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Activity changes of guanylate cyclase and cyclic GMP phosphodiesterase related to the accumulation of cyclic GMP in developing ovaries of the silkworm, Bombyx mori
Comp. Biochem. Physiol. Vol. 9311,No. 2, pp. 385-390, 1989 Printed in Great Britain
0305-0491/89 $3.00+ 0.00 © 1989 Pergamon Press pie
ACTIVITY CHANGES OF GUANYLATE CYCLASE AND CYCLIC GMP PHOSPHODIESTERASE RELATED TO THE ACCUMULATION OF CYCLIC GMP IN DEVELOPING OVARIES OF THE SILKWORM, B O M B Y X M O R I JIAN HUA CHEN and OKITSUGU YAMASHITA* Laboratory of Sericultural Science, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464-0 I, Japan (Tel: 052 781 5111) (Received 19 September 1988)
Abstract--l. Guanylate cyclase in ovaries of the silkworm, Bombyx mori, was not unusual in kinetic properties and mainly localized in the particulate fraction. Two cyclic GMP phosphodiesterases, low-Km (2.9/~M) and high-Km (125.5 tiM) enzyme, were found in the soluble fraction of ovaries. 2. All the enzymes showed the similar developmental changes in activity exhibiting a peak value in young ovaries. 3. The cyclic GMP concentrations calculated from these enzyme activities increased according to ovarian development, which resembled the developmental changes in measured values except for the first two days. 4. The calculated concentrations of cyclic GMP were high in ovaries with the removal of suboesophageal ganglion during the last period of development.
INTRODUCTION Along with adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-~yclic monophosphate (cyclic GMP) is well established to play the important roles in the control of many physiological processes in diverse organism (Goldberg and Haddox, 1977). The intracellular concentration of cyclic G M P is maintained at a steady-state value by its production from GTP by guanylate cyclase (EC 4.6.1.2) and conversion into G M P by cyclic G M P phosphodiesterase (EC 3.1.4.17). Changes in the concentration of cyclic G M P produced by physiological and pharmacological stimuli such as hormones, nervous action and metabolic inhibitors, are achieved mainly through modifications in the activity of either cyclase or phosphodiesterase, or both (LaPorte, 1984). Thus, the dynamic changes in these enzymes are finally reflected on the cellular events mediated by cyclic GMP. In the previous report, we have demonstrated that cyclic G M P behaves differently from cyclic A M P during ovarian development of the silkworm, Bombyx mori, and the level of cyclic G M P increased markedly through the last phase of vitellogenesis to oocyte maturation. Further, diapause hormone is shown to act to reduce the accumulation of cyclic G M P at this stage (Chen et aL, 1988). Such a stageand hormone-dependent accumulation of cyclic G M P seems to be associated with special properties or activity changes of cyclase and/or phosphodiesterase. Although several informations are available on the kinetic properties and developmental changes of these enzymes in insects (Bodnaryk, 1983; Smith and *Author to whom all correspondence should be addressed.
Combest, 1985), little work has appeared on the systematic analysis on the relationship between cyclic G M P levels and activities of both enzymes (Fallon and Wyatt, 1977a, 1977b). Furthermore, less attempt was done on the regulation mechanisms of these enzymes by insect hormones (Hiripi et al., 1979). In order to correlate these enzyme activities to cyclic G M P concentrations in silkworm ovaries, we have first characterized each enzyme in developing ovaries of silkworms. Referring to the properties of these enzymes, the assay condition was established and the activity was followed throughout the ovarian development. The effect of diapause hormone on developmental profile in enzyme activities was also surveyed. A simple model (Arch and Newsholme, 1976) was applied to correlate the developmental changes in enzyme activities to the accumulation profile of cyclic G M P in silkworm ovaries. MATERIALS AND METHODS
Animals A hybrid race (Shunrei and Shogetsu) of the silkworm, Bombyx mori was used. Embryonic life was maintained at 25°C under the continuous illumination to produce diapause eggs at the next generation (Yamashita, 1984). Larvae were reared on either fresh mulberry leaves or an artificial diet at 26°C under a long day condition (16hr L-8 hr D). The pupae ecdysed within 3 hr were collected to synchronize their following pupal-adult development, and kept at 26°C under the long day condition. Adult emergence occurred in females 12 days after the larval-pupal ecdysis when almost all oocytes completed their maturation (Yamauchi and Yoshitake, 1984). In some experiments, the suboesophageal ganglion (SG) which is a releasing center of diapause hormone (Yamashita, 1984) was extirpated on the day of larval-pupal eedysis (day 0) to obtain non-diapause type ovaries and eggs (Chen et al., 1988). 385
386
JIAN HUA CHEN and OKITSUGU YAMASHITA
Chemicals Cyclic GMP, creatine phosphate and creatine kinase were obtained from Yamasa Sboyu Co. (Chiba). 3-Isobutyl1-methylxanthine (IBMX) and 5'-nucleotidase were purchased from Sigma Chemical Co. (St. Louis). [3H]-GTP (10.6Ci/mmbl) and [3HI-cyclic G M P (18.3 Ci/mmol) were from Amersham International Co. (Amersham). Polyethyleneimine (PEI)-cellulose was from Seikagaku Kogyo Co. (Tokyo). Neutral aluminium oxide (grade 1) was from Woelm Pharma Co. (Eschwege). All other chemicals were of analytical grade.
Preparation of enzyme sources Ovaries were dissected out throughout pupal-adult development in cold 0.75% NaC1 and weighed quickly after the brief blotting on paper. The tissue was homogenized with 5 volumes of 20 mM triethanolamine buffer, pH 7.5, containing 350 mM sorbitol, 2 mM EDTA, 300 mM NaC1 for guanylate cyclase assay, and with 5 volumes of 50 mM Tris-HC1 buffer, pH 8.5, for cyclic GMP phospbodiesterase assay. The crude homogenate was used directly for assay of cyclase unless specified. For phosphodiesterase assay, the homogenate was centrifuged at 15,000g for 20min and the supernatant was passed through a small column of Sephadex G-25 to remove endogenous cyclic GMP. The resultant macromolecular fraction was used as enzyme source.
Assay of enzyme activities Guanylate cyclase. The conversion of [3H]-GTP into cyclic [3H]-GMP was measured in the presence of GTPregenerating system according to the method of Garbers and Murad (1979) with a slight modification. The incubation medium consisted of 50 mM triethanolamine buffer, pH 7.5, I mM IBMX, 10mM MnCI2, 8 m M creatine phosphate, 10 units creatine kinase, l mM [3H]-GTP (0.96/zCi) and 50/d enzyme solution (about 1 mg proteins) in a final volume of 200/~1. The reaction was initiated by addition of the enzyme solution and allowed to proceed for 40 min at 30°C. The reaction was stopped by addition of 0.25 ml of 200 mM zinc acetate containing 2 mM cyclic GMP. Followed by the coprecipitation of GMP, GDP and GTP with ZnCO 3 produced by addition of 0.25 ml of 200 mM Na2CO 3, the soluble fraction was applied onto a PEI-cellulose column (0.6 x 6.0cm) which was preequilibrated with 50 mM acetic acid. Cyclic GMP was eluted by 20 mM LiC1 after a sequential washing with 50 mM acetic acid and then water. Radioactivity was measured with a liquid scintillation counter (Aloka LSC-700, Aloka Co. Tokyo) after the solubilization with ACS-II (New England Nuclear Co., Boston). The recovery of cyclic G M P was estimated to be 50% by comparing with the elution efficiency of the authentic cyclic GMP.
Cyclic GMP phosphodiesterase. The activities were measured by a radiochemical procedure according to the method of Filburn and Karn (1973) with a slight modification. The incubation medium consisted of 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgC12, 1 mM dithiothreitol, 0.01 mM or I mM cyclic [3H]-GMP (0.01/~Ci) and 50#1 enzyme solution (about 1 mg proteins) in a final volume of 200/~1. The reaction was started by adding enzyme solution and continued for 10 to 30 rain at 30°C. The reaction was stopped by immersing the incubation mixture into a boiling water bath for 1 min. To the cooled mixture, 0.1 unit 5'-nucleotidase was added and the mixture was again incubated at 30°C for 30 min. The pH of the mixture was adjusted to pH 4.0 by adding 40 #1 of 1 N acetic acid and the supernatant was collected by a brief centrifugation. An aliquot of soluble fraction (200 #1) was directly applied onto an alumina column (0.6x5.0cm) which has been preconditioned with 100mM ammonium acetate, pH 4.0. The guanosine was eluted with 1.2 ml of 100 mM ammonium acetate, pH4.0. Radioactivity was measured by a liquid scintillation counter as above. By comparison with the authentic guanosine, recovery was calculated to be 87%. RESULTS
Properties of guanylate cyclase Some properties a n d cellular d i s t r i b u t i o n were determined o n ovaries f r o m day 6 p h a r a t e adults (Table 1). R e a c t i o n rates were linear with time for 100 m i n a n d protein c o n t e n t u p to 1.0 m g per assay tube. Effects o f M n 2+, Ca 2+ a n d M g 2+ were determ i n e d o n day 6 ovary extracts which were passed t h r o u g h a Sephadex G-25 column. M n z÷ was essentially required for this enzyme activity a n d the maxim u m activity was given at 10 m M . C a 2÷ a n d M g 2÷ h a d n o effects o n the activity at 1 0 m M . Therefore, 10 m M M n 2+ was included in the reaction mixture. A p H o p t i m u m was decided as p H 7.5 in the p H ranges f r o m 6.0 to 9.0. T h e activity-substrate relationship showed a s a t u r a t i o n kinetics a n d the a p p a r e n t Km value for G T P was estimated to be 0.40 m M for the particulate enzyme by the L i n e w e a v e r - B u r k plot. T h e crude h o m o g e n a t e was subjected to a differential centrifugation; 4 5 0 g for 10 min, 15,000 g for 20 min, 105,000 g for 60 m i n a n d its s u p e r n a t a n t fraction, a n d each fraction was assayed u n d e r the s t a n d a r d condition. A b o u t 9 0 % o f total activity was recovered in the 15,000g pellet. T h e t r e a t m e n t o f the 15,000g pellet with T r i t o n X-100 (0.2%) increased the activity
Table 1. Some properties of guanylate cyclase and cyclic GMP phosphodiesterases of silkworm evades Cyclic GMP Guanylate phosphodiesterase cyclase High-Kin Low-KIn Optimum pH* 7.5 8.5 8.5 Km value* 400gM 125.5 #M 2.9/zM Effect of modulators activated by inhibited by Mn2+ at 10raM and IBMX at 1 mM Triton X-100 at 0.2% Subcellular distributiont 15,000g pellet 88% 8% 7% 15,000g supernatant 11% 92% 91% Ovaries from day 6 pharate adults were homogenized and ihe ovary homogenate was used as the enzyme source for guanylate cyclase. For assay of cyclic GMP phosphodicsterases, macromolecular fraction after passing the 15,000g supernatant through Sephadex G-25 column was used as enzyme sours.
*Each value represents means of three determinations. tThe result was obtained from a typical experiment.
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Fig. 1. Hofstee plots of the rate of hydrolysis of cyclic GMP by a silkworm ovary preparation. Substrate concentrations (S) ranged from 0.15 pM to 2 raM. Velocity (V) is expressed as pmol/min per mg protein. Each point represents mean values of three determinations.
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Fig. 2. Changes in activities of guanylate cyc'lase during ovarian development of silkworms. SG was removed on the day of pupation. Each point represents mean values from three determinations consisting of 3 individual with S.D. ( 0 ) , Non-operated controls; (O), SG-removed. late cyclase throughout pupal-adult development. The activity was about 9 nmol/min per g ovaries on day 0 and increased sharply in day. 1. A sudden decrease in activity occurred on day 2 and the declined activity was maintained until day 7 with some fluctuations. There was slight decrease in activity thereafter. The similar changing pattern was observed in ovaries with SG-removed. Interestingly, activities in ovaries after day 7 remained at a slightly
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The crude homogenate of day 6 ovaries were subjected to a differential centrifugation as described above. The supernatant fraction after a 15,000g centrifugation contained about 90% of the total activity (Table 1). In the following experiments, this 15,000 g supernatant was mainly used as the enzyme source. The rate of cyclic GMP degradation was close to linearity for 60 rain. The activity was proportional to the amounts of enzyme up to 1.6 mg of proteins. The optimum pH was estimated to be 8.5 by using 100mM PIPES buffer for pH 6.0 to 7.5, 100raM E 200 Tris-HCI buffer for pH7.5 to 9.0 and 100raM glycine-NaOH buffer for pH 8.5 to 10.5 (Table 1). A slightly higher activity was found in Tris--HCl buffer compared to the other buffers used. A half maximum activity was found at pH 7.5 and pH 9.5. Indeed, when assayed at pH 7.5, activity was reduced by 58% of the maximum activity obtained at pH 8.5. The O. relationship between substrate concentrations and IE activities exhibited a complex curve having two saturation kinetics. The Hofstee plot (V against V/S) gave two straight-line portions connected by a curve, 0 2 4 6 8 I0 12 and apparent Km values were calculated for the linear portion of the graph (Fig. 1). Two apparent Km Days after larval-pupal ecdysis values for cyclic GMP were estimated; 125.5/~M as a high-Kmvalue and 2.9 p M as a low-Kin value. These Fig. 3. Changes in activities of the l~igh-/~ cyclic GMP during ovarian development of silkK= values were also found in enzymes of ovaries with phosphodiesterase worms. SG was removed on the day of pupation. The SG-removed. activity was determined on 15,000&supernatant fraction of at I mM [3HI-cyclicGMP. Each point represents Developmental changes in activities of guanylate ovaries mean values from 3 determinations consisting of three cyclase and cyclic GMP phosphodiesterases individuals with S.D. (O), Non-operated controls; (O), SG-removed. Figure 2 represents the activity changes in guany-
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Fallon and Wyatt, 1977a; Goldberg and Haddox, 1977; Morishima, 1981). The similar properties and subcellular distribution were found on guanylate 30 cyclase of silkworm ovaries (Table 1). Two types of cyclic GMP phosphodiesterases, low- Kmenzyme and high-Km enzyme, were present in silkworm ovaries > o and their properties were similar to those of other oi vertebrates and insects (Morishima, 1975; Arch and Newsholme, 1976; Fallon and Wyatt, 1977b; Filburn ~ 20 et al., 1977). Consequently, the silkworm ovary o shares the similar enzymatic system for cyclic GMP E metabolism as found in various animal tissues in their general properties. The activity of guanylate cyclase in ovaries ranged o from 2 to 15 nmol/min per g ovaries (Fig. 2), which resembles the activity found in fat body of H. ececropia (Filburn and Wyatt, 1976) and B. mori n I[ (Morishima, 1981), in brain of Locusta migratoria migratoides (Hiripi et al., 1979) and in male accessory gland of Acheta domesticus (Fallon and Wyatt, 1977a). The basal activities of cyclic GMP phosphoo i ; 6 8 diesterase of silkworm ovaries were at the range from Days after tarval-pupal ecdysis 4 to 35 nmol/min per g ovaries for low-Kin enzyme Fig. 4. Changes in activities of the low-K= cyclic GMP and 30 to 300 nmol/min per g for high'Kin enzyme phosphodiesterase during ovarian development of silk- (Figs 3 and 4). These values remain in the ranges of worms. SG was removed on the day of pupation. The values reported on several tissues from invertebrates activity was determined at 10/aM [3H]-cyclicGMP on the and vertebrates (Arch and Newsholme, 1976). Thus, same fraction used in Fig. 3. Each point represents it is likely that cyclase and phosphodiesterase in mean values from three determinations consisting of three silkworm ovaries are active enough to maintain a individuals with S.D. (O), Non-operated controls; (©), steady-state level of cyclic GMP in them. SG-removed. Fat body o f B o m b y x mori (Morishima, 1975, 1981) and Hyalophora cecropia (Filburn and Wyatt, 1976; higher level in ovaries with SG-removed than in the Filbern et al., 1977) exhibited remarkable activity changes in guanylate cyclase and cyclic GMP controls. Throughout the ovarian development, we followed phosphodiesterase according to the larval-pupalseparately the activities of both high-Kin and low-Kin adult development. During this period, however, the cyclic GMP phosphodiesterase (Figs. 3 and 4). The activities changed in the opposite directions to each high-Km enzyme activity assayed at 1 mM cyclic other. Whereas, guanylate cyclase and cyclic GMP GMP rose markedly during the first two days and the phosphodiesterase of male accessory gland of Acheta increased level was kept for one day before a steep domesticus did not undergo marked changes in decline to day 7 (Fig. 3). Thereafter, the low activity activity during its maturation, although a great remained unchanged until adult emergence of day increase of cyclic GMP took place during this period 12. The similar changing pattern in activities was (Fallon and Wyatt, 1975, 1977a, 1977b). In contrast, observed with SG-removed (Fig. 3). The low-Km silkworm ovary enzymes showed the similar temporal enzyme also showed the almost similar changing changes with a peak value at the early stage of pattern throughout the ovarian development to that ovarian development (Figs. 2, 3 and 4). The stage of found in high-Km enzyme (Fig. 4). However, the peak activity was correlated to the maturation activity of low-Km enzyme remained at 1/6 to 1/10 of of follicle cells prior to the active vitellogenesis that of high-Kin enzyme. SG-removal had no effect on (Yamauchi and Yoshitake, 1984). The active vitellothe activity changes on low-K m enzyme throughout genesis occurred accompanying with decreasing ovarian development (Fig. 4). activities. The reduced activity was related to the choriogenesis and the maturation of oocytes. It is therefore conceivable that the activity changes of DISCUSSION both guanylate cyclase and cyclic GMP phosphoAlthough cyclic GMP receives the attention as a diesterases are under the control of developmental secondary messenger for insect hormones, eclosion program of ovaries. Indeed, the mixing of enzyme hormone (Truman et al., 1979), ecdysteroids preparations from different stages of development (Courgeon and Cailla, 1981) and diapause hormone showed only the additive activity (data not shown), (Yamashita, 1983; Chen et al., 1988), a limited work indicating that the developmental changes are not has appeared on characterization and activity due to the presence of inhibitors or activators. Howchanges of guanylate cyclase and cyclic GMP ever, none of the developmental changes in activities phosphodiesterase of insect tissues (Bodnaryk, 1983; of each enzyme are correlated to the titre changes in Smith and Combest, 1985). The available data have intracellular cyclic GMP during the development demonstrated that insect guanylate cyclase is not (Chen et al., 1988). different in kinetic properties from that of the A simple enzymatic model has been developed to other classes of animals (Filburn and Wyatt, 1976; account for the maintenance of the steady-state conC
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Guanylate cyclase and diesterase in silkworms centration of cyclic A M P and the changes in its concentration from one steady-state to another (Newsbolme and Start, 1973; Davies and Williams, 1975; Arch and Newsholme, 1976). As the case of cyclic AMP, cyclic G M P is produced by only guanylate cyclase and degraded by low-K= and highK= cyclic G M P phosphodiesterase. In the steadystate situation, activity of cyclase equals the total (low-K= plus high'Kin) activity of phosphodiesterases. Since both diesterases in silkworm ovaries obeyed Michaelis--Menten kinetics (Fig. 1), the cyclic G M P concentration, s, is given by the following equation, V~=
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where V=, Vl and Vh are the activities of guanylate cyclase, low-K= phosphodiesterase and high-K= phosphodiesterase, respectively. K~ and Km refer the K= values of low-K= and high-K= phosphodiesterase, respectively. For estimation of s, Vl and Vh were corrected to 58% of the maximum activity measured at pH 8.5, because the value at pH 8.5 was reduced to 58% at pH7.5 which was the optimum pH for guanylate cyclase activity and may approximate to the intracellular pH. The concentration of cyclic G M P (s) was computed on each datum through the developmental stages (Fig. 5). Cyclic G M P levels was at around 5 nmol/g ovaries in the first two days and markedly decreased to less than 1 nmol/g on day 2. From day 4 a gradual and then steep increase occurred until day 8. Thereafter, the high level was maintained until the adult emergence. This changing pattern clearly resembled that of cyclic G M P measured on ovary homogenates, except for the first 2 days (Chen et al., 1988). Consequently, it is conceivable that the mutual changes in guanylate cyclase and cyclic G M P phosphodiesterases are responsible for the titre changes in intracellular cyclic G M P in silkworm ovaries, although the calculated values remained several times as high as those measured (Chen et al., 1988; unpublished data).
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SG-removal caused a 30% increase in calculated values o f cyclic G M P in ovaries after day 8 (Fig. 5), although the increment was less than than found on measured values (Chen et al., 1988). This increase is mainly due to guanylate cyclase which showed a little higher activity in SG-removed ovaries after day 8 (Fig. 2). However, the effect of SG-removal on guanylate cyclase activity was less than that on the calculated concentrations of cyclic GMP. An insect neurotransmitter, proctolin, causes a slight decrease (by 30%) in guanylate cyclase activity in brain of Locusta migratoria migratoides (Hiripi et al., 1979). The more obvious effect of the peptide hormone on guanylate cyclase was demonstrated in rabbit ovaries in which administration of luteinizing hormone decreased activity by 50% (Patwardhan and Lanthier, 1984). Therefore, as with some peptide hormones, diapause hormone appears to regulate guanylate cyclase activity in silkworm ovaries, although a regulatory action on cyclic G M P phosphodiesterases is not excluded.
Acknowledgements--The authors
would like to thank Professor S. Kawase and Drs. M. Kobayashi and T. Yaginuma
of Nagoya University for their encouragement. The present study was supported by a (3rant-in-Aid (No. 6030424) for scientific research from the Ministry of Education, Science and Culture, Japan. REFERENCES Arch J. R. S. and Newsholme E. A. (1976) Activities and
some properties of adenylate cyclase and phosphodiesterase in muscle, liver and nervous tissues from vertebrates and invertebrates in relation to the control of the concentration of adenosine 3',5'-cyclic monophosphate. Biochem. J. 158, 603-622. Bodnaryk R. P. (1983) Cyclic nucleotides. In Endocrinology of Insects (Edited by Downer (3. H. and Laufer H.), pp. 567-614. Alan R. Liss, New York. Chcn J. H., Yaginuma T. and Yamashita O. (1988) Effect of diapause hormone on cyclic nucleotide metabolism in developing ovaries of the silkworm, Bombyx mori. Comp. Biochem. Physiol. 91B, 631-637. Courgeon A. M. and Cailla H. L. (1981) Cyclic AMP and cyclic (3MP variations in several Drosophila melanogaster embryonic cellular clones cultured/n vitro with or without 20-hydroxyecdysone. Exp. Cell Res. 133, 15-22. Davies J. I. and Williams P. A. (1975) Quantitative aspects of the regulation of cellular cyclic AMP levels---I. Structure and kinetics of a model system. J. theor. Biol. 53, 1-30.
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/-, 6 8 10 12 Days after I.arval-pupat ec.dysis
Fig. 5. Developmental changes in cyclic (3MP concentrations calculated on the activities of guanylate cyclase and cyclic (3MP phosphodiesterases in silkworm ovaries. Calculation was done on activities shown in Figs. 2, 3 and 4 as described in the Discussion. (O), Non-operated controls; (O), S(3-removed.
Fallon A. M. and Wyatt (3. R. (1975) Cyclic guanosine 3',Y-monophosphat¢: High levels in the male accessory gland of Acheta domesticus and related crickets. Biochim. biophys. Acta. 411, 173-185. Fallon A. M. and Wyatt (3. R. (1977a) (3uanylate cyclase in the accessory gland of the cricket, Acheta domesticus. J. Insect Physiol. 23, 1037-1041. Fallon A. M. and Wyatt (3. R. (1977b) Cyclic nucleotide phosphodiesterase in the cricket, Acheta domecticus. Biochim. biophys. Acta. 480, 428-441. Filburn C. R. and Karn J. (1973) An isotopic assay of cyclic 3',5'-phosphodiesterase with aluminum oxide columns. Analyt. Biochem. 52, 505-516. Filburn C. R. and Wyatt G. R. (1976) Adenylate and guanylate cyclases of cecropia silkmoth fat body. J. Insect Physiol. 22, 1635-1640. Filburn C. R., Karn J. and Wyatt G. R. (1977) Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body. Biochim. biophys. Acta. 481, 152-163.
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Garbers D. L. and Murad F. (1979) Guanylate cyclase assay methods. In Advances in Cyclic Nucleotide Research. (Edited by Brooker G., Greesard P. and Robinson G. A.), Vol. 10, pp. 57--67. Raven Press, New York. Goldberg N. D. and Haddox M. K. (1977) Cyclic GMP metabolism and involvement in biological regulation. Ann. Rev. Biochem. 46, 823-896. Hiripi L., Rozsa K. S" and Miller T. A. (1979) The effect of proctolin on the adenylate and guanylate cyclase in the Locusta brain at various developmental stages. Experientia 35, 1287-1288. LaPorte D. C. (1984) Cyclic nucleotides---teaching an old dogma new tricks. TIBS 9, 291-292. Morishima I. (1975) Cyclic nucleotide phosphodiesterase in silkworm. Developmental changes of cyclic AMP and cyclic GMP phosphodiesterases. Bioehim biophys. Acta 403, 106-112. Morishima I. (1981) Properties and distribution of guanylate cyclase in silkworm fat body. Comp. Biochem. Physiol. 68B, 567-573. Newsholme E. A. and Start C. (1973) Regulation in Metabolism, pp. 166-167. John Wiley, Chichester, New York, Brisbane and Toronto.
Patwardhan V. V. and Lanthier A. (1984) Cyclic GMP phosphodiesterase and guanylate cyclase activities in rabbit ovaries and the effect of in vivo stimulation with LH. J. Endocr. I01, 305-310. Smith W. A. and Combest W. L. (1985) Role of cyclic nucleotides in hormone action. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 263-299. Pergamon Press, Oxford. Truman J. W., Mumby S. M. and Welch S. K. (1979) Involvement of cyclic GMP in the release of stereotyped behaviour patterns in moths by a peptide hormone. J. exp. Biol. 84, 201-212. Yamashita O. (1983) Egg diapause. In Endocrinology of Insects (Edited by Downer R. G. H and Laufer H.), pp. 337-342. Alan R. Liss, New York. Yamashita O. (1984) Control of embryogenesis and diapause in the silkworm, Bombyx mori: Role ofdiapause hormone and egg- specific protein. Adv. Inverteb. Reprod. 3, 251-258. Yamanchi H. and Yoshitake N. (1984) Developmental stages of ovarian follicles of the silkworm, Bombyx mori L. J. Morphol 179, 21-31.
0305-0491/89 $3.00+ 0.00 © 1989 Pergamon Press pie
ACTIVITY CHANGES OF GUANYLATE CYCLASE AND CYCLIC GMP PHOSPHODIESTERASE RELATED TO THE ACCUMULATION OF CYCLIC GMP IN DEVELOPING OVARIES OF THE SILKWORM, B O M B Y X M O R I JIAN HUA CHEN and OKITSUGU YAMASHITA* Laboratory of Sericultural Science, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464-0 I, Japan (Tel: 052 781 5111) (Received 19 September 1988)
Abstract--l. Guanylate cyclase in ovaries of the silkworm, Bombyx mori, was not unusual in kinetic properties and mainly localized in the particulate fraction. Two cyclic GMP phosphodiesterases, low-Km (2.9/~M) and high-Km (125.5 tiM) enzyme, were found in the soluble fraction of ovaries. 2. All the enzymes showed the similar developmental changes in activity exhibiting a peak value in young ovaries. 3. The cyclic GMP concentrations calculated from these enzyme activities increased according to ovarian development, which resembled the developmental changes in measured values except for the first two days. 4. The calculated concentrations of cyclic GMP were high in ovaries with the removal of suboesophageal ganglion during the last period of development.
INTRODUCTION Along with adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-~yclic monophosphate (cyclic GMP) is well established to play the important roles in the control of many physiological processes in diverse organism (Goldberg and Haddox, 1977). The intracellular concentration of cyclic G M P is maintained at a steady-state value by its production from GTP by guanylate cyclase (EC 4.6.1.2) and conversion into G M P by cyclic G M P phosphodiesterase (EC 3.1.4.17). Changes in the concentration of cyclic G M P produced by physiological and pharmacological stimuli such as hormones, nervous action and metabolic inhibitors, are achieved mainly through modifications in the activity of either cyclase or phosphodiesterase, or both (LaPorte, 1984). Thus, the dynamic changes in these enzymes are finally reflected on the cellular events mediated by cyclic GMP. In the previous report, we have demonstrated that cyclic G M P behaves differently from cyclic A M P during ovarian development of the silkworm, Bombyx mori, and the level of cyclic G M P increased markedly through the last phase of vitellogenesis to oocyte maturation. Further, diapause hormone is shown to act to reduce the accumulation of cyclic G M P at this stage (Chen et aL, 1988). Such a stageand hormone-dependent accumulation of cyclic G M P seems to be associated with special properties or activity changes of cyclase and/or phosphodiesterase. Although several informations are available on the kinetic properties and developmental changes of these enzymes in insects (Bodnaryk, 1983; Smith and *Author to whom all correspondence should be addressed.
Combest, 1985), little work has appeared on the systematic analysis on the relationship between cyclic G M P levels and activities of both enzymes (Fallon and Wyatt, 1977a, 1977b). Furthermore, less attempt was done on the regulation mechanisms of these enzymes by insect hormones (Hiripi et al., 1979). In order to correlate these enzyme activities to cyclic G M P concentrations in silkworm ovaries, we have first characterized each enzyme in developing ovaries of silkworms. Referring to the properties of these enzymes, the assay condition was established and the activity was followed throughout the ovarian development. The effect of diapause hormone on developmental profile in enzyme activities was also surveyed. A simple model (Arch and Newsholme, 1976) was applied to correlate the developmental changes in enzyme activities to the accumulation profile of cyclic G M P in silkworm ovaries. MATERIALS AND METHODS
Animals A hybrid race (Shunrei and Shogetsu) of the silkworm, Bombyx mori was used. Embryonic life was maintained at 25°C under the continuous illumination to produce diapause eggs at the next generation (Yamashita, 1984). Larvae were reared on either fresh mulberry leaves or an artificial diet at 26°C under a long day condition (16hr L-8 hr D). The pupae ecdysed within 3 hr were collected to synchronize their following pupal-adult development, and kept at 26°C under the long day condition. Adult emergence occurred in females 12 days after the larval-pupal ecdysis when almost all oocytes completed their maturation (Yamauchi and Yoshitake, 1984). In some experiments, the suboesophageal ganglion (SG) which is a releasing center of diapause hormone (Yamashita, 1984) was extirpated on the day of larval-pupal eedysis (day 0) to obtain non-diapause type ovaries and eggs (Chen et al., 1988). 385
386
JIAN HUA CHEN and OKITSUGU YAMASHITA
Chemicals Cyclic GMP, creatine phosphate and creatine kinase were obtained from Yamasa Sboyu Co. (Chiba). 3-Isobutyl1-methylxanthine (IBMX) and 5'-nucleotidase were purchased from Sigma Chemical Co. (St. Louis). [3H]-GTP (10.6Ci/mmbl) and [3HI-cyclic G M P (18.3 Ci/mmol) were from Amersham International Co. (Amersham). Polyethyleneimine (PEI)-cellulose was from Seikagaku Kogyo Co. (Tokyo). Neutral aluminium oxide (grade 1) was from Woelm Pharma Co. (Eschwege). All other chemicals were of analytical grade.
Preparation of enzyme sources Ovaries were dissected out throughout pupal-adult development in cold 0.75% NaC1 and weighed quickly after the brief blotting on paper. The tissue was homogenized with 5 volumes of 20 mM triethanolamine buffer, pH 7.5, containing 350 mM sorbitol, 2 mM EDTA, 300 mM NaC1 for guanylate cyclase assay, and with 5 volumes of 50 mM Tris-HC1 buffer, pH 8.5, for cyclic GMP phospbodiesterase assay. The crude homogenate was used directly for assay of cyclase unless specified. For phosphodiesterase assay, the homogenate was centrifuged at 15,000g for 20min and the supernatant was passed through a small column of Sephadex G-25 to remove endogenous cyclic GMP. The resultant macromolecular fraction was used as enzyme source.
Assay of enzyme activities Guanylate cyclase. The conversion of [3H]-GTP into cyclic [3H]-GMP was measured in the presence of GTPregenerating system according to the method of Garbers and Murad (1979) with a slight modification. The incubation medium consisted of 50 mM triethanolamine buffer, pH 7.5, I mM IBMX, 10mM MnCI2, 8 m M creatine phosphate, 10 units creatine kinase, l mM [3H]-GTP (0.96/zCi) and 50/d enzyme solution (about 1 mg proteins) in a final volume of 200/~1. The reaction was initiated by addition of the enzyme solution and allowed to proceed for 40 min at 30°C. The reaction was stopped by addition of 0.25 ml of 200 mM zinc acetate containing 2 mM cyclic GMP. Followed by the coprecipitation of GMP, GDP and GTP with ZnCO 3 produced by addition of 0.25 ml of 200 mM Na2CO 3, the soluble fraction was applied onto a PEI-cellulose column (0.6 x 6.0cm) which was preequilibrated with 50 mM acetic acid. Cyclic GMP was eluted by 20 mM LiC1 after a sequential washing with 50 mM acetic acid and then water. Radioactivity was measured with a liquid scintillation counter (Aloka LSC-700, Aloka Co. Tokyo) after the solubilization with ACS-II (New England Nuclear Co., Boston). The recovery of cyclic G M P was estimated to be 50% by comparing with the elution efficiency of the authentic cyclic GMP.
Cyclic GMP phosphodiesterase. The activities were measured by a radiochemical procedure according to the method of Filburn and Karn (1973) with a slight modification. The incubation medium consisted of 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgC12, 1 mM dithiothreitol, 0.01 mM or I mM cyclic [3H]-GMP (0.01/~Ci) and 50#1 enzyme solution (about 1 mg proteins) in a final volume of 200/~1. The reaction was started by adding enzyme solution and continued for 10 to 30 rain at 30°C. The reaction was stopped by immersing the incubation mixture into a boiling water bath for 1 min. To the cooled mixture, 0.1 unit 5'-nucleotidase was added and the mixture was again incubated at 30°C for 30 min. The pH of the mixture was adjusted to pH 4.0 by adding 40 #1 of 1 N acetic acid and the supernatant was collected by a brief centrifugation. An aliquot of soluble fraction (200 #1) was directly applied onto an alumina column (0.6x5.0cm) which has been preconditioned with 100mM ammonium acetate, pH 4.0. The guanosine was eluted with 1.2 ml of 100 mM ammonium acetate, pH4.0. Radioactivity was measured by a liquid scintillation counter as above. By comparison with the authentic guanosine, recovery was calculated to be 87%. RESULTS
Properties of guanylate cyclase Some properties a n d cellular d i s t r i b u t i o n were determined o n ovaries f r o m day 6 p h a r a t e adults (Table 1). R e a c t i o n rates were linear with time for 100 m i n a n d protein c o n t e n t u p to 1.0 m g per assay tube. Effects o f M n 2+, Ca 2+ a n d M g 2+ were determ i n e d o n day 6 ovary extracts which were passed t h r o u g h a Sephadex G-25 column. M n z÷ was essentially required for this enzyme activity a n d the maxim u m activity was given at 10 m M . C a 2÷ a n d M g 2÷ h a d n o effects o n the activity at 1 0 m M . Therefore, 10 m M M n 2+ was included in the reaction mixture. A p H o p t i m u m was decided as p H 7.5 in the p H ranges f r o m 6.0 to 9.0. T h e activity-substrate relationship showed a s a t u r a t i o n kinetics a n d the a p p a r e n t Km value for G T P was estimated to be 0.40 m M for the particulate enzyme by the L i n e w e a v e r - B u r k plot. T h e crude h o m o g e n a t e was subjected to a differential centrifugation; 4 5 0 g for 10 min, 15,000 g for 20 min, 105,000 g for 60 m i n a n d its s u p e r n a t a n t fraction, a n d each fraction was assayed u n d e r the s t a n d a r d condition. A b o u t 9 0 % o f total activity was recovered in the 15,000g pellet. T h e t r e a t m e n t o f the 15,000g pellet with T r i t o n X-100 (0.2%) increased the activity
Table 1. Some properties of guanylate cyclase and cyclic GMP phosphodiesterases of silkworm evades Cyclic GMP Guanylate phosphodiesterase cyclase High-Kin Low-KIn Optimum pH* 7.5 8.5 8.5 Km value* 400gM 125.5 #M 2.9/zM Effect of modulators activated by inhibited by Mn2+ at 10raM and IBMX at 1 mM Triton X-100 at 0.2% Subcellular distributiont 15,000g pellet 88% 8% 7% 15,000g supernatant 11% 92% 91% Ovaries from day 6 pharate adults were homogenized and ihe ovary homogenate was used as the enzyme source for guanylate cyclase. For assay of cyclic GMP phosphodicsterases, macromolecular fraction after passing the 15,000g supernatant through Sephadex G-25 column was used as enzyme sours.
*Each value represents means of three determinations. tThe result was obtained from a typical experiment.
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Fig. 2. Changes in activities of guanylate cyc'lase during ovarian development of silkworms. SG was removed on the day of pupation. Each point represents mean values from three determinations consisting of 3 individual with S.D. ( 0 ) , Non-operated controls; (O), SG-removed. late cyclase throughout pupal-adult development. The activity was about 9 nmol/min per g ovaries on day 0 and increased sharply in day. 1. A sudden decrease in activity occurred on day 2 and the declined activity was maintained until day 7 with some fluctuations. There was slight decrease in activity thereafter. The similar changing pattern was observed in ovaries with SG-removed. Interestingly, activities in ovaries after day 7 remained at a slightly
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The crude homogenate of day 6 ovaries were subjected to a differential centrifugation as described above. The supernatant fraction after a 15,000g centrifugation contained about 90% of the total activity (Table 1). In the following experiments, this 15,000 g supernatant was mainly used as the enzyme source. The rate of cyclic GMP degradation was close to linearity for 60 rain. The activity was proportional to the amounts of enzyme up to 1.6 mg of proteins. The optimum pH was estimated to be 8.5 by using 100mM PIPES buffer for pH 6.0 to 7.5, 100raM E 200 Tris-HCI buffer for pH7.5 to 9.0 and 100raM glycine-NaOH buffer for pH 8.5 to 10.5 (Table 1). A slightly higher activity was found in Tris--HCl buffer compared to the other buffers used. A half maximum activity was found at pH 7.5 and pH 9.5. Indeed, when assayed at pH 7.5, activity was reduced by 58% of the maximum activity obtained at pH 8.5. The O. relationship between substrate concentrations and IE activities exhibited a complex curve having two saturation kinetics. The Hofstee plot (V against V/S) gave two straight-line portions connected by a curve, 0 2 4 6 8 I0 12 and apparent Km values were calculated for the linear portion of the graph (Fig. 1). Two apparent Km Days after larval-pupal ecdysis values for cyclic GMP were estimated; 125.5/~M as a high-Kmvalue and 2.9 p M as a low-Kin value. These Fig. 3. Changes in activities of the l~igh-/~ cyclic GMP during ovarian development of silkK= values were also found in enzymes of ovaries with phosphodiesterase worms. SG was removed on the day of pupation. The SG-removed. activity was determined on 15,000&supernatant fraction of at I mM [3HI-cyclicGMP. Each point represents Developmental changes in activities of guanylate ovaries mean values from 3 determinations consisting of three cyclase and cyclic GMP phosphodiesterases individuals with S.D. (O), Non-operated controls; (O), SG-removed. Figure 2 represents the activity changes in guany-
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Fallon and Wyatt, 1977a; Goldberg and Haddox, 1977; Morishima, 1981). The similar properties and subcellular distribution were found on guanylate 30 cyclase of silkworm ovaries (Table 1). Two types of cyclic GMP phosphodiesterases, low- Kmenzyme and high-Km enzyme, were present in silkworm ovaries > o and their properties were similar to those of other oi vertebrates and insects (Morishima, 1975; Arch and Newsholme, 1976; Fallon and Wyatt, 1977b; Filburn ~ 20 et al., 1977). Consequently, the silkworm ovary o shares the similar enzymatic system for cyclic GMP E metabolism as found in various animal tissues in their general properties. The activity of guanylate cyclase in ovaries ranged o from 2 to 15 nmol/min per g ovaries (Fig. 2), which resembles the activity found in fat body of H. ececropia (Filburn and Wyatt, 1976) and B. mori n I[ (Morishima, 1981), in brain of Locusta migratoria migratoides (Hiripi et al., 1979) and in male accessory gland of Acheta domesticus (Fallon and Wyatt, 1977a). The basal activities of cyclic GMP phosphoo i ; 6 8 diesterase of silkworm ovaries were at the range from Days after tarval-pupal ecdysis 4 to 35 nmol/min per g ovaries for low-Kin enzyme Fig. 4. Changes in activities of the low-K= cyclic GMP and 30 to 300 nmol/min per g for high'Kin enzyme phosphodiesterase during ovarian development of silk- (Figs 3 and 4). These values remain in the ranges of worms. SG was removed on the day of pupation. The values reported on several tissues from invertebrates activity was determined at 10/aM [3H]-cyclicGMP on the and vertebrates (Arch and Newsholme, 1976). Thus, same fraction used in Fig. 3. Each point represents it is likely that cyclase and phosphodiesterase in mean values from three determinations consisting of three silkworm ovaries are active enough to maintain a individuals with S.D. (O), Non-operated controls; (©), steady-state level of cyclic GMP in them. SG-removed. Fat body o f B o m b y x mori (Morishima, 1975, 1981) and Hyalophora cecropia (Filburn and Wyatt, 1976; higher level in ovaries with SG-removed than in the Filbern et al., 1977) exhibited remarkable activity changes in guanylate cyclase and cyclic GMP controls. Throughout the ovarian development, we followed phosphodiesterase according to the larval-pupalseparately the activities of both high-Kin and low-Kin adult development. During this period, however, the cyclic GMP phosphodiesterase (Figs. 3 and 4). The activities changed in the opposite directions to each high-Km enzyme activity assayed at 1 mM cyclic other. Whereas, guanylate cyclase and cyclic GMP GMP rose markedly during the first two days and the phosphodiesterase of male accessory gland of Acheta increased level was kept for one day before a steep domesticus did not undergo marked changes in decline to day 7 (Fig. 3). Thereafter, the low activity activity during its maturation, although a great remained unchanged until adult emergence of day increase of cyclic GMP took place during this period 12. The similar changing pattern in activities was (Fallon and Wyatt, 1975, 1977a, 1977b). In contrast, observed with SG-removed (Fig. 3). The low-Km silkworm ovary enzymes showed the similar temporal enzyme also showed the almost similar changing changes with a peak value at the early stage of pattern throughout the ovarian development to that ovarian development (Figs. 2, 3 and 4). The stage of found in high-Km enzyme (Fig. 4). However, the peak activity was correlated to the maturation activity of low-Km enzyme remained at 1/6 to 1/10 of of follicle cells prior to the active vitellogenesis that of high-Kin enzyme. SG-removal had no effect on (Yamauchi and Yoshitake, 1984). The active vitellothe activity changes on low-K m enzyme throughout genesis occurred accompanying with decreasing ovarian development (Fig. 4). activities. The reduced activity was related to the choriogenesis and the maturation of oocytes. It is therefore conceivable that the activity changes of DISCUSSION both guanylate cyclase and cyclic GMP phosphoAlthough cyclic GMP receives the attention as a diesterases are under the control of developmental secondary messenger for insect hormones, eclosion program of ovaries. Indeed, the mixing of enzyme hormone (Truman et al., 1979), ecdysteroids preparations from different stages of development (Courgeon and Cailla, 1981) and diapause hormone showed only the additive activity (data not shown), (Yamashita, 1983; Chen et al., 1988), a limited work indicating that the developmental changes are not has appeared on characterization and activity due to the presence of inhibitors or activators. Howchanges of guanylate cyclase and cyclic GMP ever, none of the developmental changes in activities phosphodiesterase of insect tissues (Bodnaryk, 1983; of each enzyme are correlated to the titre changes in Smith and Combest, 1985). The available data have intracellular cyclic GMP during the development demonstrated that insect guanylate cyclase is not (Chen et al., 1988). different in kinetic properties from that of the A simple enzymatic model has been developed to other classes of animals (Filburn and Wyatt, 1976; account for the maintenance of the steady-state conC
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Guanylate cyclase and diesterase in silkworms centration of cyclic A M P and the changes in its concentration from one steady-state to another (Newsbolme and Start, 1973; Davies and Williams, 1975; Arch and Newsholme, 1976). As the case of cyclic AMP, cyclic G M P is produced by only guanylate cyclase and degraded by low-K= and highK= cyclic G M P phosphodiesterase. In the steadystate situation, activity of cyclase equals the total (low-K= plus high'Kin) activity of phosphodiesterases. Since both diesterases in silkworm ovaries obeyed Michaelis--Menten kinetics (Fig. 1), the cyclic G M P concentration, s, is given by the following equation, V~=
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where V=, Vl and Vh are the activities of guanylate cyclase, low-K= phosphodiesterase and high-K= phosphodiesterase, respectively. K~ and Km refer the K= values of low-K= and high-K= phosphodiesterase, respectively. For estimation of s, Vl and Vh were corrected to 58% of the maximum activity measured at pH 8.5, because the value at pH 8.5 was reduced to 58% at pH7.5 which was the optimum pH for guanylate cyclase activity and may approximate to the intracellular pH. The concentration of cyclic G M P (s) was computed on each datum through the developmental stages (Fig. 5). Cyclic G M P levels was at around 5 nmol/g ovaries in the first two days and markedly decreased to less than 1 nmol/g on day 2. From day 4 a gradual and then steep increase occurred until day 8. Thereafter, the high level was maintained until the adult emergence. This changing pattern clearly resembled that of cyclic G M P measured on ovary homogenates, except for the first 2 days (Chen et al., 1988). Consequently, it is conceivable that the mutual changes in guanylate cyclase and cyclic G M P phosphodiesterases are responsible for the titre changes in intracellular cyclic G M P in silkworm ovaries, although the calculated values remained several times as high as those measured (Chen et al., 1988; unpublished data).
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SG-removal caused a 30% increase in calculated values o f cyclic G M P in ovaries after day 8 (Fig. 5), although the increment was less than than found on measured values (Chen et al., 1988). This increase is mainly due to guanylate cyclase which showed a little higher activity in SG-removed ovaries after day 8 (Fig. 2). However, the effect of SG-removal on guanylate cyclase activity was less than that on the calculated concentrations of cyclic GMP. An insect neurotransmitter, proctolin, causes a slight decrease (by 30%) in guanylate cyclase activity in brain of Locusta migratoria migratoides (Hiripi et al., 1979). The more obvious effect of the peptide hormone on guanylate cyclase was demonstrated in rabbit ovaries in which administration of luteinizing hormone decreased activity by 50% (Patwardhan and Lanthier, 1984). Therefore, as with some peptide hormones, diapause hormone appears to regulate guanylate cyclase activity in silkworm ovaries, although a regulatory action on cyclic G M P phosphodiesterases is not excluded.
Acknowledgements--The authors
would like to thank Professor S. Kawase and Drs. M. Kobayashi and T. Yaginuma
of Nagoya University for their encouragement. The present study was supported by a (3rant-in-Aid (No. 6030424) for scientific research from the Ministry of Education, Science and Culture, Japan. REFERENCES Arch J. R. S. and Newsholme E. A. (1976) Activities and
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/-, 6 8 10 12 Days after I.arval-pupat ec.dysis
Fig. 5. Developmental changes in cyclic (3MP concentrations calculated on the activities of guanylate cyclase and cyclic (3MP phosphodiesterases in silkworm ovaries. Calculation was done on activities shown in Figs. 2, 3 and 4 as described in the Discussion. (O), Non-operated controls; (O), S(3-removed.
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