Insect Biochem. Vol. 14, No. 5, pp. 601-607, 1984 0020-1790/84 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1984 Pergamon Press Ltd

ESTEROLYTIC DEGRADATION OF JUVENILE HORMONE IN THE HAEMOLYMPH OF THE ADULT FEMALE OF

LEUCOPHAEA MADERAE

SONNY GUNAWAN and FRANZ ENGELMANN* Department of Biology, University of California, Los Angeles, CA 90024, U.S.A.

(Received 25 July 1983; revised 15 November 1983)

Abstract--Two JH carboxylesterases were isolated from haemolymph of vitellogenic females by anion exchange chromatography. The high degree of purification achieved by this one step procedure is inferred by the absence of any protein stainable band (Coomassie blue or silver stain) on native polyacrylamide gels, yet enzyme activity was detectable in eluates of these gels. Purified JH esterases were stable for more than eight months when stored at 5°C. Freezing at -25°C destroyed their activity. Both enzymes lost their activity upon heating to 65°C. The average apparent molecular weights of these esterases were 52,000 and 42,000. The corresponding average /(ms of the purified enzymes were approx. 0.68 x 10 -6 and 1.54 × 10 6 M. In the haemolymph of newly emerged, allatectomized, and pregnant females only one esterase (mol. wt 52,000) was identifiable. The second esterase was, however, inducible in allactectomized females by the JH analogue methoprene (ZR-515). In all cases, purification by ion exchange chro- matography resulted in an approx. 1000-fold gain in JH esterase activity. The removal of several esterase inhibitors accounts for this gain. Neither of the two idehtifiable JH esterases degraded ct-naphthyl acetate (~-NA). A population of esterases which effectively degraded ct-NA but did not hydrolyze JH-III eluted from the QAE column before the JH esterases.

Key Word Index: Leucophaea maderae, haemolymph, juvenile hormone, JH esterases, enzyme kinetics

INTRODUCTION

During recent years the research on the mode of synthesis and degradation of juvenile hormone (JH) in species of several orders has been intensified but details are known for only a few cases. F rom these efforts the conclusion emerged that titres of circu- lating JH are determined by independently changing rates of synthesis and degradation. After the first reports on the identification of JH degrading carb- oxylesterases (White, 1972; Whitmore et al., 1972), and particularly following the discovery of a specific JH esterase in last instar larvae of Manduca sexta (Sanburg et al., 1975), many attempts have been made to identify both general and specific esterases (cf. de Kort and Granger, 1981; Jones et al., 1982; Sparks et al., 1983). A combinat ion of standard protein isolation procedures has been used to purify the juvenile hormone esterases of two Lepidoptera (Coudron et al., 1981; Yuhas et al., 1983). Classes of esterases were usually described on the basis of the sensitivity to specific enzyme inhibitors and substrate utilization.

The role of JH binding compounds (carriers) in the haemolymph has been elucidated in a few species (cf. de Kor t and Granger, 1981). These JH binding compounds appear to interfere with the degradative action of certain circulating esterases. For example, the JH binders literally "protec t" the hormone from rapid esterolytic degradation in 4th instar larvae of M. sexta (Sanburg et al., 1975). Little, however, is known on the actual molecular mode of interaction of these binding compounds with the esterases.

*Correspondence to Dr Franz Engelmann at the above address.

In the course of the research on JH transport and the mode of JH action in adult females of Leucophaea maderae, knowledge on the carboxylesterases of the haemolymph and in tissues as well as their character- ization and control became essential. In the present report we will provide evidence for the existence of two specific JH carboxylesterases in the haemolymph and describe their purification. One of these esterases is under JH control and this, as will be shown, contributed to a mildly fluctuating titre of esterase activity in the haemolymph during a reproductive cycle of the female cockroach.

MATERIALS AND METHODS

Animals and haemolymph collection

For all determinations and experiments adult females of L. maderae in their first reproductive cycle were used. The animals were kept at 26°C and 70-80~ relative humidity. They were fed rat chow (Purina) and water. Haemolymph was obtained by cutting a hole into the neck membrane posterior to a ligation and centrifuging the animals head down at low speed (approx. 200 g). About 200-300 p l of serum can be collected from one animal after the blood clot has been removed.

Haemolymph fractionation

The anion exchange resin QAE (Pharmacia) was used for fractionation of the haemolymph and purification of the esterases. The resin was equilibrated in 0.08 M citrate- phosphate buffer at pH 6.8 containing 0.2 M NaCI. The initial NaC1 concentration is essential since a fraction of vitellogenin and some other haemolymph proteins precip- itate in low molarity salt solutions and the subsequent redissolution during the rising salt concentration no longer yields the "normal" protein profile. Fresh haemolymph (300 ~1) was layered on a column of 30 x 1.5 cm i.d. For elution the NaC1 molarity was linearly increased to 1.0 M

601

602 SONNY GUNAWAN and FRANZ ENGELMANN

over 400 ml of elution volume. Ten-millilitre fractions were collected at room temperature (21 °C), usually overnight. No difference in the protein elution profiles was seen when the fractionation was performed at 4°C.

Enzyme assays

For JH and general esterase activity diluted haemolymph (1:4) samples or aliquots of fractions in 0.08 M citrate- phosphate buffer pH 6.8 were assayed. For JH specific esterase activity the following procedure was adopted. The appropriate quantity of E, E [10-3H]JH-III (usually 28,600 dpm = 1.17 × 10 -8 M) in hexane was transferred to the raction tube and the solvent evaporated to dryness under a gentle stream of nitrogen. The JH was then dissolved in the desired volume of 0.08 M citrate-phosphate buffer at pH 6.8. To this JH solution the enzyme containing samples were added (final volume 100#1), cautiously swirled, and allowed to incubate at 30°C in a Forma Scientific shaker for the time periods indicated in text. The reaction was stopped by addition of 2 ml of ethyl acetate and the aqueous water phase re-extracted twice with 2ml of ethyl acetate. No radioactivity was found in the aqueous phase. The com- bined organic phases were then dried over sodium sulphate for 30 min. After evaporation, the JH-III and JH-III-acid were dissolved in 50 #t ethyl acetate-hexane (2:8, v/v) and spotted on silica gel plates containing fluorescent indicator (C. Merck, No. 5539). The tubes were rinsed three times with 30 #1 of the same solvent mixture and these washes added successively to the dried spots on the silica gel plates. The plates were then run by using chloroform-ethyl acetate (2:1, v/v) as solvent system. The zones corresponding to JH-III and JH-III-acid identified by co-chromatography of unlabelled JH-III and JH-lII-acid were cut in strips, eluted in ethyl acetate and assayed for radioactivity in a Beckman LS 7000 liquid scintillation counter. JH-acid standards were stable when refrigerated for three days. The scintillation fluid contained PPO/POPOP in toluene-methanol (3:1, v/v). The counting efficiency was approx. 35~o and recovery of radioactivity as JH-III and JH-III-acid was between 70 and 80~. Only about 3~o of diol formation occurred during the incubation with haemolymph. No more than 6~ of the radioactivity was detected on the remainder of the TLC plates. These proportions of label outside the JH and JH-acid zones were about the same in all samples.

General esterase activity was measured using ~-naphthyl acetate (~-NA) as substrate. The reaction mixture consisted of 300#1 of enzyme solution, 3ml of :t-NA at a concen- tration of 5.4 × 10-4M in 0.08 M citrate-phosphate buffer pH 6.8, and 150#1 of 0.5~ Fast Red TR salt dissolved in the same buffer (Sanburg et al., 1975). Fast Red TR salt solution was freshly made for each assay. :t-NA stock was made in acetone and diluted with buffer immediately before use (Katzenellenbogen and Kafatos, 1970). This mixture was incubated at 30°C for up to 90 min. The optical density of the colour developed by the diazonium salt-naphthol complex was read at 625 nm with a Bausch and Lomb spectrophotometer. For quantitation ~-naphthol was used as standard.

Molecular weight determinations Natural 7.5~o polyacrylamide gels (Davis, 1964) were used

for the estimation of molecular weights. Catalase (232,000), lactate dehydrogenase (140,000), ovalbumin (43,000), and carbonic anhydrase (30,000) were used as standards. Con- centrated JH esterases from the pools identified by QAE anion exchange chromatography were electrophoresed par- allel to the standards. Following electrophoresis the gels containing the standards were stained by Coomassie blue, and the gels with the enzymes were immediately frozen on dry ice, sliced into 2mm sections, eluted with 0.08 M citrate-phosphate buffer at pH 6.8, and assayed for JH esterase activity. The position of the enzyme activity on the gels was plotted semilogarithmically on a curve obtained

from Coomassie blue stained standards. In this fashion the apparent mol. wt could be estimated with reasonable accu- racy. A representative gel was also treated for silver staining (Bio-Rad).

Chemicals Juvenile hormone III (E,E[10-3H]JH-III; 11 Ci/mmol) was

obtained from New England Nuclear Corporation and unlabelled JH-III from Calbiochem. The JH analogue meth- oprene (ZR-515) was a gift of Dr Schooley, Zoecon Cor- poration. ~-Naphthyl acetate, Fast Red TR salt, p- hydroxymercuribenzoate (p-HMB) were purchased from Sigma. ~-Naphthol was purchased from Matheson- Coleman & Bell. QAE Sephadex A-50 and protein stan- dards (catalase, lactate dehydrogenase, ovalbumin and car- bonic anhydrase) were from Pharmacia. JH-III-acid was prepared by base catalyzed hydrolysis of JH-III.

RESULTS

JH degradation m the haemolymph

Incubation of J H - I | I with diluted haemolymph (0.08 M citrate-phosphate buffer pH 6.8) from both vitellogenic and non-vitellogenic females resulted in a cumulative production of JH- l l l -ac id over time (Fig. 1). The degree of JH degradation depended on the amount and source of haemolymph used. The ester- ase inhibitor p-hydroxymercuribenzoate (p -HMB) effectively inhibited the esterolytic activity of the haemolymph. Inhibition was practically complete at a concentration of 10 4 M (Fig. 1). No other enzyme inhibitors were studied.

Isolation and purification

The degree of esterolytic JH degradation in natural haemolymph may be affected by several factors (among them enzyme inhibitors), and consequently, enzyme purification and isolation becomes manda- tory for the characterization of the enzymatic reac- tions. For L. maderae column chromatography using the anion exchange resin QAE (Pharmacia) in 0.08 M citrate-phosphate buffer at pH 6.8 allowed separation and purification of the JH esterases in one step. All JH esterolytic activity in the haemolymph from vitel- logenic females eluted at a salt concentration higher than 0 .5M (Fig. 2), after all detectable proteins (measured by absorbancy at 280 nm) had eluted. Two populations of JH esterases were obtained from vitellogenic females. Both of these esterase popu- lations exhibited only JH esterase and no ~ -NA esterase activity. The enzymes of both populations had a broad pH opt imum from about 6.5-9.0 (Fig. 3) and both could be inactivated by heating to 6Y~C for 10 min. Remarkably, those isolated enzymes were stable for more than eight months when refrigerated at 5°C. However, upon freezing at - 2 5 ° C much of the esterolytic activity of the purified enzymes was lost within a few days. In contrast, repeated freezing and thawing of complete haemolymph had affected the JH esterase activity only after several months (Table 1).

General esterase activity (~-NA substrate) eluted from the Q A E column at a salt concentration of about 0.4 M and was always associated with a protein peak (Fig. 2); this peak consisted of several protein species as shown by polyacrylamide gel electro- phoresis. The same fractions from haemolymph of

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Fig. 1. Time dependent hydrolysis of JH-11I by the esterases of unfractionated haemolymph (1:4 diluted) from vitellogenic females in the absence (O Q) and presence (O--O) ofp-hydroxymercuribenzoate (p-HMB). The haemolymph was preincubated with p-HMB at 30°C for 60 rain. 28,600 dpm of [3H]JH-III

was incubated in 100 #1 incubation volume.

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Fig. 2. Anion exchange chromatography (QAE) of JH esterases from haemolymph of vitellogenic and non-vitellogenic females. Pregnant females were three to four weeks after ovulation. 25/~1 of each 10 ml fraction was diluted to 100/al and incubated with 28,600dpm of [3H]JH-III. For comparisons the JH esterase activity was recalculated and expressed per /11 of haemolymph. ( ... . . ) Representation of the

protein profiles at 280 nm absorbancy. Vg = vitellogenin.

604 SONNY GUNAWAN and FRANZ ENGELMANN

E ~8o ~3

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o--0 JHE- I I

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Fig. 3. The effect of pH on JHE-! and JHE-II activity. About 28,600 dpm of [3H]JH-III were incubated with 100 #1 (1:3 diluted) QAE purified pooled esterases from vitel- logenic females for 60 min. The following buffers were used: pH 5-6.6 citrate-phosphate 0.08 M, pH 7.2-8.2 Tr is-HCl 0.l M, pH 9.2-10.4 carbonate-bicarbonate 0.1 M. The val- ues have been corrected for base hydrolysis in the higher pH

range.

vitellogenic or pregnant females contained ~-NA esterase but no JH esterase activity. We did not assay for ~-NA esterase activity in newly emerged or allatectomized females. Molecular weight estimations of the JH esterases were made by electrophoresis on native polyacrylamide gels. Samples of QAE purified enzyme pools were electrophoresed parallel with molecular weight standards. Accordingly, for the enzymes eluting first from the QAE column we tentatively ascribed an apparent tool. wt of 51,000 and for the second enzyme one of 43,750 (Fig. 4). The position of the enzymes on the gels could only be determined by their esterolytic activity. No Co- omassie blue stainable bands were observed at the location of enzyme activity or anywhere on the gels even after concentration of the enzyme solution by 80-fold. Also, silver staining (Bio-Rad) did not yield any visible band. These observations indicate a very high degree of purification of these enzymes following a one step fractionation on the QAE anion ex- changer. We cannot provide actual data on specific activity of the enzymes or the degree of purification, because we are unable to measure protein amounts associated with the enzymes.

Table l. The effect of storage on haemolymph juvenile hormone esterase activity

Complete haemolymph 1:4 diluted '~, JH-lll-acid formed

Fresh 75.8 Frozen-thawed 4 times, 1 month later 60.3 Frozen-thawed 5 times, 2 months later 52.1 Froze~thawed 6 times, 3 months later 39.0

Purified JH esterases JHE I JHE 11

Fresh 69.0 74.7 Frozen, 2 weeks later 17.2 14.9 Frozen, 4 months later 5.4 2.9 Refrigerated, 1 month later 67.8 61.6 Refrigerated, 3 months later 64.1 58.9 Refrigerated, 8 months later 63.6 64.4

Approximately 28,600 dpm of 13HIJH-III were incubated with either 100#1 of diluted haemolymph or an aliquot (100#1) from pooled purified enzymes. The JH concentration was 1.17 × 10 8 M. The same enzyme batches were used for each category. Incubation was at 30C for 60rain.

8 0

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Fig. 4. Electrophoresis of QAE purified JHE-I and -II from vitellogenic females on 7.5% native polyacrylamide gels. Both enzymes were electrophoresed simultaneously with the s tandards in order to allow the estimation of the mol. wt. Two m m sections of the gels were eluted and assayed for

esterase activity.

JH esterase kinetics

Enzyme characteristics for the two JH esterase populations were determined for the JH-III substrate (Fig. 5; Table 3). Km values for a sample of JH esterases from vitellogenic females calculated from Lineweaver-Burk plot were 0.67 x 10 6 and 1.57 x 10 _6 M for enzymes I and II respectively. V,n,x of these particular enzymes was 5.21 and 8.44 pmol min -~ #1 -~ haemolymph (Fig. 5). Minor variations were observed between enzymes of different hae- molymph pools, but Km and Vm,x for each of the two enzymes always were in a similar range regardless of the age or physiological status of the animals from which these enzymes were obtained (Table 3).

Quantitation and the characteristics of the JH ester- ases in haemolymph o f different developmental stages

Diluted samples of haemolymph from newly emerged, vitellogenic, ovulating and pregnant fe- males were tested for the ability to degrade JH-I |I .

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3.45 x 10 6M. Incubation time was 60rain.

H a e m o l y m p h J H es terases 605

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Fig. 6. A n i o n e x c h a n g e c h r o m a t o g r a p h y ( Q A E ) o f the es terases f r o m h a e m o l y m p h o f a l l a t ec tomized ( C A - ) a n d C A - m e t h o p r e n e t r e a t ed females . 400 # g o f the J H a n a l o g u e h a d been top ica l ly app l i ed ten d a y s p r i o r to t e r m i n a t i o n . H i g h vi te l logenin t i t re a n d egg g r o w t h to h a l f m a t u r e size i nd i ca t ed the

effectiveness of methoprene. Assay conditions were identical to those in Fig. 2.

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The same was done with samples from alla- tectomized and methoprene (ZR-515) treated fe- males. For comparisons of the enzyme activities between various samples, substrate concentrations and enzyme quantities were empirically chosen to yield linear degradations over a given time period. As is seen in Table 2 (values represent data from pools of haemolymph from three to four animals each), the level of esterolytic activity fluctuated somewhat with the physiological status of these females but always was at a substantial level.

Aliquots of these same haemolymph pools were fractionated on QAE resins and the esterase profiles determined (Figs 2 and 6). Two important aspects emerged from these determinations. First, only vitel- logenic females either during mid-vitellogenesis or at ovulation (shown in Tables 2 and 3) had two esterase populations, thus suggesting that JH may control the synthesis of the second esterase species. Second, if one computes the total JH-esterase activity contained in two purified enzyme populations (300#1 hae-

molymph distributed over 80-180 ml of elution vol- ume) and compares this to that of one pl of natural haemolymph an approx. 1000-fold gain in total en- zyme activity is calculated (Table 2). Enzyme in- hibitors (unpublished data) contained in the complete haemolymph apparently have been removed in this purification step. With the exception of the newly emerged females, esterase I is found at similar levels regardless of the reproductive status of the females. The modestly fluctuating total esterase activities are thus mostly due to the presence or absence of esterase II (Figs 2 and 6).

The correlation of the appearance of a second JH esterase population with vitellogenesis (high JH activ- ity) suggests endocrine control. This was tested by treating allatectomized females with 400 p g of the JH analogue methoprene. Methoprene was used because of its stability. Ten days later when the terminal oocytes of these animals were about half grown (2.5-3.0 mm), haemolymph was taken and fraction- ated on QAE resins. Esterase II could be identified

Table 2. Juvenile hormone esterase activity

JH-IIl-acid formed (pmolmin ~ #1 ~ haemolymph)

Purified esterases Natural - - -

haemolymph JHE total JHE I JHE II

Newly emerged females 2.15 x 10 -3 0.969 0.969 - - Vitellogenic females 0.74 x 10 -3 0.875 0.395 0.480 Ovulating females 1.78 x 10 _3 1.097 0.433 0.664 Pregnant females* 0.48 × 10 _3 0.696 0.696 - - Allatectomized females 0.41 × 10 _3 0.395 0.395 - - Allatectomized females* 0.65 × 10 -3 1.030 0.546 0.484

methoprene treated

Figures are averages from two determinations except* indicating only one determination. For the natural haemolymph the figures are averages from pools of three to four animals each. Haemolymph (300 pl) containing a known amount of JHE activity per pl haemolymph had been applied to the QAE column. All fractions (10 ml) were assayed for JHE activity (Fig. 2). Those fractions with JHE activity were pooled and the activity quantitated by adjusting substrate and enzyme quantities to yield a linear reaction time. For comparison of JHE activity of the purified enzymes with that of natural haemolymph the activity was calculated per equivalent t~l of haemolymph.

606 SONNY GUNAWAN a n d FRANZ ENGELMANN

Table 3. Characterist ics o f the purified JH esterases

J H E I J H E 1I

K~ (M) V~., Moi. wt K~ (M) V~ , (pmol min 1 #1-i ) (pmol min i u l - i )

Mol. wt

Newly emerged females 0.70 × 10 -6 4.46 53,000* - - - - Vitellogenic females 0.67 x 10 6 4.58 51,000" 1.56 × 10 6 8.40 43,750 Ovulat ing females 0.69 × l0 6 4.29 52.000 1.51 × 10 6 8.79 40.750 Pregnant females 0.67 × 10 -6* 4.13" t - - Allatectomized females 0.66 × 10 -6 3.28 52,500* - - - - Allatectomized females 0.68 × 10 -6* 4.84* 52,000* 1.54 × 10 6, 8.13" 42,250*

methoprene treated

Figures are averages f rom two determinat ions except *indicat ing only one determination. Haemolymph pools were f rom three to four animals, t N o t determined.

(Fig. 6). We can thus conclude that the second esterase was indeed induced by the circulating JH analogue and this correlated well with findings from normal vitellogenic females.

For all purified JH esterases the Km and Vmax values were established by Lineweaver-Burk plots. JH ester- ase I values were comparable irrespective of the reproductive status of these animals (Table 3). For JH esterase n the values were different from that of JHE I, but similar in all cases where it occurred. JHE II is thus distinguished by its K m and Vm,~ value. K m values for JHEs from complete haemolymph were higher (lower affinity for the substrate) possibly be- cause several enzyme inhibitors and JH binders con- tained in the haemolymph interfere with the enzyme kinetics. Since the concentration of these enzyme inhibitors fluctuate and thus affect the determinations of the Kin, the values obtained with unfractionated haemolymph can vary considerably and are not representative for the correct enzyme kinetics.

Differences between the two JH esterases are seen not only in their kinetics but also in the respective molecular weights. The average mol. wt of JHE I is about 10,000 higher than that of JHE II (Table 3).

D I S C U S S I O N

Esterolytic degradation of JH in the haemolymph of larvae and pupae of several species, and in a few cases also of adults, has been studied (cf. de Kort and Granger, 1981). Attempts to isolate pure enzymes were often laborious and associated with loss of activity due to the instability of the esterases. In spite of these difficulties an apparently pure JH esterase has been obtained from last instar larvae of M . sex ta (Coudron et al., 1981) and from Trichoplusia ni (Rudnicka and Hammock, 1981; Yuhas et al., 1983). Substantial losses of activity have been reported in both cases during the several isolation steps. From this point of view it is remarkable that the L. maderae esterases could be purified in one step by QAE anion exchange chromatography. No loss of activity was seen and instead an approx. 1000-fold gain in activity was obtained when recalculated per /d of hae- molymph assayed (Table 2). Several esterase in- hibitors (i.e. JH binders) have been removed by the anion exchange chromatography (data to be pub- lished). The failure to identify stainable (Coomassie blue or silver) protein bands on native poly- acrylamide gels, even after 80-fold concentration of the solution, leads us to believe that our enzyme preparations were highly purified relative to the

starting materials. Since we were unable to measure protein, we cannot report purification in terms of specific activity. In contrast to our findings, the reported purfications of JH esterases in other species was associated with losses of enzyme activity (in one case more than 90~) but stainable protein bands on PAGE were identified. Both enzymes isolated from vitellogenic females had JH esterolytic activity only and no ~-NA esterase activity was detectable. They were present in similar quantities, and were inhibited by p-HMB (an esterase inhibitor). The ~-NA ester- ases of vitellogenic and non-vitellogenic females do not degrade JH-III. In other words, for the adult cockroach L. maderae the term JH esterase is based solely on substrate specificity, and not on inhibitor characteristics as reported for M . sex ta (Sanburg et al., 1975; Coudron et al., 1981) or for several other species of insects (cf. de Kort and Granger, 1981). A complete separation of the two classes of esterases is achieved by the one anion exchange chromatography step.

The esterases of L. maderae are remarkably stable after purification and we could not detect any loss of activity when kept refrigerated at 5°C for more than eight months. This is in contrast to most published reports. Our two purified JH esterases have pH optima, molecular weights, and substrate affinities similar to those reported for other species (cf. de Kort and Granger, 1981). The two haemolymph esterases of L. maderae can be distinguished from each other by differences in the Kin, Vm~x and apparent molecular weights.

Fluctuating titres of haemolymph esterase activity may be the result of two independently varying parameters: esterase production and the occurrance of enzyme inhibitors (which may be identical with JH binders), or both. Enzyme production, at least as determined by esterase levels in intact haemolymph, appears to fluctuate with developmental progression in all species examined (cf. de Kort and Granger, 1981). However, in any one of the reported cases no distinction can be made as to which of the two possible causes may determine these fluctuations. Interestingly, in the adult cockroach Diploptera punc - tata esterolytic degradation of JH-III in the hae- molymph during periods of vitellogenesis (high JH titres) is lower than at other times, yet it is also reported that an exogenously applied JH analogue raised the esterase levels (Rotin et al., 1982). Since unfractionated haemolymph had been used in these assays it is unclear whether the changing esterase activities reflected changes in enzyme levels per se or

Haemolymph JH esterases 607

changes in titres of "JH protecting" agents. If we permit ourselves to compare D. punctata with data obtained from L. maderae the latter possibility be- comes a likely component. To be sure, with regard to JH degradation in vivo, the effective esterase activity of the whole haemolymph is the biologically mean- ingful information. However, for an analysis of the esterase effectiveness in any animal at various devel- opmental stages and for research on the potential controls, both esterase production and the presence of inhibitors need to be studied simultaneously. For these types of studies pure enzymes are required.

The first suggestive evidence for JH induction of haemolymph esterases was given for M. sexta larvae (Whitmore et al., 1972) and the idea was followed up by others using primarily additional Lepidopteran species (cf. de Kort and Granger, 1981; Hammock et al., 1981; Sparks et al., 1983). For these reported species no conclusive evidence for induction of addi- tional esterases could be given, since whole hae- molymph, had been used for the assays. JH binders or inhibitors of the haemolymph may inversely fluctuate with the JHE activity and may be under control of JH. A corresponding apparent rise or fall in the activity can thus be recorded when whole haemolymph is assayed. By a simple isolation pro- cedure we now can show for L. maderae that a new esterase is induced by JH (Figs 2 and 6). A remote alternative possibility exists, namely, that JH causes the cleavage of a 10,000 dalton unit from the JHE I, thus converting it into JHE II. With this change in molecular size the enzyme kinetics, i.e. Km and Vm,x, has changed as well.

The observations contained in this report describe several novel aspects of esterolytic degradation of JH in an adult insect, but the question has to be asked: do they have any functional significance for the intact animal? A major port ion of the JH esterases are present at high levels at all times of the adult life of L. maderae. On the other hand, in several immature species correlations between JH titres, esterase levels, and developmental stages suggest that JH induced esterases function to remove the remaining circu- lation JH and thus allow metamorphosis to occur (Hammock et al., 1981). Certainly, if we look at the JH esterolytic activity in the intact haemolymph of L. maderae, as well as the levels of the purified esterases, we see that JH only modestly raises the effective enzyme activity. It is peculiar that we observe the highest levels of JH esterase activity when circulating JH concentrations are presumably highest. Clearly, in L. maderae these esterases do not adequately remove JH from the circulation, evidenced by the fact that JH effectively induces vitellogenin production and vitel- logenesis proceeds (Engelmann, 1969). In L. maderae the JH esterases apparently do not influence JH levels in the circulation, because practically all JH appears to be bound to JH esterase inhibitors of high affinity (to be published). The observed events signal the necessity for a more complex interpretation than hitherto available. This new picture has to incorpo- rate information on the circulating esterase in- hibitors, such as JH binding compounds. In the haemolymph of L. maderae several such JH binders

with high affinity for JH (Ko of 10 S M) can be identified (unpublished). An analysis of the role of these compounds which bind JH and their effect on the esterolytic activity in the haemolymph will be given in a subsequent publication. From the biologi- cal point of view, JH esterases and inhibitors cannot be separated, both are essential for the functional interpretation.

Acknowledgements--The research reported here was sup- ported by a grant from the US National Institute of Health (HD-15530). We would like to thank Dr E. Mundall and Dr G. della-Cioppa for critically reading this paper and making valuable suggestions for improvement.

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