Further characterization of the juvenile hormone binding protein from the cytosol of a Drosophila cell line

Insect Btochem Vol 15, No 2, pp 197 204, 1985 Printed m Great Britain All rights reserved

0020-1790/85 $3 00+0 00 Copyright © 1985 Pergamon Press Ltd

F U R T H E R CHARACTERIZATION OF THE JUVENILE HORMONE BINDING PROTEIN FROM THE CYTOSOL OF A DROSOPHILA CELL LINE USE OF A PHOTOAFFINITY

LABEL

ERNEST S. CHANG,* MARILYN J. BRUCE* and GLENN D. PRESTWICHt *Bodega Marine Laboratory, Umverslty of Cahfornm, P O Box 247, Bodega Bay, CA 94923 and tDepartment of Chemistry, State Umverslty of New York, Stony Brook, NY 11794, U S A

(Recetved 25 April 1984) Abstract--Further characterization of the juvenile hormone (JH) blndmg protein from the cytosol of Drosophda melanogaster Kc cells has been accomphshed with the use of a photoaffimty analogue of JH The analogue, 10,I l-epoxy(2E,6E)farnesyl dlazoacetate (EFDA), is trmated m the 10-position Following photolysls wlth short-wave ultraviolet hght, it can be demonstrated that [JH]EFDA binds speofically to the cytosohc JH binding protein This bmdmg is inhibited if irradiation occurs m the presence of either unlabelled JH I or JH III Both JH homologues protect the binding site equally against [JH]EFDA No protection ~s observed with either methoprene or farnesyl acetate, a close structural analogue of EFDA that lacks the dlazo photoactlvatable group The cytosohc JH binding protein, following covalent labelhng with trltlated EFDA, was characterized by gel filtration column chromatography, velocity sedimentation through sucrose gradients, both native and denaturing gels, and binding to DNA cellulose The binding protein has a molecular weight of approx 49,200 and may consist of two subumts

Key Word Index Juvende hormone, photoaffimty label, hormone binding protein, hormone receptor, cell hne, Drosophda INTRODUCTION Juvenile hormones are sesqulterpene denvatwes that regulate a number of developmental and reproductive functions in insects (Granger and Bollenbacher, 1981). Investigations into the mode of action of juvenile hormone (JH) at the cellular level is hampered m whole tissues by the relatwely high concentrations of endogenous hormone present and by mixed populations of cell types. The Drosophda melanogaster Kc cell hne IS a useful model system in which to investigate the action of insect hormones ( O ' C o n n o r and Chang, 1981) Although Drosophda cells have been particularly useful m examining the mode of action of ecdysteroid hormones, there is increasing evidence that JH also has a number of physiological effects upon the cells. These effects include the specific inhibition of the ecdysterold-mduced alterations in cell morphology and biochemistry ( O ' C o n n o r and Chang, 1981, Chang et al., 1982; L Cherbas, personal communication) The molecular mode of action of this hormonal antagonism and the molecular achon of JH at the cellular level are largely known There are, however, several reports indicating that J H action may be mediated by binding to a cytoplasmic hormone binding protein (Chang et al., 1980, Engelmann, 1980, Klages et al., 1980; Koeppe et al, 1981; Roberts and Wyatt, 1983). These studies demonstrate the presence of saturable macromolecules that specifically bind radiolabelled juvenile hormones with relatively high affinity. However, only preliminary physical data on the hormone binding proteins were obtained, due in part to the rapid 197

dlssooatlon and metabohsm of JH from the binding proteins In the systems employed The recent development of 10,11-epoxyfarnesyl dlazoacetate (EFDA), a photoaffinity analogue of J H III (Prestwlch et al., 1982, 1984), permits the formation of a stable covalent bond between the J H binding protein and a radlolabelled hgand This analogue thus permits a more extensive characterization of the binding protein than can be obtained with noncovalently bound llgands. In this report, we describe the use of the radiolabelled photoaffimty analogue, [JH]EFDA, in the further characterization of the intracellular JH binding protein in the cytoplasm of Drosophtla melanogaster Kc cells. MATERIALS AND METHODS

Cells and btochemwals The 7-D-11 clone of the Kc hne of Drosophda melanogaster was maintained as previously described (Chang et al., 1980) Radlolabelled and unlabelled 10,11-epoxyfarnesyl dlazoacetate (EFDA) and farnesyl acetate (a competltwe inhibitor of esterase actlwty, Fxg 1) were synthesized as prewously described (Prestwlch et al, 1982, 1984) Racemlc juvende hormone I and III (JH I methyl cts(2E,6E)10,11-epoxy-7ethyl-3,11-dlmethyl-2,6-trldecadlenoate, JH III" methyl (2E, 6E)- 10, 11-epoxy-3, 7, 11-tnmethyl-2, 6-dodecadlenoate) were from Calblochem Methoprene 0sopropyl(2E,4E)llmethoxy-3,7,11-tnmethyl-2,4-dodecadlenoate) was also used as a racemlc preparation and was generously provided by Dr G B Staal (Zoecon Corp.) Hormones and hormone analogues were purified by high-performance hquld chromatography when necessary (Chang, 1983) Phenylmethylsulphonyl fluoride (PMSF) and bactenal (Streptomyces grlseus)

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Fig 1 Chemical structures of (A) naturally occurring juverole hormone III (JH III), (B) [3H]I0,11-epoxyfarnesyl dmzoacetate ([3H]EFDA), (C) farnesyl acetate and (D) methoprene

protease were from Sigma Electrophoresls chemicals were from BIo-Rad Photolysis Cells were harvested (density approx 3 x 106cells/ml) by centrffugatlon (800g, 8 m m , 4°C) The cells were resuspended in TM buffer ( 1 0 m M Tns/5 m M MgCI 2, pH 6 9, at 4°C) and then repelleted They were disrupted with 20 strokes of a Dounce homogenizer (A pestle) The homogenate was then centrifuged (122,000g, 1 5 hr, 4°C) to obtain the supernatant fractmn designated as cell cytosol For photolysts time course studies, cytosol (350pl) as incubated for 90 m m at 25°C with [3H]EFDA (5 4 Cl/mmol) at 1 x 10 6M, with and without a 3 0 m m pre-mcubatlon with 5 x 10 5M JH III The soluUons were placed m an ice water bath and ahquots transferred to quartz tubes (8 x 100 mm) and photolyzed for various times from 0 25 to 1 2 m m Control cytosol was left unphotolyzed Photolysls was carried out m a Rayonet Photochemical Reactor (Southern New England Ultravmlet) equipped with four 253 7 n m lamps. Ahquots (100/11) were then transferred to test tubes, precipitated with 3 vol of ethanol for 15 mln, and cenmfuged at 800g for 4 m m at 4°C The resulting pellet was mixed with 0 4 m l of 75% ethanol, refrigerated overmght, and cenmfuged the following day This washed precipitate was dissolved in water (50/tl) and scintillation fluor (Beckm a n Ready-Solv EP) and analyzed by scintillation spectrometry (Beckman LS 7800) Results were corrected for non-covalently bound [3H]EFDA by subtracting radloactwlty b o u n d at zero photolysts time All tubes were mltmlly coated with 1% polyethylene glycol 20,000 (Sigma) Photolyzed (radmlabelled) cytosol was occasionally stored for several days at - 7 5 " C prior to use Characterzzatton o f bm&ng protein For c o m p e t m o n studies of [3H]EFDA with unlabelled JH I and III, 2 x 10 7 M of the radlolabel was incubated with cytosol (100/d) and unlabelled JH in various concentrations from l x l 0 ~ M t o e l t h e r 4 x l 0 5Mor3xl0 4M f o r J H I

and JH lII, respectively The tubes were incubated for 90mln, then photolyzed (8 mm), and assayed by ethanol precipitation as described above Parallel tubes that were not photolyzed were also assayed to determine background radloactwlty This activity was subtracted from the values obtained for the photolyzed tubes Concentrations of methoprene from I x 10 6 to 1 x 10-4M were similarly tested for protection of the JH b m d m g site from reaction with [3H]EFDA upon photolysls For polyacrylamlde gel electrophoresls (PAGE) studies of the JH b m d m g protein, cell cytosol was mcubated with [3H]EFDA ( 2 x 10 7M) for 9 0 m m with and without a 30ram pre-mcubatlon with 50 times excess JH II1 The cytosol was then chilled m an ice water bath and photolyzed for 8 r a m U n b o u n d hgand was removed by addition of 1 5 vol of 1 25% dextran-coated charcoal (DCC), allowed to adsorb for 15mln at 25°C, and centrifuged at 4500g for 10min at 4°C The supernatant was stored at - 7 5 ° C for later analysis on P A G E The photolyzed cytosol was separated on both denaturing and natwe gels Slab gels (0 75 m m ) were prepared with a 12% separating and a 5% stacking gel Denaturing gels had 0 1% (w/v) sodium dodecyl sulphate (SDS) added to the gel and the samples were prepared m sample buffer according to the method of L a e m m h (1970) Proteins were stacked at 15 m A and the amperage was then increased to 20 m A when the dye front reached the separating gel Following electrophoresls at 4°C, gels were stained for l hr m 50% (w/v) mchloroacetlc acid and 0 1% Coomassle blue and destamed overnight with 7 5% acetic acid on a reciprocating rocker platform Gels to be analyzed by scintillation spectrometry were shced into 2 m m sectmns and the radloactlwty eluted for at least 6 hr m 0 4 ml of 1% SDS at 37°C In a shaker bath prior to addition of scmtlllatmn fluor Initial experiments utilized 0 1 m M phenylmethylsulphonyl fluoride (PMSF) in the homogemzatlon buffer (TM buffer, 10 m M Tns-HCI/5 m M MgCI 2, pH 6 9) to prevent proteolysls Comparisons of gels with and without the inhibitor indicated that its presence was not necessary Proteolytlc digests of the photolyzed cytosol were conducted using the method of Cleveland et al (1977) Ten percent of the sample volume of a 0 0 1 % solution of bacterial protease was added to the cytosol sample The protease was added to the cytosol sample just prior to the m m a t m n of electrophoresls When the dye front had almost entered the separating gel, the current was turned off for 40 m m Then the current was turned back on and the gel electrophoresed as described above For compeUtlon studies by farnesyl acetate, 1 x 10-TM [3H]EFDA was incubated for 90 mln with cytosol at 250C with and without a 30 mln pre-lncubatmn with 10, 100 and 1000 times excess farnesyl acetate followed by 8 m m of photolysls in an ice water bath Non-covalently bound and u n b o u n d hgand were removed either by treatment with 75% ethanol as described above or by D C C adsorption (50% volume of 1 25% DCC) Gel filtration column chromatography was performed with Sephadex G-100 (Pharmacla, 2 4 x 1 6cm, l d , bed) usmg T M K buffer (10 m M T n s - H C I / 5 m M MgCI2/150 m M KCI, pH 7 4) as the eluant Effluent was monitored at 2 8 0 n m by means of a flow through absorbance m o m t o r (Pharmacm) Fractions were subsequently analyzed by means of scintillation spectrometry Cytosol incubated w~th or without a pre-mcubatton with unlabelled .IH III was chromatographed DNA-cellulose (Sigma) column chromatography was performed with a 9 5 x 1 6 cm 0 d ) column and eluted with a step gradient of 0.0, 0 1, 0 3, 0 5 and I 0 M KCI m TE buffer (10 m M Tns-HC1/1 m M dlsodlum EDTA, pH 7 4) Effluent was momtored at 280 n m Unlabelled cytosol as fractionated by the KC1 step-gradient elutlon and then incubated with [3H]EFDA In these fractions, the a m o u n t of specific b m d m g was measured by means of pre-lncubatmn with or

Cellular JH binding protein w~thout unlabelled JH III, photolysls and ethanol precipitation as described above The amount of total protein m these fractions was determined by the method of Bradford (1976) Velocity sedimentation centrffugat~on was conducted through preformed sucrose gradients (10-30%) in TMK buffer by centrffugation at 190,000 g for 22 hr at 4°C in a SW 50 1 rotor (Beckman) Fractions were collected and analyzed by scintillation spectrometry Protein standards were from Bio-Rad (electrophores~s) and Pharmacia (column chromatography and centrffugation)

O f the several methods that were used to separate covalently bound [3H]EFDA from non-covalently bound llgand, we observed that precipitation of the protein-hgand complex with 75% ethanol resulted in the most consistently reproducible results. This method was found to be superior to either precipitation with polyethylene glycol (PEG) or a m m o n i u m sulphate, or adsorption with dextrin-coated charcoal F~gure 2 shows a typical experiment. Although there 2O

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Fig 3 Protection of the JH binding site from covalent attachment to [3H]EFDA by unlabelled JH I (circles) and JH III (triangles) Cytosol was incubated with the radiolabel (2 × 10 7M) and the unlabelled hormones for 90 min, photolyzed for 8 min, and assayed by ethanol precipitation Percentage bound radioactwlty is relative to the amount of [3H]EFDA bound m the absence of any compeUtors

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identical, J H I and J H III were measured for their relatwe abilities to compete with [3H]EFDA (Fig. 3). It can be seen that the two JH homologues equally inhibit binding of [3H]EFDA with the binding protern. Slmdar results have been observed ff the radioactive hgand is either [SH]JH I or [3H]JH III.

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Drosophtla Kc cell cytosol Following a pre-mcubatlon of cytosol with (triangles) or without (circles) 5 x 10 5M JH III, cytosol was photolyzed for the indicated lengths of time Radlolabelled EFDA (l x 10 -6 M) was pre-incubated with the cytosol for 90 mm prior to ultraviolet irradiation at 254 nm. Means ( 4- SD) for triphcates of a typical experiment are shown is a relatwely large amount of covalent non-specific binding, there is still a slgmficant amount of protection from covalent binding of the radlolabel by preincubation lth unlabelled J H III. In order to demonstrate that the lower amount of binding of [3H]EFDA m the presence of unlabelled J H is not simply due to absorption by JH of the ultrawolet hght (which would then not be available to react with the photoactlvatable dlazo group of E F D A ) , experiments were conducted with the JH analogue methoprene (Fig 1) The conjugated ester chromophore of methoprene also absorbs ultrawolet light strongly m the 220-260 nm region, where both JH and E F D A absorb. At concentrations as high as 1 × 10 -4 M, methoprene was never observed to protect the J H binding s~te from covalent attachment to E F D A (no decrease m binding of [SH]EFDA). To obtain information that the E F D A and JH binding sites on the J H binding protein were indeed

Since there ~s sigmficant esterase and other enzyme acUvlty present in Kc cell cytosol ( O ' C o n n o r and Chang, 1981), it was necessary to demonstrate that the [3H]EFDA was not simply a substrate binding to an esterase. A close structural analogue of E F D A lackmg the t0,11-epoxlde and the photoactivatable dlazoacetate, farnesyl acetate (Fig 1), was therefore tested for competlhon with E F D A . At farnesyl acetate concentrations of 10, 100 and 1000 times the amount of [3H]EFDA present m the cytosol incubations, no protectmn of the active s~te from covalent binding was ever observed Farnesyl acetate has been observed, however, to inhibit cytosohc esterase actlwty by about 40% when present at a concentratxon of 10 -3 M (unpubhshed observations). Several experiments were then conducted to determine the molecular weight of the J H binding protem Following photolysls, radlolabelled cytosol was chromatographed on a Sephadex G-100 column. A major peak of radioactivity eluted in the fraction that had an eluUon behavlour similar to that of an ovalbumln standard (Fig. 4). By means of a standard curve of Kay (Vo-Vo divided by I/",-II0) versus log mol. wt, a molecular weight of 49,200 + 1600 ( + SD) was obtained from three separate analyses. This peak of radmactw~ty was not present ff the cytosol was mmally incubated with excess unlabelled J H III prior to incubation with [3H]EFDA and ultraviolet lrradmtlon. When cytosol that had been photolyzed following lncubauon with [3H]EFDA was analyzed on sucrose gradients, again a single peak of radioactivity assocrated with a macromolecule was observed (Fig. 5). This peak was greatly diminished ff the cytosol was prewously incubated with excess unlabelled J H III. By means of a standard curve of relatwe mlgratmn into the gradient versus log mol. wt a molecular weight of 51,000±5800 ( ± S D ) was obtained for three separate analyses.

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Fig 4 Sephadex G-100 column (24 × 1 6 cm) chromatography of 1 0 ml ofDrosophda Kc cell cytosol that had been incubated with [3H]EFDA prior to photolysls Eluant was TMK buffer (10 mM Trls-HC1/5 mM MgCI2/150mM KCI, pH 7 4) at a flow rate of 0 42ml mln ' Fractions (2 1 ml) were analyzed for radioactivity (circles) and absorbance at 280 nm (solid line) at 0 5 AUFS External standards (Blo-Rad) were gamma globulin (GG), ovalbumln (OA), myoglobln (MG) and vitamin B-12 (B-12) BSA

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Fig 6. Dastnbutmn of radioactivity m 2 mm shces of an electrophoresed polyacrylamade gel (12% separating, 5% stacking) with a sample of Kc cytosol that was photolyzed with [3H]EFDA (1 x l0 6 M) that had been incubated either with (triangles) or without (circles) excess unlabelled JH III (5 × 10 5 M) Fractmn 1 represents the top of the gel

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Flg 5 Sucrose gra&ents (10~30% m TMK buffer) of 0 2 ml of photolyzed (1 x 10 6M [3H]EFDA) Kc cell cytosol Gra&ents (4 5 ml) were preformed and centrifuged for 22 hr (190,000g, SW 50 1 rotor) Cytosol was previously incubated with (triangles) or without (circles) a 50-fold excess of unlabelled JH III External standards (Pharmacm) were bovine serum albumin (BSA), ovalbumln (OA), chymotrypsmogen A (CT) and nbonuclease A (RNa) To further characterize the J H binding protein, photolyzed cytosol was analyzed by m e a n s of polyacrylamlde gel electrophoresis (PAGE). Figure 6 shows the radioactivity distributed in a n o n - d e n a t u r i n g gel following electrophoresls of photolyzed cytosol. It shows t h a t there IS essentially only one peak of radioactivity, again suggesting that a single protein species is being covalently b o u n d . This peak o f radioactivity is not present if the cytosol is initially

incubated with unlabelled J H IlI Analysis with S D S - P A G E u n d e r d e n a t u r i n g condltions enabled a more precise molecular weight d e t e r m i n a t i o n of the binding protein As seen in Fig 7, there is essentially only one m a j o r b a n d o f ra&oactIvlty. However, the a p p a r e n t molecular wexght of the protein with the radlolabel was 24,600 + 1100 ( + SD) for six determinations. This is a b o u t half of the molecular weight o b t a i n e d from the n o n - d e n a t u r i n g determinations These data suggest t h a t the J H bindmg protein m a y consist o f two subunIts, each with a molecular weight o f a p p r o x 24,600. Proteolytlc digests of the photoaffinity labelled cytosol were also electrophoresed on S D S - P A G E In all cases, only a single radioactive peak was observed (data not shown) The resulting peak was never present if the photolyzed cytosol h a d previously been incubated with excess unlabelled J H III F u r t h e r evidence t h a t the macromolecule to which E F D A was covalently b o u n d h a d properties characterlsUc o f a receptor for J H was o b t a i n e d from D N A cellulose column c h r o m a t o g r a p h y Unlabelled,

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Fig 7 Distribution of radloactwlty m 2 mm slices of a SDS-polyacrylamide gel that had been prepared and treated as described m Fig 6 Cytosol was pre-mcubated with (triangles) or without (circles) excess unlabelled JH III Molecular weight ( x 10 3) markers (B~o-Rad) are shown below the gel (stained with Coomassie blue)

201

Cellular JH binding protein unphotolyzed cytosol was apphed to the column and eluted with several volumes of buffer. Increasing concentrations of KC1 in buffer were then applied to the column. After lncubataon of these KC1 fracttons wlth [3H]EFDA and photolysas, an aliquot was removed and assayed for total protein and also analyzed for bound [3H]EFDA by ethanol precipatataon. In a representatwe experament (Table 1), the protean Table 1 DNA-cellulosecolumnchromatographyof Kc cellcytosol NoKC1 01 MKCI 03MKC1 [3H]EFDAbound (ng/mgprotein) 11 89 00 that was not retained by the column (eluted in buffer wlthout KC1) was able to band 1.1 ng of [3H]EFDA per mg of protein. The protean that was eluted with 0.1 M KC1, however, was able to brad 8.9 ng of label per mg of protein. This m&cates that there was a significant enrtchment of the specific binding acttvity following affinity chromatography with DNA-cellulose DISCUSSION The data presented m th~s report tn&cate that [3H]10,11-epoxyfarnesyl daazoacetate (Krafft et al., 1982; Prestwich et al., 1984) is a useful analogue of JH for binding protean studies These observations are m addation to the reports of Prestwich et al (1982) and Koeppe et al. (1984), working with the cockroach, Leucophaea maderae, on the utflaty of [3H]EFDA in characterlzmg mtracellular JH binding protems. The protectaon experaments detailed by Prestwich et al. (1985) were not possible an the Drosophtla Kc cell system. These experiments consast of first premcubatmg the binding protein wath excess unlabelled JH. Unlabelled EFDA is then added followed by photolysls. If the JH and EFDA banding sites are adentlcal, then the JH banding site should remain unoccupied by the covalently attached analogue (EFDA). Separation of the non-covalently bound hgand from the protean is then necessary prior to the demonstration of blndang by radlolabelled JH In all cases, however, we were unable to efficiently remove the non-covalently bound hgand from the bmdmg protein without losing most of the binding actawty. Dassociatlon and dialysis with 0.5 M phosphate or 0.75 M KC1 were not successful Precipitation of the protein with 75% ethanol, 22% PEG, 50% ammonium sulphate or 20% trlchloroacetac acad all resulted m a sagmficant loss of binding actiwty when the protean was resolubtlized. In additton, neither concentrated DCC nor hydroxylapatate were effective tn the separation of non-covalently bound hormone or analogue from the bmdmg protein. For these reasons, we concentrated on the use of [3H]EFDA. Throughout the course of the experaments, unlabelled excess JH III was always used to compete [3H]EFDA m a parallel series of mcubatlons. The results were always consistent wath the conclusion that the EFDA and the JH III bmdmg sates were an fact the same site. Use of [3H]EFDA suggests that the juvende hormone banding protein from the cytosol of Drosophila Kc cells may be composed of two subunlts, each of

203

which has an approximate molecular weight of 24,000. There appears to be only a smgle protein that specifically binds the labelled hgand. Additional suggestions supported by these studtes are that there ts only a single hormone binding site per subunit as lndtcated from the PAGE analyses of the protease dtgests of the cytosol. Also, the bmdmg protem appears to be relatwely stable since ~t was not cleaved m the absence of a protease inhibitor (phenylmethylsulphonyl fluoride) or by the process of freezing and thawing. The molecular weight data on the JH bmdmg protein presented in th~s report are somewhat different than those obtained previously by this laboratory (Chang et al., 1980) We believe that the current data are more accurate since the previous determinations depended upon non-covalent bmdlng of [3H]JH III with the possibility of subsequent exchange of the label to other proteins. In ad&tton, the covalent nature of the binding m the present experaments permitted molecular weaght determanations by SDS-PAGE, a much more accurate method (Weber and Osborn, 1969; Laemmla, 1970) that was impossible wathout the use of the photoaffintty label. The results of the DNA-cellulose column chromatography are addlt~onal evidence that the macromolecule that is covalently bound by [3H]EFDA as mdeed a receptor for JH. It has been shown that most receptors of non-peptade hormones do bmd to DNAcellulose and that th~s type of affimty chromatography may be useful in subsequent purification of the receptor (Schrader, 1975). It thus appears that [3H]EFDA lS a useful tool to examme the mode of actton of juvenile hormone at the cellular and molecular levels. The covalent nature of the banding will permit investigation into the ultimate role of JH In medmting dafferentml gene expressaon m the nucleus Prehmmary evtdence does an fact indacate that there are nuclear receptors for JH (Rid&ford and Mitsu~ 1978; Goodman and Chang, 1985; Chang, 1985). We are presently contmuing our anvestlgataon of nuclear JH receptors with the photoaffintty label. Acknowledgements--We thank Dr D W Borst and Dr J K

Koeppe for helpful comments We are grateful to the National Institutes of Health (GM-30899), the Alfred P Sloan Foundation and the Camille and Henry Dreyfus Foundation for awards to G D P m partial support of th~s work REFERENCES

Bradford M M. (1976) A rapid and sensltwe method for the quantification of m~crogramquantities of protein utlhzmg the principle of protein-dye binding Analyt Blochem 72, 248 254 Chang E S (1983) Analysis of insect hormones by means of a ra&al compression separation system J Ltqutd Chromat 6, 291-299 Chang E S. (1985) Cellular juvende hormone binding proterns In Methods m Enzymology (E&ted by Law J. H and Rllllng H C), Vol III Academic Press, New York In press. Chang E S, Coudron T A, Bruce M J, Sage B A, O'Connor J D and Law J H (1980) Juvende hormonebinding protein from the cytosol of Drosophda Kc cells. Proc natn Acad Sct U S A 77, 4657~4661

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