to October. The activity of asparagine synthetase reached a maximum in roots of sugarcane in June or July. The high activity of asparagine synthetase seemed to depend on additional fertilizer which was given to the roots because its activity increased rapidly in June and/or July. Asparaginase activity was lower than asparagine synthetase in the basal part in August. In N:CO', the activity of asparaginase increased ' in the middle and basal parts of stalk in August and then decreased. In 'Chikusha', asparaginase reached the highest activity in the basal part of the stalk in September. In both Fig. 2 Seasonal changes of the activities of varieties, the activity of asparaginase increased asparagine synthetase and aspara- rapidly in the upper part of stalk at harvest ginase in stalks of 'Chikusha' season. In the basal part of the stalk in both Symbols as shown in Fig. 1. varieties asparagine synthetase showed the highest activity in August, whereas aspara- 676 日本食品工業学会誌第34巻第10号 1987年10月 ( 48) ginase showed the highest activity in Septem- ber. Since NH3 produced by nitrogen fixation and nitrogen assimilation is toxic, it is converted to the nontoxic amide, asparagine, by asparagine synthetase at high ammonium levels10). The asparagine is converted to aspartic acid by asparaginase. In sugarcane, the highest activity of asparagine synthetase reached one month earlier than that of asparaginase. In both varieties, asparagine synthetase reached high activity at roots in June and/ or August and then it decreased suddenly. The activity of asparaginase was almost constant throughout the experiment. Glutaminase and glutamine synthetase: Glutaminase catalyzes the hydrolysis of amides and produces NH3 The physiological role of the glutaminase is not clear and it may be the same as asparaginase. Glutamine synthetase binds ammonia to glutamic acid at a low con- Fig. 5 Seasonal changes of the activities of centration. Fig. 4 shows the activities of the glutamine synthetase and gluta- glutamine synthetase from three equally divid- minase in roots of both varieties ed parts and glutaminase in the basal parts of stalks. In both varieties, the activity of gluta-

mine synthetase was highest in the basal part of stalks in August and then it decreased suddenly. It showed almost the same profile as the activity of asparagine synthetase in the stalks. On the other hand, the glutaminase reached the highest activity in September for both varieties, and it showed also the same tendency as the asparaginase in the stalk. Glutamine synthetase also reached the highest activity one month earlier than glutaminase. Fig. 5 showed the activities of glutamine synthetase and glutaminase in the root for both varieties. In 'N:CO', the activity of glutamine synthetase decreased suddenly in the root from July, whereas in 'Chikusha' it increased in August and then decreased. According to ARI- MA et al.11), the activity of glutamine synthetase is much lower in rice seedling root above Fig. 4 Seasonal changes of the activities of the ground than under the ground. glutamine synthetase and glutami- The difference of activities of asparagine syn- nase in stalks of both varieties thetase between stalk and root does not depend on the condition of nitrogen nutrition located on above or under the ground, but it depends on ( 49) MATSUI and KITAGAWA: Seasonal Changes of Activities of Asparagine Synthetase 677

the functional properties of the enzyme itself. November. In both varieties, aspartic acid

In 'N:CO', glutamine synthetase showed high- contents decreased in stalk until August, and

er activity of 20μmol/min per mg protein in then they were almost constant until harvest

root, conpared with the activity of 1.25μmol/ season. The fact that asparagine showed

min per mg protein in stalk. higher content than aspartic acid at harvest Amides, aspartate, and glutamate contents season means that asparagine may act as Asparagine and glutamine are used to trans- storage type of amino acids. Asparagine, port and store NH3 in a nontoxic form before aspartic acid, glutamine and glutamic acid it is excreted. Glutamine plays a significant contents in the root are shown in Fig. 7. In 'N:CO' role in nitrogen metabolism as a precursor of , asparagine showed the highest con- amino group, but asparagine does not appear tent in August, whereas in 'Chikusha' it to serve as a source of nitrogen in biosynthetic showed the highest content in July. Aspartic reactions8). Asparagine contents from equally acid showed the highest content of the stalk in divided three parts from the stalks as well as July for both varieties. Asparagine in root of aspartic acid contents in the basal parts of both varieties also showed higher content than stalks are shown in Fig. 6. In 'N:CO', as- aspartic acid. In 'N:CO', glutamine and paragine content in the basal part of stalk glutamic acid showed higher contents in May, continued to decrease until October, whereas it whereas in 'Chikusha' they did in July. in the upper parts of stalk increased in Sep- Glutamine and glutamic acid contents in the tember to November. In 'Chikusha', aspara- stalks are shown in Fig. 8. In both varieties, gine showed the highest content in the stalk in glutamine showed the lowest content in the July, and its content increased in the middle and top parts of the stalk in September to

Fig. 7 Seasonal changes of asparagine , aspartic acid, glutamine and gluta- Fig. 6 Seasonal changes of asparagine and mic acid contents in roots of both aspartic acid contents in stalks of varieties both varieties 678 日本食品工業学会誌第34巻第10号 1987年10月 ( 50)

Fig. 8 Seasonal changes of glutamine and Fig. 9 Seasonal changes of stalk and root glutamic acid contents in stalks of weights in both varieties both varieties

top of stalk in August. Asparagine and glutamine are considered to be consumed during rapidly growing period. basal part of stalk in August and increased The activities of asparaginase and glutami- steadily. Glutamic acid content increased nase were higher in the root in June and/or from November in the middle of stalk. July, whereas asparagine and glutamine con- Seasonal changes of weights tents reached higher value in July and/or Aug- Fig. 9 shows the seasonal changes of stalk and ust (Fig. 7). The glutamine and asparagine root weights. Stalk weight increased rapidly contents seemed to depend on the activities of in July to September in both varieties. In 'N: glutaminase and asparaginase. CO', it increased until November, whereas in The activities of the two synthetases were Chikusha' it was almost the same in Sep- ' paralle with those of deamination in sugarcane. tember to November. Root weight was almost The synthetase activities in basal part of stalks constant in both varieties. The weight of 'N: reached maximum value in August, whereas the CO' sugarcane was about two times as large as deaminase activities in the basal part of stalk that of 'Chikusha' in November. The activities were higher in September. Asparagine at first of asparagine synthetase and asparaginase was accumulated at internode in succession to showed the same profile as those of glutamine upper one. Thus, its accumulation was similar synthetase and glutaminase in both varieties. to that of sucrose1). In 'N:CO', asparagine The activity of asparagine synthetase was showed higher content in apical part of the higher than that of glutamine synthetase, stalks which is called a growing point of sugar- whereas asparagine content was higher than cane. This is considered to be the result of glutamine content. The activities of aspara- translocation of the amides to a growing gine and glutamine synthetases reached the point, because amino compounds were consum- highest value in August, whereas asparagine ed at this point. and glutamine contents were the lower in the Although asparagine showed the highest con- ( 51) MATSUI and KITAGAWA: Seasonal Changes of Activities of Asparagine Synthetase 679 tent in stalk in both varieties in October in the York), p. 467 (1976). 9) BONNER, J. and VARNER, J.E.: Plant Bio- previous paper1)3), sugarcane 'Chikusha' is con- chemistry (Academic Press, New York), sidered to be harvested in November on the Third Edition, p. 525 (1976). basis of asparagine, stalk weight and sucrose 10) GIVAN, C.V.: Phytochemistry, 18, 375 contents reported in this paper. (1979). 11) ARIMA, Y., HORINOUCHI,T. and KUMAZAWA, Acknowledgements: The authors wish to thank K.: Nippon Dojo-Hiryogaku Zasshi, 47, Prof. Sin'itiro KAWAMURA of Kagawa-Ken Mei- 198 (1976). zen Junior College for his suggestions, and also (Received Oct. 28, 1986) thank Prof. Masaru KURETANI of Kagawa Uni- versity for his helpful suggestions in cultivation of sugarcanes. We also thank Mr. Kazuo IKEDA of Chusho Kigyo Doyukai for his technical サトウキビのアスパラギン合成・分解酵素 assistance. 活性の季節変化について

References 松井年行*・北川博敏* *香川大学農学部農業生産学科 1) MATSUI, T. and KITAGAWA, H.: Nippon Shokuhin Kogyo Gakkaishi, 32, 135 (1985). (〒761-07 香川県木田郡三木町池戸)

2) MATSUI, T.: Tech. Bull. Fac. Agric. サトウキビの2品種,'竹蔗'と'N:CO310'の根と Kagawa Univ., 32, 135 (1981). 茎を使って,アスパラギン,アスパラギン酸,グルタミ 3) MATSUI, T.: Nippon Nogeikagaku Kaishi, ン,グルタミン酸含量とアスパラギン,グルタミン合成 61, 29 (1987). ・分解酵素活性の季節変化を調べた 4) DUNLOP, P. and ROON, R.: J. Bacteriol. .アスパラギン合成 122, 1017 (1975). の最大活性は,8月に茎の下部で認められ,アスパラギ 5) RAVEL, J., NORTON, S., UMPHREYS, J. and ン分解酵素の最大活性は9月であった .一方,アスパラギ

SHIVE, W.: J. Biol. Chem., 237, 2845 (1962). ン合成酵素は,6月か7月の根に最大活性を示した .'N: 6) ROPER, J. and MCILWAIN, H.: Hoppe- CO310'において,茎の下部のアスパラギン含量は10 Seylers. Z. Physiol. Chem., 42, 485 (1958). 7) LOWRY,O.H., ROSERBROUGH,N.J., FARR, 月まで減少し続け,一方上部では9月から11月まで増 A.L., and RANDALL, R.J.: J. Biol. Chem., 大した.'竹蔗'において,アスパラギン含量は7月に 193, 265 (1951). 茎で最大を示し茎の中部,下部ではその含量は9月から 8) CONN, E.E. and STUMPF, P.K.: Outline of 11月まで増大した,'竹蔗'の最適収穫時期は,茎の重 Biochemistry, (John Wiley & Sons, New 量,アスパラギン含量から11月であると考えられる.