Kanehisa, Katsuo

Tsumuki, Hisaaki

Okayama University Scientific Achievement Repository

Ber. Ohara Inst. landw. Bio1. 18: 31-41 (1980) ENZYME ACTIVITIES ASSOCIATED WITH GLYCOGEN METABOLlSM IN DIAPAUSING AND DEVELOPING LARV AE OF THE RICE STEM BORER， CHILO SUPPRESSALIS W ALKER Hisaaki TSUMUKI and Katsuo KANEHISA Trehalose and polyols were shown to be the carbohydrates that ac-cumulated in diapausing insects (Wyatt 1967). Glycogen was converted to sorbitol and glycerol at the beginning of diapause， and these polyols were reconverted to glycogen at the termination of diapause in eggs of the si1kworm， Bombyx mori (Chino 1958， Yaginuma and Yamashita 1978). Tsumuki and Kanehisa (1978) have also shown in the rice stem borer， Chilo suppressalis Walker， that accumulation of glycerol in hae-molymph was associated with a dec1ine of glycogen content in the fat body during PI・e・diapausingto diapausing stage， whi1e the quantity of glycerol in haemolymph decreased progressively with increased glycogen in the fat body during post-diapausing stage. However， indeveloping lar-vae these phenomena have never been observed (Tsumuki and Kanehisa 1978). Such changing patterns of carbohydrates may be correlated with enzyme activities in tissues of insects (Wyatt 1967， Sacktor 1970). In haemolymph， alimentary canal， fat body and carcass of the rice stem borer， changing patterns of the enzyme activities associated with glycogen metabolism during hibernation were revealed (Tsumuki and Kanehisa 1979). It may likewise be important problem to reveal the changing patterns of enzyme activities in the whole body of rice stem borer larvae during hibernation. Furthermore， changes of enzyme ac-tivities in developing larvae of the rice stem borer are stil1 obscure. This study was undertaken to determine the kinetic properties of enzymes associated with glycogen metabolism and differences in the enzyme activities in the tissues and whole body of larvae between dia-pausing and developing stage in the rice stem borer. MATERIALS AND METHODS Experimental animals Hibernating larvae of the rice stem borer， Chilo suppressalis Walker， were collected at Okayama Agricultural Experiment Station in Okayama Prefecture. Developing larvae were reared on rice seedlings at 25士20Cunder 16ーhourlight，j 8-hour darkness， as described by Tsumuki and 32 H. Tsumuki and K. Kanehisa Kanehisa (1978). Chemica1s Glucose-1-phosphate (G・1-P)，nicotinamide adenine dinuc1eotide phos-phate (NADP)， glucose-6・phosphatedehydrogenase (G・6-P Dase)， phos-phoglucomutase (PG-mutase) and uridine-5'-diphospho glucose (UDPG) were purchased from Sigma Chemical Co. (St. Louis， Missouri). UDP・E・C-G(273 mCi/mM) was purchased from the Radiochemical Centre (Amersham， England). Al1 other chemicals were of reagent grade. Prepaγ'ations 01 enzyme sources and enzyme assays Whole bodys of developing and hibernating larvae were homogenized in 40 volumes of ice cold water. Mature larvae during development were divided into four ti鎚 U回， haemolymph， fat body， alimentary canal and carcass， and homogenized according to the method described pre-viously (Tsumuki and Kanehisa 1979). The homogenates were cen-trifuged at 3，500 r. p. m. for 15 minutes and the resulting supernatants were used for enzyme sources. Hexokinase， PG-mutase， UDPG pyrophosphorylase， phosphorylase， gly-cogen synthetase and trehalase activities were assayed according to the method described previously (Tsumuki and Kanehisa 1979). He玄okinase，PG-mutase， UDPG pyrophosphorylase and phosphorylase activities were determined by production rates of NADPH from NADP. Glycogen syn-thetase activity was determined by incorporation of "C-glucose from UDP-1・C-Ginto glycogen. Trehalase activity was determined by pro-duction of glucose from trehalose. Protein concentration was estimated by the method of Lowry et a1. (1951). RESULTS Time course The relationship was examined between incubation time and the activities of six tissue enzymes (Fig. 1). The duration of linearity dif・fered with the kind of enzyme and organ under these assays. Optimum pH Fig.2 shows that the enzyme activity depends on pH. All enzymes showed bel1-shaped curves against pH and there were not any significant differences in optimum pH between the tissues examined， but the optimum pH differed significant1y among enzymes， as e玄pected.33 60 Glycogen Metabolism in the Rice Stem Borer 30 Time(min) 2 0.3 0.2 0.1 -.・・・..・..・..._.30 60 Time(min) -・.-・"〆〆.〆-". 〆〆"，' . 〆• 〆F 0.3 0' o 0.2 0.1 ??????。ノ〆〆〆〆， 〆/ ，・4 ノ〆..... ~ ~_.. ・..・-f.:，..ζ孟60 30 Time(min) 4 0.1 0.05， 0 0 3 60 • • • • • ，?????• ???， ， ??• •... ?， ， ??? ? ?? ????? ? ? ???? ?30 Time(min) 0.3 0.2 0.1 ????? ?6 一_.....-もー‘Fョιニニ;2 . o lddrf.二H・H・中…..・...・H・.-. 30 60 0 30 60 . 90 Time (min) Time (min) Time course of enzyme activity in tissues of rice stem borer larvae. 8 4 5 ??• ?，?• ???10 5 ? ? ????4) phosphory-3) UDPG pyrophosphorylase 6) trehalase. ・:fat body ー・ー・ー・:carcass FIG. 1. 1) hexokinase 2) PG-mutase lase 5) glycogen synthetase -一一:alimentary canal 34 H. Tsumuki and K. Kanehisa /"-一J・'，dFa-一〆r仲，町~・hR.丸¥? 下、h • 、一¥、、一-、~ l /1 " . 50 〈~ 3〈# u 50ト。100 ~ 350 0 〈7.8 7.4 07.4 . . 8.6 pH 代;、8.2 pH 8.2 pH • 。9.4 7.8 -、• 50 。9.0 7.0 100 50 9.0 0'5.a 8.6 pH 7.8 pH 6.6 pH 9.4 8.6 7.4 FIG. 2. Effect of pH on enzyme activity in tissues of rice stem borer larvae. 1) hexokinase 2) PG-mutase 3) UDPG pyrophosphorylase 4) phosphory-lase 5) glycogen synthetase 6) trehalase. 一ー一:alimentary canal ・・・・:fat body -・ー・→:carcass K明 valuesThe enzyme activity was corrected with the control in which sub-strate was omitted. The enzyme activities increased according to increasing concentrations of the corresponding substrates and attained 60ト1Ac Glycogen Metabolism in the Rice Stem Borer 35 、r、30 20 40 。 km=0.02ImM 50 100 20ト3Ac、r、10 。 Km=0.066mM 園開園_._・圃圃圃圃園田・‘・・20 40 4Ac y-I 40 100t 6 Ac y-I 50 40 200ト1Fb 10 20 Glucose(mM-') Km=0.030mM 01 50 100 G-1-P(mM-I) 20 50 100 UDPG(mM叶)4 Fb Km=0.86mg 01 o 2 4 Glyc勾・n(吋 -1)8 ~ 5 Fb 4 50 100 UDPG(mM-I) 1.000t 6 Fb 80 50 100 2Ca 附n=0.03命，，1'.1。20 40 500 0.5 1 Tr・h・Iose(mM-1)3 Ca 5 。 ??????????4Ca ・附n=0.42mg。2 4 5Ca 40 20 40~ 6 ca OL O 10 20 FIG. 3. Effect of substrate concentration on enzyme activity in tissue of rice stem borer larvae. 1) hexokinase 2) PG-mutase 3) UDPG pyrophospho・rylase 4) phosphorylase 5) glycogen synthetase 6) trehalase Ac: alimentary canal Fb: fat body Ca: carcass 36 H. Tsumuki and K. Kanehisa saturation levels following typical Michaelis-Menten type curves. The Km value of each enzyme was calculated by the method of Lineweaver and Burk (Fig. 3). Since phosphorylase activity in the alimentary canal was very low， the estimation of Km value for glycogen was fai1ed. Km value for trehalase from the fat body was the highest of al enzymes tested. Changes in enzyme activities in hibernating larvae Fig. 4 shows the changes in enzyme activities in the whole body of hibernating larvae. Hexokinase， PG-mutase， UDPG pyrophosphorylase and trehalase activities decreased during pre-diapausing to diapausing stage and increased during post-diapausing stage， but UDPG pyrophos-phorylase activity decreased abrupt1y before pupation. Glycogen syn-30 .~ 20 、E • +'o a E 帥@ E "l る 10h .ゼ長。《0 Oct Nov Oec -Pre-diapausa -ーー-Oiapause-一一+←一向st・diapause-_ Month of collection FIG. 4. Changes in enzyme activities in the whole body of rice stem borer larvae during hibernation. ・:hexokinase ..: PG-mutase ロ:UDPG pyrophosphorylase x: phosphorylase t;，.: glycogen synthetase 0: trehalase 37 phos-and Glycogen Metabolism in the Rice Stem Borer thetase activity was at a high level in November and Apri1 phorylase activity in December and in Apri1 to June. Changes in enzyme activities in developing laγvae Fig. 5 shows the changes in enzyme activities in the whole body of developing larvae. Hexokinase， PG-mutase and trehalase activities inc-reased progressively with advances in larval age， then， these activities ¥ ? ? ? ? ? ? ? ? ?， ? ， ????70 .~ .5 e # E4O E 同2 2 :s. E 〉、:~ 20 0 《。40 30 20 10 しarvalage in days FIG. 5. Changes in enzyme activities in the whole body of rice stem borer larvae during development. .: hexokinase .: PG-mutase 口:UDPG pyrophosphorylase x: phosphorylase ，t:，.: glycogen synthetase 0: trehalase decreased before pupation. On the other hand， UDPG pyrophosphorylase activity decreased abrupt1y with advances in larval age. Glycogen synthetase activity decreased progressively， whereas phosphorylase acti-vity increased. The activities of glycogen synthetase and phosphorylase were lower compared to these of the other four enzymes. 38 H. Tsumuki and K. Kanehisa Enzyme activities in the tissues 01 hibernating and developing larvae Table 1 shows the enzyme activities in the tissues of diapausing and developing larvae. The enzyme activities differed according to the exa-mined tissues of diapausing and developing larvae. Hexokinase， trehalase and phosphorylase were higher in developing larvae than in diapausing TABLE 1 Enzyme activities in tissues of diapausing and developing lavae Enayme舵 tivity(mμmole/mg protein/min) H偽記吋品問。細川lase79.6 11.3 28.0 2.5 0.2 152.8 canal Developing ~ Fat body 8.4 72.3 66.4 8.2 8.8 4.5 larvae Carcass 35.2 28.5 25.2 4.7 0.5 18.1 (…y 29.9 8.6 26.9 1.3 0.3 67.1 canal Diapausing~ Fat bOdy 1.7 51. 9 130.0 4.7 2.0 0.6 larvae Carcass 25.3 24.5 20.4 2.9 0.3 7.2 larvae， and especially， high in the alimentary canal of developing lar-vae. The enzyme activities in the haemolymph of hibernating and developing larvae were recognized only as“trace" or could not be detected. DlSCUSSION Changing patterns of hexokinase， PG-mutase， glycogen synthetase， phosphorylase and trehalase activities in the fat body， alimentary canal and carcass have been demonstrated during hibernation of the rice stem borer (Tsumuki and Kanehisa 1979). 1n the present study， simi1ar results were obtained in the tissues and whole body of rice stem borer larvae. Hexokinase， PG-mutase， UDPG pyrophosphorylase and trehalase activities decreased during pre-diapausing to diapausing stage and in-creased again during post-diapausing stage. The enzyme activities in the tissues and whole body of developing larvae were higher than those in diapausing larvae except for UDPG pyrophosphorylase activity， and resembled the enzyme activities of hibernating larvae before pupation (Tsumuki and Kanehisa 1979). These results demonstrate that the rate of carbohydrate metabolism in developing larvae is higher than that of diapausing larvae. Glycogen Metabolism in the Rice Stem Borer 39 Other study of the rice stem borer (Tsumuki and Kanehisa 1979) have shown that the alimentary canal trehalase may uti1ize the hae-molymph trehalose. Simi1ar results were obtained in this study. Tre-halase and hexokinase activities in the alimentary canal of developing larvae were higher than those of diapausing larvae (Table 1). Fur-thermore， these activities in the fat body of diapausing larvae were at low levels. Trehalose concentration in the haemolymph of diapausing larvae collected from fields was higher than that of developing larvae (Tsumuki and Kanehisa 1978). Consequent1y， trehalase in the ali-mentary canal appears to uti1ize the haemolymph trehalose. However， the trehalase in the carcass may also be related to the uti1ization of the haemolymph trehalose (Tsumuki and Kanehisa 1979). Glycogen synthetase activity was high in November and Apri1， and phosphorylase activity was high in December and in April to June. The changing patterns of these activities were coincident with those of glycogen contens during hibernation (Tsumuki and Kanehisa 1978). On the other hand， in developing larvae the changing patterns in these two activities were also coincident with those of glycogen contents (Tsumuki and Kanehisa 1978). Concerning to glycogen metabolism in some insects， a key enzyme regulated glycogen uti1ization seems to be trehalase (Yamashita 1969) and phosphorylase (Wright et al. 1973， Yamashita et al. 1975)， and hexokinase and glycogen synthetase are important in glycogen synthesis (Murphy and Wyatt 1965， Jungreis 1976). In this study， hexokinase， PG-mutase and trehalase activities in the whole body of hibernating and developing larvae were at high levels， and glycogen synthetase and phosphorylase activities were low. From these results， itis suggested that glycogen synthetase and phosphorylase seem to be rate-limiting steps in glycogenetic and glycolytic pathways in rice stem borer larvae. SUMMARY The enzyme activities associated with glycogen metabolism were investigated in hibernating and developing larvae of the rice stem borer， Chilo suppressalis Walker， to determine the carbohydrate metabolic pathway. Hexokinase， phosphoglucomutase， uridine-5'-diphosphoglucose pyro咽phosphorylase， phosphorylase， glycogen synthetase and trehalase were detected in homogenates of the tissues and whole body of hibernating and developing larvae. Hexokinase， phosphoglucomutase， uridine-5'-diphosphoglucose pyro-phosphorylase and trehalase activities decreased during pre-diapausing 40 H. Tsumuki and K. Kanehisa to diapausing stage and then increased during post-diapausing stage. On the other hand， these activities in developing larvae increased with advances in larval age， and then decreased before pupation except for uridine-5にdiphosphoglucose pyrophosphorylase. The changing patterns of enzyme activities were coincident with those of carbohydrate con-tents in larvae during development and hibernation. Changes in glycogen synthetase and phosphorylase activities were coincident with those of glycogen contents in hibernating and developing larvae. The activities of hexokinase， phωphoglucomutase， uridine-5'-diph関 phoglucose pyrophosphorylase and trehalase in hibernating and developing larvae were high， and glycogen synthetase and phosphorylase activities were low. From these results， itis suggested that glycogen synthetase and phosphorylase are the rate司limitingsteps in glycogenetic and glycoJytic pathways in larvae of the rice stem borer. Acll1towledgllments The authors wish to thank Prof. Y. Yasue and Dr. K. Kawada for their helpful suggestions throughout this investigation. Thanks are also extended to Dr. O. Yamashita， Laboratory of Sericultural Science， Faculty of Agriculture. Nagoya University， for his kind advice. The assistance of Mr. T. Shiraga in the collection and breeding of the rice stem borer is acknowledged. REFERENCES Chino， H. 1958. Carbohydrate metabolism in‘the diapause eggs of the silkworm， Bomby'" morI-II. Conversion of glycogen to sorbitol and glycerol during diapause. J. Insect Physiol. 2: 1-12. Jungreis， A. M. 1976. Regulation of均aloplwracecropia fat加dyhexokinase by hexose phosphate common to the pathways glycolysis， glycogen and trehalose synthesis. Comp. Biochem. Physiol. 53B: 405-413. Lowry， 0.， Rosebrough， N. J吋 Farr，A. L. and Randall， R.J. 1951. Protein measurement with the Folin phenol reagent. J. biol Chem. 193: 265-275. Murphy， T. A. and Wyatt. G. R. 1965. The enzymes of glycogen and trehalose synthesis in silk moth fat body. J. biol Chem. 193: 265-275. Sacktor， B. 1970. Regulation of intermediary metabolism， with special reference to the control mechanisms in insect fiight muscle. Adv. Insect Physiol. 7 : 267-347. Tsumuki， H. and Kanehisa， K. 1978. Car加hydratecontent and oxygen uptake in larvae of the rice stem borer. Chilo suppressalis Walker. Ber. Ohara Inst. landw. Biol.， Okayama Univ. 17: 95-110. Tsumuki， H. and Kanehisa， K. 1979. Enzymes associated with glycogen metabolism in larvae of the rice stem borer， Chilo suppressalis Walker: Some properties and changes in activities during hibernation. Appl. Ent. Zool. 14:幻0-277.Wright， J.E.， Crookshank， H. R. and Rushing， D. D. 1973. Glycogen phosphorylase activity in pharate adults of the stable fiy and the effects of a juvenile hormone analogue. J. Insect Physiol. 19: 1575-1578. Wyatt. G. R. 1967. The biochemistry of sugars and polysaccharides in insects. Adv. Insect Physiol. 4: 287-3ω. Yaginuma， T. and Yamashita. O. 1978. Polyol metabolism related to diapause in Bomb.， eggs: Different behaviour of sorbitol from glycerol during diapause and post-Glycogen Metabolism in the Rice Stem Borer 41 diapause. ]. Insect Physiol. 24: 347-354， Yamashita， O. 1969. Studies on the glycogen metabolism in pupal overies of the silk-worm， Bomb， mori L.， (In Japanese with English summary). J. Sericult. Sci. Japan 38: 329-339. 

ENZYME ACTIVITIES ASSOCIATED WITH GLYCOGEN METABOLlSM IN DIAPAUSING AND DEVELOPING LARVAE OF THE RICE STEM BORER, CHILO SUPPRESSALIS WALKER

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