Time interval measuring enzyme for resumption of embryonic development in the silkworm, Bombyx mori

J. Insect Physiol. Vol. 41, No. 10, pp. 905-910, 1995 Copyright 0 1995 Elsevier Science Ltd

0022-1910(95)00024-O

Pergamon

Printed in Great Britam. All rights reserved 0022-1910195 $9.50 + 0.00

Time Interval Measuring Enzyme for Resumption of Embryonic Development Silkworm, Bombyx mori HIDENORI

KAI,*§ YOSHIHIKO

KOTANI,*t

YUNGEN

MIAO,*f

MASAAKI

in the

AZUMA*

Received 18 July 1994: revised 4 January 199.5

Time measurement by esterase A4 (Ease A4) purified from Bombyx diapausing eggs has been studied using ATP as a substrate. At 5’C, the ATPase activity of Ease A4 was very low initially, but in 2 weeks activity increased sharply and then rapidly fell. The period required to activate the enzyme was equivalent to that observed in uivo and was coincident with the chilling period indispensable for diapause termination. Ease A4 extracted from the eggs that had been chilled for 10 days reached a peak in a few days which implies that activation took place 2 weeks after chilling even if the chilling resulted from a combination of 10 days in uiuo and a few days in vitro. The duration of chilling was the critical factor in its activation. The re-inactivated enzyme could be regenerated by guanidine_HCl (GuHCl) treatment. The GuHCl effect was also observed during the initial activation of the enzyme. GuHCl reset the Ease A4 so that another 2 weeks was needed for activation. It is concluded that the Ease A4 may undergo conformational changes by an identical mechanism both in uiuo and in uitro, resulting in a time-interval activation of the enzyme which is crucial for diapause termination by cold. Biological duration

clock

Interval

timer

Timer

molecule

Esterase

A4

DNA-dependent

ATPase

Diapause

the cold (YC), the general esterase activity of Ease A4 suddenly increases at a certain time during the chilling period and then falls rapidly (Kai et al., 1984). This transitory burst in activity can also be observed in vitro (Kai et al., 1987): when the purified inactive Ease A4 was chilled at 5°C in vitro general esterase activity was very low initially, then was suddenly elevated after a certain period of chilling and then dropped rapidly. The sudden elevation of Ease A4 activity in vitro was equivalent to that observed in vivo and was coincident with the chilling period that is known to be indispensable for diapause termination (Kai and Nishi, 1976; Kai et al., 1984, 1995). Although those observations reinforce the validity of Ease A4 for research on diapause development, a number of important questions remain unanswered. Some of the problems that remain are: (1) the use of a non-physiological substrate, (2) the long intervals over which the enzyme assay was run and (3) it is not known whether an identical mechanism was occurring in vivo and in vitro (Kai et al., 1987). In the present experiment, we find that Ease A4 has an intrinsic ability to hydrolyze adenosine-.5’triphosphate (ATP) in the presence of DNA, and we detect the elevation of ATPase activity at short intervals. Experiments were also performed to test whether identical mechanisms occur in vivo and in vitro. Our

INTRODUCTION Diapause, once switched on photoperiodically or by a temperature shift, cannot be immediately broken by the return of favourable conditions: a fixed minimum period in the dormant state must first elapse. This feature distinguishes diapause from mere quiescence which can be broken at any time by placing the organism in favourable conditions. In general, exposure to cold is essential for the termination of diapause (Denlinger, 1985). We have been studying the mechanism of diapause termination by cold temperature (Kai and Nishi, 1976; Kai et al., 1995) and our attention has been focused on a time-interval activation of an enzyme, esterase A4 (Ease A4) (Kai et al., 1984, 1987). Ease A4 appears to possess the capacity to measure cold duration. Ease A4 is inactive in unchilled diapausing eggs of the silkworm, Bombyx mori. When the eggs are exposed to

*Insect Biochemistry and Biotechnology, Department of Bioresource Science, Faculty of Agriculture, Tottori University, Koyama, Tottori, 680 Japan. tPresent address, Diagnostics Research Laboratories, Nippon Shoji Kaisha Ltd., Sho 2-24-3, Ibaraki, Osaka, 567 Japan. IPresent address, Sericultural Department, Zhejiang Agricultural University, Hangzhou, P.R. China. $To whom all correspondence should be addressed. 905

HIDENORI

906

principal conclusions follow: (1) the duration of chilling, irrespective of whether the enzyme is in situ or is in solution, is the critical factor in its activation, (2) Ease A4 can measure the duration and (3) the timer function is built into the protein structure. MATERIALS

PuriJication

AND METHODS

of Ease A4

The Ease A4 was purified from C 108 Bombyx silkworm diapause eggs by the method of Kai et al. (1986) with several modifications as follows. Unless otherwise noted, all procedures were carried out in a cold room (4°C) or in an ice-water bath. Eggs laid within 3 h were collected to obtain synchronous egg batches. Two days after oviposition, the eggs (usually 5 g) were immersed in cold acetone overnight. After discarding the acetone, the eggs were crushed completely with another 30-vol. of cold acetone in a glass mortar. The precipitate obtained by centrifugation at 10,000 g for 30 min was suspended in 15 ml of 25 mM Verona1 buffer (pH 7.5; barbital-Na 5.155 g, CHCOONa 3.402 g, 6N HCl c. 2 ml to adjust pH in a final volume of 1000 ml). The remaining powder in the glass mortar was also suspended in a few millilitres of buffer. The combined suspension (approx. 20 ml) was heated at 85°C for 15 min (17 min of heating because 2 min was needed to raise the temperature of the cold sample to 85C). The heat-stable supernatant was obtained by centrifugation. The precipitate thus remaining was re-suspended in another 5 ml of 25 mM Verona1 buffer (pH 7.5) and re-centrifuged. The supernatants were combined (c. 25 ml) and brought to 50% saturation with ammonium sulphate (adjusted exactly to pH 3.30 by HCl). After standing for more than 3 h, the solution was centrifuged at 10,000 g for 30 min, and solid ammonium sulphate was added to the supernatant with gentle stirring to give 80% saturation. The purification was carried to the step of the 80% saturated ammonium sulphate in 1 day and the following step was completed in another day. The precipitate from the 80% saturated ammonium sulphate was further washed with acetone and then 80% saturated ammonium sulphate. The washed precipitate was re-dissolved in 1 ml of the equilibrating buffer (25 mM piperazine-HCl, pH 5.25) and applied to a 1.5 x 30 cm Sephadex G-25 (medium) column, which had been equilibrated in a cold room with the piperazine-HCl buffer. The flow rate of the buffer was adjusted to 0.40.5 ml/min. The Ease A4 was eluted later than the void volume and earlier than the ammonium sulphate (Fig. 1). The Ease A4 fractions were collected and concentrated to less than 10 ml with Centricon-10TM (Amicon). The Centricon-10TM was used at less than 1OOOg (2500 rpm) in all experiments. The concentrated sample was further submitted to high pressure chromatofocusing on a Mono-P column (FPLC) (Pharmacia). The piperazineHC1 buffer (pH 5.25) was

KAI et al

used as an initial buffer and the poly-buffer (Pharmalite 2.555, 1.0 ml; distilled deionized water, about 26.5 ml; 0.1 N HCl, about 2.5 ml to adjust to pH 3.25; final volume 30.0 ml) was used as an elution buffer. The isoelectric point of Ease A4 is pH 3.85; fractions from pH 3.95 to 3.75 (c. 3.5 ml) were used as the enzyme preparation. The final preparation (c. 3 pg) displayed a single band with silver stain on native and denaturing polyacrylamide gel electrophoresis (Kai et al., 1986, 1987) and a single peak on Toyo TSK-Gel3000 SW HPLC column with sodium phosphate buffer, pH 7.5 (Kai, unpublished result). Over a period of a few months, the Ease A4 was crystallized from 80% saturated ammonium sulphate at pH 3.85 and 4°C.

In vitro chilling of Ease A4 and ATPase

assay

3.5 ml (equivalent to about 3 pg protein) of the enzyme solution eluted with the poly-buffer (pH 3.85) was incubated at 5°C in sterilized plastic tubes. At appropriate intervals, 40 ~1 aliquots were assayed for ATPase. All incubation tubes were coated with silicon. The ATPase assay was a modification of Carter and Karl (1982). Solution A was made by mixing 4 vol. of 2 N HCl and 3 vol. of 0.23 M NazMo04. Solution B was 0.042% (w/v) malachite green oxalate in 1% (w/v) polyvinyl alcohol. Solution C was 8.0% (v/v) HzS04. The adenosine 5’-triphosphate Tris(hydroxymethyl)aminomethane vanadium free salt (Sigma) was used as the substrate, and 50 mM solution was made with the reaction buffer pH 7.4 [25 mM HEPES, 12.5 mM Trizma Base, 50 mM NaCl, 20 mM KCl, 1.O mM ethylenediamine tetra acetic acid disodium salt (EDTA.2Na) and 100 pg/ml salmon testes DNA]. The salmon testes DNA was effective for Ease A4 activation. A 20 ~1 sample of the 50 mM substrate solution was added to 340 ~1 of the reaction buffer (pH 7.4), and then 40 ~1 of the enzyme solution was added to this reaction mixture (2.5 mM ATP in final concentration). The reaction mixture was incubated at 25°C for 30 min, and the liberated inorganic phosphate was determined as described below. To the 400 ~1 reaction mixture, 280 ~1 of solution A was added, followed immediately by 120 ~1 of solution B. It was mixed and allowed to stand for exactly 2 min at 25°C for colour development, then 800 ~1 of solution C was added and mixed thoroughly. This mixture was allowed to stand at room temperature for 1 h to complete the colour development, after which the absorbances were read at 625 nm.

Treatment

of Ease A4 with guanidineeHC1

The chilled Ease A4 was further treated by 6 M guanidine-HCl (GuHCl) with modifications from Kai et al. (1987). The denaturation buffer was 25 mM HEPES buffer (pH 7.4) containing 12.5 mM Trizma Base, 50 mM NaCl, 20 mM KCl, 1.0 mM EDTA.2Na, 8M GuHCl and 10 mM DTT. To the Ease A4 preparation,

TIME

INTERVAL

MEASURING

3 vol. of the denaturation buffer was added and incubated at 25°C for 10 min. For renaturation, 10 vol. of cold renaturation buffer, 25 mM ATPase reaction buffer (pH 7.4) with 10 mM DTT, 5 mM ATP and 0.2 mg/ml Z-3-14 (Calbiochem), was added and filtered through a Centricon- 1OTMat 5°C. Further dilutions and filtrations were performed 4 times by the renaturation buffer to reduce GuHCl to less than 6 mM. The remaining Ease A4 was again chilled at 5°C. At appropriate intervals, an aliquot was withdrawn from the chilled enzyme solution and the SH-reagents were reduced to less than 10 PM by repeated dilutions and filtrations at 5°C. The dilution buffer eliminated DNA from the ATPase reaction buffer. Although the SH-reagent is an inhibitor to Ease A4, the Ease A4 is likely to aggregate during chilling without it. The remaining Ease A4 was assayed after the final filtration

ENZYME

907

RESULTS Time-interval

activation

of the DNA-dependent

ATPase

To determine the ATPase activity of Ease A4 at short intervals, the inactive Ease A4 was first purified from eggs 2 days after oviposition and chilled at 5°C. A typical result is illustrated in Fig. 2. While the activity remained low for l&11 days, it suddenly increased reaching maximal activity on day 13 and then again dropped to its basal level. The assays were run at intervals of a few hours, especially during the activity peak. The result establishes the one-time activation of Ease A4. The results demonstrate that one of the physiological substrates is ATP, and that the activity change is such a rapid transition that enzyme activity could be detected only for a brief period.

A

FIGURE 1. Separation of Ease A4 by gel permeation chromatography through a Sephadex G-25 column. Panel (A) shows the elution profile of crude Ease A4 preparation through the Sephadex column. Panel (B) shows the SDS-PAGE separation of each effluent fraction through the column. The numbered lines are keyed to the fractions shown in panel (A). Molecular weight markers are shown in lane M.

HIDENORI

908

KAI et al

r

1

0

a

1

c

a

’

10

5 Days

20

15

after

in vitro

25

chilling

FIGURE 2. Effect of in ritro chilling on the activity of Ease A4 purified from eggs 2 days after oviposition. The purified enzyme solution was incubated at 5°C in sterilized plastic tubes for 24 days. At appropriate intervals, aliquots were assayed for ATPase activity.

Duration ofchilling is the criticalfactor activation of Ease A4

in the time-interval

of Ease A4 increased suddenly and reached a sharp peak in a few days. Such an early activation does not seem to be consistent with the result mentioned above. One explanation for the early activation could be that the activation takes place after about 2 weeks of chilling, such as 10 days in vivo and a few days in vitro (Fig. 3). When the eggs were chilled beginning 2 days after oviposition and Ease A4 was extracted on days 4 and 7 of chilling, enzyme activity increased at 9 and 6 days, respectively,

To investigate the activation mechanism, in vivo and in vitro combination experiments were designed. First, eggs were chilled 2 days after oviposition, and 10 days after the commencement of chilling Ease A4 was extracted. Then, the purified enzyme was chilled at 5°C. In these in vitro chilling experiments, ATPase activity

41

in

vivo

in

-;

vitro

0-O I

5

I

I

I

.

I

I

15

10 Days

after

20

25

chilLing

FIGURE 3. The effect of a combination of in uiuo and in vitro chilling on Ease A4 activity. Diapausing eggs were first transferred to 5°C 2 days after oviposition and kept in the cold for 10 days. The inactive Ease A4 was purified from the lo-day-chilled eggs, and was further chilled at 5°C. The changes in ATPase activities during chilling were determined at the intervals indicated.

TIME

INTERVAL

MEASURING after

Days 0

5

ENZYME GuHCl 10

I s 8 ’ c I r s s c I

15

10 Days

after

in vifro

909 treatment 15

20

8 8 c ’ r s ’ ’ ’

20

25

I

22

’

I

30

chilling

FIGURE 4. Effects of preliminary chilling of Ease A4 on activation by GuHCI. The Ease A4 was first purified from 2-day-old eggs, and then chilled at 5’C for 8 days. The &day-chilled enzyme was treated with 6 M GuHCl and then re-chilled.

during the following in vitro chilling (data not shown). Thus, the duration of chilling, irrespective of whether the enzyme was in situ or was in solution, was the critical factor in its activation. In contrast, the Ease A4 extracted from eggs that had been chilled for 15 days never showed a peak of activity (data not shown). The Ease A4 seems to measure the chilling period, and once activated and subsequently re-inactivated, Ease A4 appeared to be incapable of re-activation by chilling. GuHCl resets the Ease A4so that another 2 weeks is needed for activation Once the enzyme had gone through its cycle of activation, it could no longer be activated by chilling but could only be re-activated by a short treatment with 6 M GuHCl (Kai et al., 1987). Thus, two additional GuHCl treatments were carried out. First, GuHCl was used to treat the Ease A4 that had been re-inactivated by the in vivo and in vitro combination chilling for 25 days. The treated Ease A4 was re-chilled at 5°C and demonstrated changes in activities that were essentially the same as that shown in Fig. 2 (data not shown). No differences were observed from the results of Ease A4 without the combination chilling (Kai ef al., 1987). In the next experiment, the effect of GuHCl re-naturation on the initial activation of the enzyme was compared with its effect on the re-inactivated enzyme. Ease A4 purified from eggs 2 days after oviposition was first chilled for 8 days and then exposed to GuHCl treatment, followed by successive chilling (Fig. 4). Enzyme activity remained low for a longer period. The enzyme was not active on days 12-14, but only became active on about day 19 with the

maximal activity on day 20. As shown by the upper abscissa, the time of activation corresponds to 12 days after GuHCl treatment. The preliminary chilling of the enzyme for 8 days did not help to shorten the period required to activate the enzyme. Apparently, the GuHCl treatment reset the Ease A4 so that another block of nearly 2 weeks was needed for activation. These findings suggest that the duration of the pre-activation period may depend on a structural change in Ease A4 that can be disrupted by GuHCl; the GuHCl apparently erased the structural change in the Ease A4 molecule that had occurred during the S-day-chilling period.

DISCUSSION

The timing of active resumption of Bombyx embryonic development is dependent on the time that elapses after the eggs are exposed to the cold (Kai and Nishi, 1976; Kai et al., 1995). The period of cold indispensable for the resumption of embryonic development coincides with the period required for Ease A4 activation in eggs and with the period required for Ease A4 activation in vitro (Kai et al., 1984, 1987). In this study we addressed the mechanism by which the activation is promoted in Ease A4 using ATP as a substrate. Crystallization, a single HPLC peak and a single band on an electrophoretic gel are not sufficient evidence for purity of Ease A4. Nevertheless, a possible artifact due to in vitro chilling may be ruled out by the present experiments, i.e. the timing of.activation in response to a combination of in viva and in vitro chilling periods and the GuHCl effect on the initial activation. The fact that

910

HIDENORI

GuHCl treatment can lead to repeatable activation of Ease A4 excludes the possibility that protein processing such as a limited hydrolysis is occurring during the chilling period. Possibly a protein conformational change is the built-in time measuring mechanism. That the duration of the chilling, irrespective of whether the enzyme was in situ or was in solution, was the critical factor in its activation indicates that in vitro activation occurs at the same rate as in uivo activation. In addition, no differences were observed between the GuHCl effects in cases with and without combination chilling (a mixture of in uivo and in vitro chilling). It is most likely that Ease A4 possesses some sort of time-measuring activity inherent in the molecule and the molecule may undergo a series of conformational changes with time. The re-naturation of Ease A4 after acid-denaturation (Kai et al., 1988a), rapid activation of Ease A4 in alkaline conditions (Kai et al., 1988b), the GuHCl effect on the Ease A4 (Fig. 4) and the feature that Ease A4 is likely to aggregate (Kai et al., 1987) may suggest that changes from a molten globule state to a partly folded state might be involved in the time interval activation; one specific conformation must be the active form. Organisms have evolved various forms of time-keeping. Many important contributions, genetic, biochemical and molecular biological, have been made to such time-measuring mechanisms (Edmunds, 1988; Takahashi et al., 1993; Kyriacou, 1994; Page, 1994). However, a mechanism for a long-term interval timer type of biological clock is unknown. The interval timer is quite a different type of biological time-keeping mechanism from any of the types of periodic clocks that have been considered so far. Such timers may be involved in the accurate timing of a developmental switch, such as the active resumption of development at the end of diapause after a fixed duration. Ease A4 is the first protein reported to have the possible capability of measuring a time interval in accordance with development. We thus refer to Ease A4 as a time interval measuring enzyme (TIME). The mechanism by which Ease A4 is activated still remains unknown, and the question of how ATPase activation is related to the resumption of embryogenesis is still unresolved. It is also unknown why activation of the enzyme does not take place when the diapause eggs are incubated at 25°C. Cold is essential for both Ease A4 activation and diapause termination. The fact that treatment of crude Ease A4 preparations at 85°C for 15 min (the second step in the Ease A4 purification) does

KAI et al.

not disturb time measurement might be important. The crude preparation may contain thermostable inhibitor(s), and the Ease A4 may remain inactive until this is removed by the cold. The characterization of a time-zero setting mechanism, the mechanism of enzyme activation and the physiological function of the enzyme, are now under investigation.

REFERENCES Carter S. G. and Karl D. W. (1982) Inorganic phosphate assay with malachite green: an improvement and evaluation. J. Biochem. Biophys. Meth. 7, 7-13. Denlinger D. L. (1985) Hormonal control of diapause. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 3533412. Pergamon Press, Oxford. Edmunds L. N. Jr (1988) Cellular and Molecular Bases of Biological Clocks, 1st edn. Springer, New York. Kai H., Kawai T. and Kaneto A. (1984) Esterase A4 elevation mechanism in relation to Bombyx (Lepidoptera: Bombycidae) egg diapause development. Appl. Ent. 2001. 19, 8-14. Kai H., Kawai T. and Kawai Y. (1986) Purification of esterase A4 from Bombyx eggs. J. Seric. Sci. Jpn 55, 77-78. Kai H., Kawai T. and Kawai Y. (1987) A time-interval activation of esterase A4 by cold-relation to the termination of embryonic diapause in the silkworm, Bombyx mori. Insect Biochem. 17,367-372. Kai H., Kawashiri K., Nakashima S. and Azuma M. (1995) Fixed minimum duration of chilling critical to the termination of diapause, and effects of longer chilling on hatching mode in Bombyx eggs. J. Seric. Sci. Jpn 64, 132-141. Kai H., Miao Y., Xu P. X. and Kawai T. (1988a) Effective acid-treatment in vitro to elevate the time measuring esterase A4. J. Seric. Sci. Jpn 57, 313-317. Kai H., Miwa T., Doi S. and Kawai T. (1988b) pH-effect on esterase A4 activation by chilling in vitro. J. Seric. Sci. Jpn 57, 531-532. Kai H. and Nishi K. (1976) Diapause development in Bombyx eggs in relation to esterase A activity. J. Insect Physiol. 22, 1315-1320. Kyriacou C. P. (1994) Clock research perring along-its about time. Trends Genet. 10, 69971. Page T. L. (1994) Time is the essence: molecular analysis of the biological clock. Science 263, 157&1572. Takahashi J. S., Kornhauser J. M., Koumenis C. and Eskin A. (1993) Molecular approaches to understanding circadian oscillations. A. Rev. Physiol. 55, 729-753.

Acknowledgements-The authors wish to thank Professor D. L. Denlinger, Department of Entomology, Ohio State University, U.S.A. for valuable suggestions and reading the manuscript. The authors also thank Mr K. Higashi, Faculty of Agriculture, Tottori University, Japan for his help on GuHCl treatments and Professor Dr I. Morishima of Tottori University, Japan, for his encouragement.