J. Biochem. 84, 1495-1500 (1978)

Adenylate Cyclase in Silkworm Properties of the Enzyme in Pupal Fat Body1 Isao MORISfflMA Department of Agricultural Chemistry, Tottori University, Tortori 680 Received for publication, May 29, 1978

Adenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg 1+ or Mn 1+ , and the degree of stimulation by Mn 1+ was 2 times greater than that by Mg 1+ compared at the saturating concentrations. In the presence of Mg 1+ , the enzyme was inhibited by both EGTA and high concentrations of Ca a+ , showing biphasical response to Ca 1+ . The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13 mM for MgATP and 0.086 mM for MnATP.

In vertebrate animals, adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] that catalyzes the formation of adenosine 3',5'-monophosphate (cyclic AMP) from ATP has been intensively studied, and evidence has accumulated that the enzyme has a central role in mediating cellular responses to a variety of stimuli (7, 2) In insect, however, little is known about the role of cyclic AMP and there is little information about adenylate cyclase, although the existence of cyclic AMP, phosphodiesterase, adenylate cyclase, and protein kinase has been demonstrated (see Ref. 3 for a listing). In previous papers I have reported that a lepidopteran insect silkworm (Bombyx mori) has a phosphodiesterase system comparable to the 1

This work was supported in part by a Scientific Research Grant 256064 from the Ministry of Education, Science and Culture of Japan. Abbreviations: Cyclic AMP, adenosine 3',5'-monophosphate; EGTA, ethyleneglycol-bis(/)-aminoethyl ether>Ar,A''-tetraacetic acid; Me»+, Mg«+ or Mn*+. Vol. 84, No. 6, 1978

vertebrate system and that the activity greatly changes during development of the insect (4-6). These facts suggest that cyclic AMP system plays an important role in regulating cellular events in insect as well, and led me to study adenylate cyclase, the other enzyme controlling intracellular levels of cyclic AMP. In this paper I describe the properties of adenylate cyclase in silkworm pupal fat body, an organ analogous in many functional aspects to mammalian liver in playing a central role in lipid, carbohydrate, and protein metabolism (7, 8). MATERIALS AND METHODS 2-{'H]ATP ammonium salt (19-27 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, and 8-["C]cyclic AMP (53 mCi/mmol) from New England Nuclear. [*H]ATP was used without any further purification. Neutral alumina (activity grade I) was obtained from E. Merk, washed with water to remove fine particles and dried in an

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1496 oven at 100°C (9). Dowex 50W x 4 H+-form, 200400 mesh) was the product of Dow Chemicals. Non-labeled nucleotides, creatine phosphate and creatine phosphokinase (rabbit muscle) were from Sigma Chemicals. Silkworm (Bombyx mori) of strain Daizo was reared on mulberry leaves or artificial diet (70) at 25°C in our laboratory. Preparation of Silkworm Fat Body Extract— Fat body was removed from silkworm pupa, rinsed in 0.14 M KC1-0.01 M phosphate buffer, pH 6.8, and blotted on filter paper. After weighing, the tissue was sonicated in 10 vol. of 0.05 M TrisHC1, pH 7.2, containing 0.25 M sucrose with a sonicator fitted with a microprobe (Type 5201D, Ohtake Works, Tokyo) at 5 watt for 10 s. The sonicated tissue was immediately used for adenylate cyclase assay unless otherwise indicated. In the experiments to determine subcellular distribution of the enzyme, the tissue was homogenized in 5 vol. of the buffered sucrose with 6 strokes in a glass homogenizer. The homogenate was centrifuged at 1,000 g for 15 min, the supernatant drawn off, and the sediment resuspended in the original volume of the homogenizing buffer.

I. MORISHIMA with 20 ml of 0.1 M ammonium acetate, pH4.0. The eluate obtained from the alumina column was discarded, and the Dowex column was removed. The alumina column was then washed with two successive 2.5-ml lots of 0.01 M ammonium acetate, pH 4.0, followed by 0.5 ml of 0.4 M ammonium acetate, pH 4.0. Both eluates were discarded. Cyclic AMP was then eluted with 2.5 ml of 0.4 M ammonium acetate, pH4.0, and collected in a scintillation vial. One-tenth volume of the final eluate was taken for measurement of absorbance at 260 nm, after appropriate dilution with 0.01 N H O , to assess and correct for recovery of cyclic AMP. Recovery was also determined using the reaction tube in which [uC]cyclic AMP was substituted for PH]ATP; both methods gave equivalent values. The radioactivity was measured with toluene-Triton X100 scintillator. The assays were run in duplicate or triplicate, and the variation in values was usually less than 5 %.

Analysis by paper chromatography developed with two different solvents (isopropanol : cone. NH 4 OH : H,O=7 : 1 : 2 and ethanol : 1 M ammonium acetate, pH 7 . 5 = 7 : 3) showed the triAdenylate Cyclase Assay—Adenylate cyclase tiated product recovered from the column to be activity was determined by a method based in part cyclic AMP. By this procedure, 80-90% of cyclic on an improvement of the alumina chromato- AMP was consistently recovered in the final eluate. graphy at acidic pH originally described for phos- Assay blanks were prepared by omitting tissue phodiesterase assay by Filburn and Karn (77). extract or by adding heated tissue extract; both The standard incubation mixture contained 40 raM methods gave equivalent values, which routinly Tris-HCI, pH7.2, lOmM MgCl, or 2 mix MnCl,, ranged from 0.003 to 0.005 % of initially added SH 10 raM creatine phosphate, 3 units of creatine counts. The blank value did not significantly phosphokinase, 0.08% bovine serum albumin, increase during storage of [3H]ATP in 50% ethanol 0.4 mM l-methyl-3-isobutylxanthine, 1 mM cyclic at -20°C for at least 6 months. AMP, 0.2 mM PHJATP (50-60 cpm/pmol) and Miscellaneous—Protein was determined by a 50-400 pg protein of tissue extract in a total modification (72) of the method described by Lowry volume of 100 p\. The incubation was for 15 min et al. (13) with bovine serum albumin as standard. at 30°C unless otherwise indicated. The reaction The concentrations of MeATP (Me is Mg or Mn) was stopped by heating for 2 min in a boiling and free divalent cations were determined by water bath, following the addition of 100 [i\ of a employing a cubic equation similar in form to mixture containing 5 mM cyclic AMP, 4 mM ATP, that derived by Moe and Butler (14). The stability and 0.2 M acetic acid. The reaction mixture was constants used in calculation were those reported then diluted to 1 ml with 0.05 M Tris-HCI, pH 7.2, by Khan and Martell (75). and applied to a column (0.7x13 cm) containing l m l of Dowex 50Wx4 (H + form) resin. The RESULTS eluate from the column and 2.5-ml HjO wash was discarded. To each column 3.5 ml of H,O were Eniymic Formation of Cyclic AMP by Silkworm then added, and the eluate was passed directly Pupal Fat Body Homogenates—When [3H]ATP was into a column (0.7x13 cm) containing 0.75 g of incubated with sonicated preparation of pupal fat neutral alumina that had been previously washed body under standard assay conditions, the rate of J. Biochem.

ADENYLATE CYCLASE IN SILKWORM FAT BODY cyclic AMP production was linear for at least 30 min both in the presence and absence of 5 mM N a F (Fig. 1). The activity was proportional to the amount of enzyme up to 400 /*g protein per assay. In contrast to adenylate cyclase of cecropia silkmoth fat body (16), addition of 2 HIM dithiothreitol to the reaction mixture had no effect on the silkworm enzyme activity (data not shown).

10

20

30

45

Time (min)

Fig. 1. Adenylate cyclase activity as a function of time. The standard reaction mixtures containing silkworm pupal fat body extract of 106 ^g protein and 10 mM MgCl, were incubated in the presence ( • ) or absence (O) of 5 mM NaF.

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Subcellular Distribution—Pupal fat body homogenates were centrifuged at 1,000 g for 15 min and activities in the resulting sediment and supernatant were determined in the presence of Mg l + or Mn T+ . Typical results are shown in Table I. Though the total recovery of adenylate cyclase was variable and low (35-63 % in 4 repeated experiments), the ratio of Mg 1+ -stimulated and Mn !+ -stimulated activity in each fraction was nearly constant in repeated experiments, and 7 5 8 3 % of the recovered activity appeared in the particulate fraction. Similar variable recovery has been observed with cecropia silkmoth adenylate cyclase (16). Effect of pH—The optimum pH range for the enzyme was 7.2 to 7.6 in the presence of 10 mM MgCl, (Fig. 2). In the presence of 2 mM MnCl t , the activity was maximum at slightly lower pH, and sharply decreased above pH 7.5. Fluoride Stimulation—Silkworm adenylate cyclase activity was stimulated by fluoride ion as in vertebrate enzyme (1). In the presence of Mg t + , the stimulation was maximum at NaF concentrations of 3 to 7.5 mM, and higher concentrations were rather inhibitory (Fig. 3). In the presence of Mn 1+ , in contrast, NaF concentration of 10 mM or higher was required for maximum stimulation,

TABLE I. Subcellular distribution of adenylate cyclase. Homogenate of pupal fat body was centrifuged as described in Methods. The sediment was resuspended in the original volume of the buffered sucrose. The reaction mixtures contained 25 it\ of each fraction, 10 mM MgCl, or 2 mM MnCl,, and other reagents as described in Methods. Adenylate cyclase activity pmol/min/mj ! wet wt (%)

Homogenate Supernatant Paniculate Recovery Recovered activity in paniculate

Vol. 84, No. 6, 1978

Mg'+

Mn1*

1.00 (100) 0.098 (9.8) 0.489 (48.9) (58.7)

2.05 (100) 0.215 (10.5) 1.06 (51.7) (62.2)

(83. 3)

(83.1)

Fig. 2. Effect of pH on adenylate cyclase activity. The standard reaction mixtures containing 230 fig protein were incubated in the presence of 10 mM MgCl, ( • , • ) or 2mM MnCl, (O, A). A, A, Tris-maleate; • , O, Tris-HCl.

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I. MORISH1MA

-

A

t 1

4 i

i / /

3 5 / 1

1

5 10 NaF (mM)

I

20

Fig. 3. Effect of NaF on adenylate cyclase activity. The standard reaction mixtures containing 120 fig protein were incubated in the presence of 10 mM MgCl, ( • — • ) or 2 mM MnCli (O--O) with varying concentrations of NaF.

and no inhibitory effect was observed up to 20 mM. Effect of Divalent Metal Ions—The adenylate cyclase required Mg1+ or Mn1+ for activity. The activity as a function of varying Mg1+ and M a " concentrations is shown in Fig. 4. The optimum Mg8+ concentration was 10 mM both in the presence and absence of 5 mM NaF. Mgt+ concentrations higher than 10 mM were rather inhibitory. Activation by Mn s+ occurred over a concentration range approximately 10 times lower than that of MgJ+. The activity obtained with saturating concentration of Mn1+ was approximately 2 times higher than that with Mg t+ . The effect of saturating concentrations of Mg1+ and Mn t+ were not additive (Fig. 4). Caa+ could not activate the enzyme (data not shown), but strongly inhibited the Mg1+stimulated activity (Table II). The concentration of free Ca1+ which gave 50% inhibition of the activity was 1.2 mM determined by dose-response curve for free Ca l+ (not shown). A Ca1+-chelating agent, EGTA, also inhibited the Mg1+-stimulated cyclase; the addition of 0.1 mM EGTA caused 35% inhibition. The same concentration of EDTA, however, had no effect on the activity; higher concentration was slightly inhibitory. In contrast, Mn*+-activated enzyme was less affected by Caf+ and EGTA.

if if •/

y

* *

4 /

* */

I** I

0 0.2

1 2 10 20 Me ! t (mM) Fig. 4. Effect of MgI+ and Mn1+ on adenylate cyclase activity. The standard reaction mixtures containing 216 fig protein were incubated in the presence of varying concentrations of MgCl, or MnCl!. • — • , MgCl,; A--A, MgClt plus 5mM NaF; o — O , MnCl,; A--A, MnCl, plus 10mM NaF; • , 2mM MnQ, plus lOmM MgQ,. TABLE n. Effect of Ca*+ and chelating agents on adenylate cyclase activity. The sonicated fat body was centrifuged at 1,000 g for 15 min, the sediment resuspended in the original volume of buffered sucrose, and 150 /ig protein used for assay under the standard conditions. Specific activities in the absence of additions were 6.3 and 13.2 pmol/min/mg protein with 10 mM MgCl, and 2 mM MnCl,, respectively, and designated as 100%. Adenylate cyclase activity (%) Additions None FreeCa

EGTA EDTA

1+

Mg'+

Mn'+

100

100

0.08 mM

102

98

0.83 mM

67

102

2.5 mM

21

77

0.1 mM

65

102

1 mM

55

95

0.1 mM

98

102

1 mM

76

91

/. Biochem.

ADENYLATE CYCLASE IN SILKWORM FAT BODY

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has been considered by others (19, 20) and was beyond the scope of this study. However, the observations described here suggest that the adenylate cyclase in silkworm is subject to a conformational change depending upon the species of cation that binds to it, the change being the cause for the observed difference in enzymic properties. This postulate is based on the assumption that both cations activate the same adenylate 0 10 20 -10 0 10 20 (MgATP)-'(mM- ) cyclase system. The fact that combination of the (MnATP) (mM' ) Fig. 5. Lineweaver-Burk plots of adenylate cyclase two cations at saturating concentrations does not activity. Reciprocal velocities (expressed as pmol have an additive effect supports the assumption cyclic AMP formed/min/mg protein) are plotted as a and rules out the possibility that silkworm fat body function of reciprocal MgATP (left) or MnATP (right) contains multiple adenylate cyclase systems, some concentration. Assays were performed under the activated by Mga+ but not Mn1+, and vice versa. standard conditions using 230 fig protein per assay. The following observations also support the • , Basal activity; O, NaF-stimulated activity, where assumption. The ratio of Mg1+-stimulated and NaF concentrations were 5 mM for Mg*+-activity and MnI+-stimulated activities is constant in each of 10 mM for Mn1+-activity. subcellular fraction (Table I), in spite of variable recoveries of the activity. The constant ratio of Kinetic Properties—The enzyme demonstrated the activities is also obtained regardless of the typical Michaelis-Menten kinetics. Lineweaver- methods used for tissue disruption, although the Burk plots of the activity show that basal and level of activity and the degree of stimulation by NaF-stimulated activities exhibited no difference NaF vary as a function of strength of tissue in their affinity for both substrate MgATP and sonication or homogenization (Morishima, L MnATP, and that the primary action of NaF was unpublished results). to increase F m a x (Fig. 5). The Km values were High concentrations of Ca1+ exhibited a determined by linear regression analysis of the marked inhibition of Mga+-stimulated adenylate slopes and intercepts of s/v versus s plots. The cyclase of silkworm fat body, but little if any mean values ±S.E. of Km obtained from three inhibition of the Mn1+-stimulated activity. This independent experiments were 0.13 ±0.005 mM for inhibition by Cal+ can not be attributed solely to MgATP both in the presence and absence of NaF, apparent decrease of the activity due to the reducand 0.086±0.006 and 0.083±0.005 mM for MnATP tion of the concentration of the substrate MgATP in the presence and absence of NaF, respectively. by the addition of high concentration of Caa+, since the addition of 3 mM Ca s+ to the reaction mixture containing 10 mM MgO t and 0.2 mM DISCUSSION ATP, for example, causes reduction of the MgATP Silkworm pupal fat body contains adenylate concentration from 0.199 mM to 0.170 mM, but cyclase comparable to the mammalian enzyme in this reduction would cause only 6% decrease of plots). its properties, the activity depending on either the activity (calculated from ATP kinetics similar selective inhibition of MgI+-activity by Mg t+ or Mn1+. The degree of stimulation of the A f+ activity by Mn l+ is greater than that by Mg1+. Ca has been reported in mammalian tissues (21, Similar results have been reported with adenylate 22). The activity of silkworm adenylate cyclase cyclase of rabbit heart (17) and rat cerebral cortex was also inhibited by EGTA. The observation cyclase requires low con(18). The Mg1+-stimulated and Mnt+-stimulated indicates the adenylate t+ activities exhibit marked differences in some centrations of Ca for the full activity. Thus 1+ enzymic properties: pH-activity curve, NaF stimu- the enzyme exhibits a biphasic response to Ca . 1+ lation, affinity for substrate and inhibition by Ca . This behavior has been observed for the enzyme of A detailed analysis of the mechanism by which mammalian brain and some other tissues (23-25), f+ Mgt+ and Mnf+ interact with adenylate cyclase where the activation by low concentrations of Ca 1

Vol. 84, No. 6, 1978

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1

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I. MORISHIMA

is attributed to a Cal+-dependent regulator. The results observed here may indicate the existence of Cal+-dependent regulator in silkworm fat body, yet so far such a regulator has not been detected in insect.

10. Nihmura, M. (1973) Nippon Ndgei Kagaku Kaishi (in Japanese) 47, 241-249 11. Filburn, C.R. & Karn, J. (1973) Anal. Biochem. 52, 505-516 12. Schacterle, G.R. & Pollack, R.L. (1973) Anal. Biochem. 51, 654-655 13. Lowry, O.H., Rosenbrough, NJ., Farr, A.L., & I wish to thank Professors Dr R. Nakamura and Dr S. Randall, R.J. (1951) /. Biol. Chem. 193, 265-275 Hirano for their encouragement and support. I am 14. Moe, O.A. & Butler, L.G. (1972) / . Biol. Chem. 247, very grateful to Dr T. Kawai for supplying silkworm 7315-7319 eggs and to Mr H. Tanaka and Mr Y. Kimura of Totton Sericultural Experimental Station for technical advice 15. Khan, M.M.T. & Martell, A.E. (1966) / . Am. Chem. Soc. 88, 668-671 on rearing silkworm on artificial diet and for supplying 16. Filburn, C.R. & Wyatt, G.R. (1976) /. Insect Physiol. materials of the diet. 22, 1635-1640 17. Drummond, G.I., Severson, D.L., & Duncan, L. REFERENCES (1971) /. Biol. Chem. 246, 4166-4173 18. Perkins, J.P. & Moore, M.M. (1971) / . Biol. Chem. 1. Perkins, J.P. (1973) in Advances in Cyclic Nucleotide 246,62-68 Research (Greengard, P. & Robison, G.A., eds.) 19. Garbers, D.L. & Johnson, R.A. (1975) J. Biol. Chem. Vol. 3, pp. 1-64, Raven Press, New York 250, 8449-8456 2. Maguire, M.E., Ross, E.M., & Gilman, A.G. (1977) in Advances in Cyclic Nucleotide Research (Green- 20. Londos, C. & Preston, M S. (1977) /. Biol. Chem. 252, 5957-5961 gard, P. & Robison, G.A., eds.) Vol. 8, pp. 1-84, 21. Steer, M.L. & Levitzki, A. (1975) /. Biol. Chem. 250, Raven Press, New York 2080-2084 3. Catalan, R.E., Castill6n, M.P., & Municio, A.M. 22. Hanski, E., Sevilla, N., & Levitzki, A. (1977) Eur. (1976) Biochem. Biophys. Res. Commun. 69, 914-919 J. Biochem. 76, 514-520 4. Morishima, I. (1975) Biochim. Biophys. Acta 391, 23. Von Hungen, K. & Roberts, S. (1973) Nature New 75-83 Biol. TA1, 58-60 5. Morishima, I. (1975) Biochim. Biophys. Acta 403, 24. Brostrom, CO., Huang, Y.-C, Breckenridge, B.M., 106-112 & Wolff, D.J. (1975) Proc. Natl. Acad. Sci. U.S. 72, 6. Morishima, I. (1975) Biochim. Biophys. Acta 410, 64-68 310-317 25. Brostrom, M.A., Brostrom, CO., Breckenridge, 7. Kilby, B.A. (1963) Adv. Insect Physiol. 1, 111-174 B.M., & Wolff, D.J. (1976) /. Biol. Chem. 251, 8. Wyatt, G.R. (1975) Verh. Dtsch. Zool. Ges. 1974, 4744-1750 209-226 9. Krishnan, N. & Krishna, G. (1976) Anal. Biochem. 70, 18-31

J. Biochem.

Adenylate cyclase in silkworm. Properties of the enzyme in pupal fat body.

J. Biochem. 84, 1495-1500 (1978) Adenylate Cyclase in Silkworm Properties of the Enzyme in Pupal Fat Body1 Isao MORISfflMA Department of Agricultural...
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