J Comp Physiol B (1984) 154:213-223 Journal of ~Systemic, .... Comparative and Environ- Physiology B ~,-~, Springer-Verlag 1 9814

Juvenile hormone esterases of Lepidoptera II. Isoelectric points and binding affinities of hemolymph juvenile hormone esterase and binding protein activities

Keith D. Wing ~, Maria Rudnicka 2, Grace Jones 3, Davy Jones 3, and Bruce D. Hammock Departments of Entomology and Environmental Toxicology, University of California, Davis, California 95616, USA

Accepted May 30, 1983

Summary. The juvenile hormone esterase (JHE) tal biology of the Lepidoptera are biochemically and juvenile hormone binding protein (JHBP) ac- similar to a variety of other species in this order. tivities from the last larval instar of 14 species of Lepidoptera (Pieris rapae, Colias eurytheme, Dan- aus plexippus, Junonia coenia, Hemileuca nevaden- sis, Pectinophora gossypiella, Spodoptera exigua, Introduction Trichoplusia hi, Heliothis virescens, Orygia vetusta, Ephestia elutella, Galleria mellonella, Manduca Levels of circulating juvenile hormones are regu- sexta and Estigmene acrea) were analyzed by ana- lated, in part, by their interaction with juvenile lytical isoetectric focusing (IEF). While the multi- hormone esterases (JHEs; E.C. 3.1.1; Hammock plicity and isoelectric point of these proteins var- and Quistad 1981) and specific juvenile hormone ied, all of them were mildly acidic (pI 4.0-7.0), and binding proteins (JHBP's; de Kort and Granger a large number of the species possessed only a sin- 1981; Kramer and Law 1981). The titer of JHE gle JHE and/or JHBP activity. The Michaelis con- activity has been measured in several species dur- stants (Km'S) of the whole hemolymph JHE activi- ing development, and in many of the species exam- ties from selected species for JH III were in the ined there are two peaks of hemolymph JHE activi- range of 10 -v M. The equilibrium dissociation ty, before and after wandering stage (Vince and constant K d of the JHBP was determined by Scat- Gilbert 1977; Reddy etal. 1979; Sparks etal. chard analysis for selected species as well, with the 1979; Wing et al. 1981; Jones et al. 1982). In the majority of species.having a Ke near 10 -7 M. This cabbage looper, Trichoplusia ni (Hfibner), the es- information is consistent with JHE acting as a terase activity has been hypothesized to be due to scavenger for JH at various times during develop- a single protein which is present during different ment and relying entirely on mass action to remove days of the last larval stadium or which can be JH from its protective JHBP complexes. The JHBP artificially induced, and which exists in the hemo- should limit nonspecific binding and thus facilitate lymph and fat body (Sparks and Hammock 1979a; the rapid transport of the intact hormone through- Wing et al. 1981). However, there is some contro- out the hemocoel. These data indicate that the spe- versy regarding the number of JHE's involved in cies currently used in the study of the developmen- other species (Sandburg et at. 1975; Kramer and Childs 1977; Rudnicka et al. 1979; Coudron et al. Abbreviations. JH juvenile hormone; JHE juvenile hormone es- terase; JHBP juvenile hormone binding protein ; IEF isoelectric 1981 ; Roe et al. 1983; Sparks et al. 1983). focusing; EPPATO-ethyl-S-phenyl phosphoramidothiolate; The specific JHBP activity in M. sexta has also DFP O,O-diisopropyl phosphofluoridatc been shown to be due to a single protein (Kramer Current addresses: 1 Research Laboratories, Rohm and Haas et al. t976), which is present in the hemolymph Comp., Spring Housc, PA 19477 of fourth (penultimate) stadium larvae at different 2 Institute of Organic and Physical Chemistry, Technical Uni- stages (Goodman and Gilbert 1978). High specific- versity of Wroclaw, Wybreze, Wyspianskie 27, 50-370 Wro- claw, Poland ity carriers for JH occur widely among the Lepi- 3 Departmem of Entomology, University of Kentucky, Lex- doptera (Ferkovich et al. 1975 ; Kramer et at. 1976; ington, KY 40546 Hammock et al. 1975, 1977; Kramer and Childs 214 K.D. Wing et al. : pl's and binding affinities of .I H esterases and binding proteins

1977). In particular, the T. ni JHBP activity seems activities are highly reproducible among subsequent genera- to be due to a single protein in the hemolymph tions. However, the activity gradually fluctuates over a period and fat body of both penultimate and ultimate lar- of months (maximum prewandering JHE activity in nmole/min- ml: February, 13: May, 64; August, 28). val stadia. In both M. sexta and T. ni, the JHBP is thought to have primarily a distributive role Assays. JHE activity was assayed by the partition method of (Gilbert et al. 1978; Sparks et al. 1979; Wing et al. Hammock and Sparks (1977) using geometrically pure JHI 1981) while JHE is able t0rid the animal of hor- or tlI. For routine analysis a final substrate concentration of mone at appropriate developmental times either 5 x 10 .6 M JH Ill was used and incubated for 15 rain at 30 ~ C unless otherwise specified. Esterase assays with JH I as sub- in the presence or absence of the JHBP. strate were run using the same procedure, except that carbo- Despite the fact that both JHE and JHBP activ- waxed (polyethylene glycol MW 20,000, Calbiochem Aquaci- ities have been demonstrated in hemolymph from de 11) tubes were used to prevent adhesion of JH I to glass various Lepidoptera, few rigorous studies have (Hammock et al. 1975; Kramer et al. 1976). K,,'s (apparent Michaelis-Menten constant) and V~,ax's been done to determine the multiplicity of these (maximal enzyme velocity) for JHE activity in whole hemo- proteins, except in the case of a few select species. lymph from 7". ni, S. exigua, tI. virescens and G. mellonella were More information on the nature and number of determined by Lineweaver-Burk double reciprocal analysis these regulatory macromolecules would help deter- (Lineweaver and Burk 1934). Jtl concentrations varied from mine to what extent findings in these species could 1.6x10 -~ to I xl0-SM, and each assay contained about 30,000 DPM of 3H-JH I or lit. Michaelis plots were used to be extrapolated to the order Lepidoptera. Towards insure that the concentrations employed were appropriate for this end, analytical stab gel isoelectric focusing has Lineweaver-Burk analysis. been used to resolve JHE and JHBP activities from Binding constants (Ka) and concentrations of binding sites a variety of lepidopteran hemolymph. In addition, of whole hemolymph from selected species were determined by the method of Scatchard (1949). JH I concentrations were relatively little is known regarding the interaction varied from 8.0x 10 -s M to 2.4x 10 .6 M, and binding was of these two proteins and their effect on free JH measured by a modification of a charcoaPdextran assay de- concentrations. While it has been assumed that the scribed previously (Korenman 1969; Hammock etal. 1975; esterase can metabolize the hormone even in the Kramer et al. 1976; Goodman et al. 1976; Goodman and Gil~ presence of the high affinity binding protein, equi- bert 1978; Wing et al. 1981). The hemolymph of T. ni and H. virescens (pool of 15 animals each, diluted 20 x for assay), librium measurements using well-timed animals in C. eurytheme and M. sexta (pool of 15 and 5, respectively, di- the same laboratory have not been previously pub- luted 10 x), and E. acrea and G. mellonella (pool of 15 and lished. Thus, estimates of binding affinities for 20 animals, respectively, diluted 50 x ) were taken from last sta- both the esterase and binding protein were made dium prewandering larvae having a weight concomitant with maximal hemolymph JHE activity. and their implications on regulation of circulating The dissociation rate constant k s for the JH-JHBP complex hormone titers will be discussed. was measured using a modification of the charcoal-dextran as- say as described by Chang et al. (1980), Early day two fifth instar 7". ni hemolymph was collected and diluted 20 x with Materials and methods buffer, and 4 ml of the diluted hemolymph was then added to a carbowaxed 8 ml vial to which 40 lal of 1 x 10-~ M, O- Experimental animals and hemol),mph preparation. Trichoplusia ethyI-S-phenyl phosphoramidothiolale (EPPAT) had pre- ni (Hfibner) and ICefiothis vireseens (Fab.) were reared accord- viously been added and evaporated under N 2 to dryness, giving ing to Shorey and Hale (1965). Manduca sexta (L.), Spodoptera a 1 x 10 -3 M solution. This mixture was allowed to incubate exigua (Hiibner), Galleria mellonella (L,), Junonia coenia 10 rain at 30 ~ C, and then 40 I*l of I x 10 -7 M 3H-JH I in etha- (Hiibner), Danaus plexippus (L.), Orygia vetusta (Boisduval), nol was added slowly. After very gentle vortexing the vial was Pieris rapae (L.). Ephestia elutella (Hiibner), Pectinophora gos- held for 1 h at 0.5 ~ From this stock of bound, radiolabeled sypiella (Saunders) and Hemileuca nevadensis (Stretch) were JH, 50 gl aliquots were placed into carbowaxed 6 x 50 mm cul- raised as described previously (Jones et al. 1982). The Colias ture tubes in which 2 x l0 -s M JH had been previously dis- eurytheme (Boisduval) used were last stadium larvae collected solved by sonication in 50 pl of buffer. These tubes containing from alfalfa fields in Yolo County, California during July and 1 x 10 -5 M cold JH 1 were vortexed immediately and then held August 1981. for specified times at a known temperature before addition of Hemolymph was collected by piercing a proleg and drawing 100 gl charcoal-dextran to stop the reaction. The tubes were the blood into glass capillary tubes. The hemolymph was di- again held for 5 rain at 0.5 ~ before centrifugation (3,200 g, luted 2 x with buffer (sodium phosphate pH 7.4, I =0.2 M, con- 4 ~ and 100 pl of supernatant were removed for liquid scintil- taining 0.01% phenylthiourea to inhibit tyrosinases) or double lation counting. distilled water containing 0.01% phenylthiourea (for isoelectric To measure the association rate constant k~, 100 lal of 20 x focusing), and frozen until further use. Hemolymph was collect- diluted early fifth instar (day-two) hemolymph was pipetted ed from animals having weights and physiological markers indi- into 6x50mm carbowaxed tubes, the JHE inhibited with cative of the peak of prewandering hemolymph JHE activity EPPAT and 1 lal of t x 10 -7 M ~H JH I in ethanol was added during development (Sparks et al. 1979; Jones etal. 1981, to initiate binding. After selected time intervals at 0.5 ~ bound 1982), as it has been shown in T. ni that the JHBP titer under- hormone was separated from free by the addition of charcoal- goes less fluctuation during the last larval stadium than does dextran, vortexed, centrifuged and the supernatant analyzed JHE (Wing et al. 1981 ). This procedure made removal of endog- by liquid scintillation counting. enous JH unnecessary. In our culture of T. ni the peak JHE Alternatively, a modification of an assay using hydroxyla- K.D. Wing et al.: pl's and binding affinities ofJH esterases and binding t)rotcins 215 patite as the separatory reagent was utilized to develop a Scat- surcd by eluting gel slices in 0.4 ml double distilled water over- chard plot (Goodman et al. 1978). One hundred microliters of night; the pH of the eluate was measured using a Corning early (day 2) fifth instar hemolymph diluted 80 x in Tris buffer 125 pH meter with a Corning Triple Purpose Ag/AgCt Refer- (0.01 M Tris, 0.01 M KCI, pH 7.5) were delivered into ence Electrode at 10 ~ All hemolymph samples were assayed 6 x 50 mm carbowaxed culture tubes, to which was added 1 ~tl for JItE and JHBP at least once on wide range and twice on of I x 10 -l M EPPAT in ethanol. After being incubated at narrow range gels, except where noted. 30 ~ for 10 rain, 3H-JH I solutions ranging from 1.0 x 10 -3 M to 1.0 x 10 -6 M .wcrcladded in 1 gl of ethanol, and incubated Partition co~ffficients. Partition coefficients were determined by for 1 h at either 0.5 or 30 ~ At this time, 300 p.l of a slurry vigorously vortexing radiolabeled JH I or JH Ill (1 • 10 -5 M) of hydroxylapatite (Biorad HT; 20 ml settled wet bed per 30 ml with equal volumes of water saturated n-octanol and n-octanol total volume) in Tris buffer with 1% Triton X-100 (Rohm and saturated water (Fujita et al. 1964). Following centrifugation Haas; to eliminate nonspecific binding ofJH I to hydroxylapa- samples of the respective phases were analysed by liquid scintil- rite) was pipetted into each tube and then vortexed, After hold- lation counting. The integrity of the JH in each phase was ing the tubes for 5 min, they were centrifuged at 515 g for 5 min determined by thin layer chromatography on silica gel plates at 4 ~ and the supernatants removed by vacuum. Another developed in hexane: ether 5 : 1. 300 lal of the Triton X buffer was then added, and the tube was again vortexed and centrifuged. This washing procedure was repeated three times, and in each case the supernatant was aspirated off. The entire tube was then placed into a plastic Results scintillation vial (Wheaton Omni-Vial), and 3 m! of scintillation cocktail (Amersham ACS) were added to this vial. The pellet Isoelectric points of the hemolymph JHE activities was distributed evenly throughout the tube by vigorous vortex- are shown in Table 1. The pIs of the lepidopteran ing, and the sample analyzed by liquid scintillation counting. JHE's in this study were mildly acidic, ranging Independent experiments showed that 300 p,l of the above hyd- from pH 3.6 to 7.8. Six of the thirteen species had roxylapatite slurry provided at 1.5 x excess over that necessary only one major JHE activity in their prewandering to pellet >95% of the protein-bound radioactivity when 80 x diluted hemolymph was allowed the equilibrate with hemolymph (T. hi, S. exigua, M. sexta, G. mellon- 1.0 x 10 -s M labeled JH I. It was also shown that under these ella, E. elutel[a, C. eurytherne). Another four of the conditions three washes with 300 gl of the Triton X buffer were species had two separable JHE activities (H. vires- adequate to reduce radioactivity in the supernatant to 5% of cens, E. acrea, P. gossypiella, J. coenia), two the total counts. Protein concentrations were determined using a commercial (0. vetusta and H. nevadensis) had three activities solution of Coomassie Brilliant Blue G-250 (Biorad Laborato- and in one case (P. rapae) there were four detect- ries) and bovine serum albumin fraction V (Sigma) as a stan- able JHE activities. To determine whether the mul- dard, as described previously (Bradford 1976; Spector 1978). tiplicity of JHE's in H. virescens could have been due to intraspecific variation, the hemolymph of lsoelectricfocusing (IEF). Narrow range (pH 4.0-6.0) IEF gels were cast according to established procedures (Winter et al. individual larvae weighing 64:2 + 50 rng (n = 5) were ! 977) to a 2 mm thickness using commercially available ampho- analyzed for JHE as usual on narrow range IEF. lines and electrofocusing-grade acrylamide and bis-acrylamide In four out of five cases, results were similar to (LKB). Wide range (pH 3.5-9.5) PAG plate IEF gels were pur- findings with pooled hemolymph in that a signifi- chased pre-cast from LKB (1 mm thickness). For analysis of JHE activity 15 111 of hemolymph diluted 2 x in buffer were cant minor JHE peak accounting for 11.1+ 1.9% applied to the gel after determination of native JHE activity. (n=4) of the total activity recovered on the gel It was found later thatdiluting hemotymph samples in double was present, which focused at pH 4.8, while a ma- distilled water with 0.01% phenylthiourea produced better pH jor peak was found in all individuals at pH 5.9. gradient uniformity due to decreased salt concentration while In agreement with results reported earlier, a single leading to no loss of JHE activity. For JHBP experiments a 50 gl aliquot of the hemolymph diluted 2 x was placed in a JHE activity was found in the prewandering hemo- 6 x 50 mm culture tube. The sample was preincubated with EP- lymph of both T. ni (pi=5.5; Sparks and Ham- PAT (10-4 M, added in 1 p.l ethanol, 10 rain, 30 ~ to inhibit mock 1979a; Wing et al. 1981) and M. sexta (pI= JHE activity and then transferred to a Sigmacote| treated 5.6; Coudron et al. 1981). There seemed to be no 6 x 50 mm culture tube to which had been added 132,000 dpm of 3H-JH I delivered in 2 gl ethanol which was evaporated to relationship between either pI or the number of near dryness under N z immediately before hemolymph addi- separable JHE activities on IEF, and a species' tion. The JH-JHBP complex was allowed to form for 15 rain prewandering JHE titer or number of hemolymph at 30 ~ and then 30 rain at 4 ~ Immediately before the gets JHE peaks the animal experienced during the last were run 15 gl of this solution was applied to the gel surface in sample wicks. All JHBP samples for IEF were found to larval stadium. The single JHE activities of the have < 5% their original JHE activity after EPPAT incubation. two pyralids tested, G. mellonella and E. elutella, Gels were run over 8 cm (Winter et al. I977) and analyzed focused at more acidic pH's than in most other for JH Ill esterase and JH [ BP activity, as described previously species, but possessed widely diverging JHE titers. (Sparks and Hammock 1979a; Wing et al. 1981). For both The majority of the species which had high JHE JHE and JHBP analysis, replicate sample wicks were placed in different positions on the gel to prevent artifacts due to titers had only a single JHE, but H. virescens and the wicks from being interpreted as biological activity. The gels O. vetusta comprise exceptions. O. vestusta in par- were sliced into 0.5 cm sections, and pH gradients were mea- ticular demonstrated remarkably high prewander- 216 K.D. Wing e~ aL: pl's and binding a~lqnities of JH esterascs and bin~iing proteins

Table I. Isoelectric points and percent of total enzymatic activity of J H esterases from various species of Lepidoptera

Species pl ~ Percent of Original hemolymph activity activity Common name Scientific name (family) on the get b (nmoles/min.ml HL) c

Cabbage looper Trichoplusia ni 5.5 97 • 2.9 29-104 (Noctuidae) Beet armyworm Spodoptera exigua 5.6 • 0.1 91 • 4,0 38-163 (Noctuidae) Tobacco budworm Heliothis virescens 4.8 28 • 1.4 t 34-170 (Noctuidae) 5.9 75 • 4,9 Tobacco hornworm Manduca sexta 5.6 91 +__4.0 38--163 (Sphingidae) Greater waxmoth Gatleria mellonelta 4.5 89 • 4.9 t 9-147 (Pyralidae) Tobacco moth Ephestia elutella 5.0 • 95 • 5 4.3-10.1 (Pyralidae) Western tussock moth Orygia vetusta 5,3 14.5• 27-34 (Liparidae) 5,55 • 0.2 64 • 9.9 5.9 • 21.5• Salt marsh caterpillar Estigrneneacrea 5.3 75 4.2 (Arcliidae) 5.6 25 Pink bollworm Pectinophora gossypiella 3,6 60 2.4 (Gelechiidae) 5.9 40 Nevada buck-moth Hemileucanevadensis 6.2 69 8.9 (Saturniidae) 6.9 10 7.9 21 Buckeye Junonia coenia 5.4 84 • 17 1~ - 7,2 (Nymphalidae) 5.63 _+ 0.l 16 • 17 Cabbage white Pieris rapae 4.1 20 3.8 (Pieridae) 5.8 • 63 6.2 • 3.1 7.7 i9 Alfalfa bmterfly Col• eurytheme 4.8 80 19,3 (Pieridae)

Data is mean • where three or more determinations on narrow range tEF were made b Indicates activity in that peak divided by total CPM in the gel track, times 100. Data are means+_SD Hemotymph JHE activity befbre it was applied to the gel. Data are ranges of original hemolymph JHE activity, from minimum to maximum

ing JHE titers (350 nmoles JH III cleaved/min-ml; fast stadium, and with Wing et al. (1981) who dem- Jones et al. 1982) and yet had 3 separable activities onstrated that a single JHBP activity of the same on IEF, although the hemolymph used in this pI was detectable in the fat body. The pI described study was not at peak levels. Two of the butterflies here for M. sexta (4.94) is in agreement with that which were examined, J. eoenia and P. rapae, both described by other researchers for partially purified possessed very low hemolymph JHE levels com- JHBP (pi=4.95; Kramer et al. 1976). Analysis of pared to the moths, and P. rapae in particular hemolymph JHBP samples from four D. plexippus had four separable JHE's ranging from pI 4.1 to and five S. exigua individuals showed that only a pI 7.7. However, prewandering field-collected single binding activity was present in all cases, fo- C. eurytheme larvae had higher hemolymph JHE cusing at pH 6.2 and 5.9, respectively. activity and only a single JHE detectable on IEF. In order to demonstrate the saturability of The results from IEF of various lepidopteran binding protein activity on the gel, the pI 4.8 T. ni binding proteins were more uniform, as shown in JHBP activity was used as a model. Preincubation Table 2. In general, the JHBP activities were usual- of the hemolymph with either carrier-free 3H-JH ly more acidic than the corresponding JHE's, and I or with 6.7 • t0 -8 M JH 1 led to a constant 69% no species examined had more than two JHBP's. of the total counts bound, as measured by liquid The pI for the T. ni binding protein activity is in scintillation counting of the pH 4.8 gel slice after agreement with Sparks and Hammock (1979a, b), narrow range IEF. Preincubation of the hemo- who found that the same protein may be present lymph with 6.7 • 10 -6 and 6.7 • 10 -* JH I (above in several stadia and during several stages of the true solubility) resulted in a reduction to 44% and K.D. Wing el aI.: pl's and binding affinities ofJH esterases and binding protcins 2t7

Table 2. Isoelectric points and percentages of radioactivity due Kmand V~ vatues tar hen~olymph JHF_ r ~ol JH Qc,~ ] ' to .Ill binding proteins from various species of Lcpidoptera acbv,t~es from prewar'der,rg l/V lm'n-~Tb~-m~ leptdopterous larvae 010~ d Species pl" Percent of CPM on the gel b Spec*e5 ~( M ,10 "~) V~.(ncr',o[ JH 008~ f aad / rn,n- mL } j T n, 1 2 L,1 / A/ T. ni 4.8-t-0.05 70.2_+ 10.7 T n, 32 52 006-~ /././.o" S.e.v~q, ua 5.9 88.3 • 6.6 S ex,gua 1 0 60 /r ...9~ m H virescens 31 159 / [ ~.'t"- " . H. vh'escens 5.31 +0.10 54 __+ 19.0 M. sexta 4.94_+0.05 73 + 8.5 G. melhmelta 6.3-+0.08 69 _+ 14.0 E. elutella 4.8-+0.07 90.5+ 6.4 O. vetusta 4.45 22 _% 2.8 7U 4.8 52 + 4.2 -96 -80 -6& 4.8 -32 -16 0 16 32 L,8 6& E. acrea 5.2 95.3 1/(jH)~ 106(M) -1 P. gossypietla 4.9 55.3 _+ 30.7 H. netmdensis > 5.7 83 Fig. 1. Lineweaver-Burk analysis of JH esterase activity in J. coenia > 6.5 71 whole hemolymph of several Lepidoptera. Correlation coeffi- P. r apae 5.1 44 cients for all lines were 0.97 or above. Each dose of JH was C. eurytheme 4.8 92.6 run in duplicate, and for each species the points are averages D. plexippus 6.2 70.9 + 11.0 from two separate experiments. All incubations were performed at 30 ~ but incubation times were varied to give between Data are means-+SD where three or more determinations 10-40% substrate conversion. JH I estcrasc activities in 7". ni on narrow range IEF were made hemolymph were run in carbowaxed tubes. Protein concentra- b Total CPM in peak divided by total CPM in gel track, times tions run in duplicate using the method of Bradford (1976) 100. Data are means-+SD where three or more determina- are T. ni - 44.2; S. exigua - 26.4; H. virescens - 58,7; and tions on narrow range IEF were made. The majority of the G. mellonelta - 64.1 mg protein/ml hemolymph. The ranges of radioactivity which was not associated with a major peak the averages were +0.02 (nmoles JH [ acid/min-ml)-~. Subse- was found immediately below the sample wick. Greater than quent experiments using substrate concentrations above and 90% of the radioactivity was accounted for in each case below the K m indicated K='s of 3 and 6xl0-Sm for Jlt III (Wing 1981) and 1, respectively from T. ni (Abhel-Aal, unpublished)

J.6% of the total counts bound after IEF, respec- as fast as JH Ill, as has been observed in the sugar- tively, with concomitant increases in nonbound ra- cane borer Diatraea saccharalis (F.) (Roe et al. diolabeled JH I as estimated by the radioactivity 1983) and M. sexta (Coudron et al. 1981). remaining under the sample wick which did not Scatchard analysis of JH I binding by the he- focus with a macromolecule. The counts remaining molymph of several species of Lepidoptera is on the sample wick after IEF remained relatively shown in Fig. 2. In each case the dissociation con- constant for all concentrations of cold JH I stant K d for JH I binding is near 10 -v M. The ex- (1.7-3.6% of total CPM). Because the binding con- perimentally obtained K s for M. sexta is similar ditions used in the IEF experiments make use of to the previously reported value of 1.26 x l0 -7 M only very low concentrations of JH I and focusing for partially purified JH binding protein using a times of 2.5 h are used, detection of non-specific, DEAE-filter disc assay (Kramer et al. 1976) and low affinity lipoprotein binding is extremely un- for whole hemolymph high affinity binding activity likely. Thus, by far the most prevalent case in the from fourth stadium larvae of different ages (1.5 Lepidoptera examined is the presence of a single, to 2.5 x 10 -7 M; Goodman et al. 1976; Goodman slightly acidic, high affinity JH carrier in the pre- and Gilbert 1978). The total number of binding wandering hemolymph (13 of 14 species). How- sites obtained for M. sexta hemolymph is in excel- ever, Orygia vetusta was unique in having two de- lent agreement with previous findings (Goodman tectable JHBP activities. and Gilbert 1978). Total binding site concentration The results of the Lineweaver-Burk double re- in other hemolymph samples ranged from ciprocal analysis are shown in Fig. 1. For all spe- 8.] • 10 -6 M (G. mellonella) to 1.0 x 10 .6 M for cies r values for the lines were 0.97 or above. V,,,x M. sexta. On a per mg protein basis, there was values for the different species varied considerably, a wide variation in specific binding activity ranging however K m values for all species were quite simi- from 1.7x 10 -tt (C. eurytheme) to 11.7x lar, ranging from 1.0x 10 -7 M ['or S. exigua to 10- 11 moles binding site per mg protein (E. acrea). 3.1 x 10-VM for H. virescens. Velocities utilizing Three separate pools of hemolymph from 15 T. ni 5 x 10-6 M substrate as in standard assay condi- larvae each showed little variation in Scatchard tions showed that JH I was hydrolyzed about twice binding behavior, as indicated by the standard de- 218 K.D. Wing ct at.: pl's and binding affinities ofJH esterases and binding proteins

024- H virescen5 - r= 0,97 l 1\\ Kd :61 x 10-7M 061 (:~ Binding sites :41x 10-6M r = 095 -~ Kd :6.0x10-TM | X(~O M sexta - r = 0.98 | O\ Kd = 1.5 ~ ~0-TM ~--I~~~~Z 016-es0.08_ = 80x10"6M O,L --\ Binding sites : 10 x IO-6M

z 02

I l ~ " l I 0 -- I I I I C. eurytheme- r=0.96 0,2/,- 8 Kd=3.8x10-TM r = 097 Binding sites = 13 x t0-6N Kd = 66 x]0-TM o"0( ~ z .. ------G meitonel[o - r=0.97 " " = x05M1 - Kd = 26 x 10-7N T 0.16-- 0.4 "" t . O\ Binding sites = 8.1 • 10-6N

X3 i 0.08- "-'L o 0 ..... I I" -- I ~ 0 ~ -- I -I 0.8- -- T. ni- r =0,94 4.0 8.0 12.01 16.0 Kd =6.9 • 0.33 x 10_7N -1.6 (JH bound) x 10-8M \~ Binding sites = 354 • 10-6M Fig. 3A, B. Scatchard analysis of JH binding protein activity 06- \ ------E.acrea- r:0g7 -1.2 in whole T. ni hemolymph, using a hydroxylapatite assay. Each & \ \ Kd =7.6 x Io-BM dose of JH I was run in duplicate, and the experiment was :Z ~) Binding sites =6.3 x 10-6M performed twice using two separate pools of hemolymph. A o~- \ -08 experiment run at 30 ~ C; B experiment run at 0.5 ~ k a[] Ok

02- ~0 ~.~i0 -0,4 400- { 200- 100 - 0 I I -- I N I L 0 0 4,0 80 120 16.0 200 24.0 50- --.-___._ .s (JH bound) , ~0-8M i 25- Fig. 2. Scatchard analysis of JH binding protein activity in 10 whole hemolymph of several Lepidoptera, using the charcoal- I I I~ I I I dextran technique. Each dose of ]H I was run in duplicate, 0 t 2 3 & 5 6 and for all species the experiment was performed tiwce using Time (min) a single pool of hemolymph, except in the case of T. ni where Fig. 4. Determination of the dissociation rate constant k n in three separate pools were run and the mean Kd and binding whole T. hi. hemolymph at 0.5 ~ using a charcoat-dextran site concentrations is presented (• binding site concentra- technique. Each time point was run in triplicate. In absence tion is 3.54• 10-6M). When calculated on a per mg of nonradiolabelled JH I as a competitor, the amount of JH protein basis; the binding site concentrations are - for T. ni bound was 350 pmolar; however this point is not included in 5.9; E. acrea - 11.7; H. virescens - 5.2; G. mellonella - 9.0; the regression since it does not represent a true zero time due M. sexta- 5.0; and C. eurytheme- 1.7 x 10- ~ moles binding to mixing effects. Data are plotted as the mean of the 3 repli- site per mg hemolymph protein. The ranges of the averages cales; standard deviations at each time point are < 10%_ r value were _+0.03 JH bound/JH free for the line shown is 0.98

viations of less than 5% of the mean for both K d analysis using the modified hydroxylapatite assay and binding site concentration determination. For on dilute T. ni hemolymph at 0.5 ~ (Fig. 3B) any particular dose of JH within an assay run, agree very well with those same values generated the coefficient of variation for the replicates was using the charcoal-dextran technique. As expected, never more than 10% and usually less than 4%. the charcoal-dextran assay gave a slightly higher The dissociation constant K d and molarity of dissociation constant and a lower binding site con- high affinity binding sites obtained by Scatchard centration, which may be due to the nonequili- K.D. Wing ct al.: pl's and binding affinities ofJH cstcrascs and binding protcins 219

brium situation present when charcoal is added pear that a single enzyme is probably responsible resulting in some stripping of JH from the JHBP. for the majority of the JH ester hydrolysis during The coefficient of variation for replicates of a sin- the periods of rapid JH turnover in the last larval gle dose of JH within a hydroxylapatite assay run stadium. was usually less than 10% and typically closer to Similarly, earlier work by Rudnicka et al. 5%. When this experiment was repeated with all (1979) indicated the possibility of multiple JHE's operations carried out at 30 ~ C (Fig. 3A), a similar .in G. mellonella. However, in this study a single Kd value was obtained which indicates that the JHE was detected in G. meltonella hemotymph by kinetic on-rate k a and the off-rate k d either remain IEF, and recent research on purification supports about the same or change proportionally with tem- the presence of a single enzyme (Rudnicka, unpub- perature. lished). However, it is apparent from our results The dissociation rate constant k a was deter- that other species do indeed have several JHE's. mined to be 0.308 min-* at 0.5 ~ using the char- H. virescens provides an example of a species coal-dextran assay, indicating a fairly rapid disso- whose whole hemolymph displayed very high JHE ciation phenomenon in the presence of excess JH activity which on IEF could be separated into at (Fig. 4). The dissociation of radiolabelled JH I least two different proteins. The other members (1 x 10 -9 M) from JHBP was also studied by dilut- of the noctuid family tested (7". ni; S. exigua) had ing the hemolymph from 2 to 200 x. This tech- only a single peak of activity, demonstrating differ- nique indicated that dissociation is much slower ences even within a family. The pyralids tested in the absence of excess JH (data not shown). Us- (G. mellonella, E. elutella) both possessed only a ing the values for Ka and k d determined empirically single JHE peak, but the remaining moths repre- in this study, one may calculate a theoretical value senting the families Liparidae, Arctiidae, Gelechii- for k, of 4.5 x 105 M - 1 min- 1. Attempts to deter- dae and Saturniidae all displayed multiple JHE's. mine the on-rate empirically resulted in what The butterflies (J. coenia, P. rapae) also gave evi- seemed to be instantaneous association of hor- dence of several enzymes. Another pierid tested mone with binding protein, which was not amen- (C. eurytheme), however, gave evidence of only a able to kinetic analysis. single JHE on IEF, again illustrating that differ- ences are possible within a single family. Because of their ease of analysis, esterase Discussion isozyme patterns have been studied extensively in The biochemical characteristics and multiplicity of many species and notably in insects. The electro- the JHE's and JHBP's of 7". ni, G. mellonella and phoretic patterns of hemolymph esterase isozymes M. sexta have been studied in some detail, but little are among the most variable of all enzymes studied work has been done on other species of Lepidop- possibly because these esterases hydrolyze ~argely tera. Even in these key species the number of ester- extrinsic substrates (Johnson 1973; Wagner and ases involved in JH catabolism remains in ques- Selander 1974; Poweli 1975). Even within this tion. In particular, Sanburg et al. (1975) described study, the patterns of the esterase isozymes stained the presence of three separate JHE activities in with ~-naphthyt acetate varied dramatically, M. sexta hemolymph after analysis by column iso- among individuals, among species, and among de- electric focusing (pI 5.4, 5.5 and 5.6), two of which velopmental stages within a species. The apparent were inseparable from 0~-naphthyl acetate esterase similarity among the esterases hydrolyzing JH may activity. Recently, however, Coudron et al. (1981) further indicate that their evolution has been con- have purified to reported homogeneity a single ju- servative and that these are specialized, constitu- venile hormone esterase from M. sexta hemo- tive enzymes, hydrolzying an important intrinsic lymph which is inactive on o~-naphthyl acetate and substrate. An investigation of the relative rates of Sparks et al. (1983) find a single peak of activity structural changes in JH esterases vs other hemo- on gel filtration. Similarly, the accumulated evi- lymph esterases could be of evolutionary interest. dence in T. ni is consistent with the presence of It is apparent from this and other comparative a single JHE enzyme which is responsible for studies that macromolecular binding of JH I is >95% of the JH ester cleavage, is relatively inac- fairly widespread across several families and spe- tive on ~z-naphthyl acetate, and has a pl of about cies of Lepidoptera (Kramer et al. /976; Kramer 5.5 (Sparks etall 1979; Sparks and Hammock and Childs 1977). Because a binding protein gel 1979a; Rudnicka and Hammock 1981 ; Wing et al. fraction from T. ni hemolymph could be saturated, 1981). Our results are in agreement with the later use of IEF is probably a valid technique for quali- findings. In both M. sexta and T. ni it would ap- tative identification of high affinity JH binding. 220 K.D. Wing et al. : pl's and binding affinities of JH esterases and binding proteins

Although the majority of the species examined here mine K S as well as rates of acylation and deacyla- displayed a single JHBP, O. vetusta yielded data tion in order to develop a model of JH catabolism indicating at least two seParate proteins which in the hemolymph. bound JH on IEF. However, the results for JHBP JHBP equilibrium dissociation constants for six in the Lepidoptera seem to be more uniform than of the Lepidoptera showed almost a 10 x range those found for JHE, and one could surmise that in apparent binding affinities as measured in the based on this study a single hemolymph high affini- whole hemolymph. In all cases only a single slope ty carrier is the usual case. was obtained, indicating a single major class of The apparent Michaelis constant K,~ for JH IiI high affinity binders, a result which is in agreement was in the range of 10-7 M for the three noctuids with the IEF data. The E. acrea JHBP was charac- and G. mellonella. In all cases only a single slope terized by having a very high affinity and, in addi- was obtained, supporting evidence for a single class tion, was present at high concentrations on both of JHE activity. Although IEF analysis of H. vires- a per volume and per mg protein basis. The values cens indicated the presence of 2 JHE's, only a sin- obtained for binding protein equilibrium dissocia- gle slope in the Lindweaver-Burk analysis was ob- tion constants are higher than those typically ob- tained for this species as well. These results are served for mammalian steroid binding globulins, an order of magnitude lower than findings de- which are in the range of 10 -9 M at 4 ~ (West- scribed previously for M. sexta (K,, JH III = 1.4 x phal 1980). 10-6 M; Vince and Gilbert 1978). However, recent Measurement of the equilibrium dissociation findings by Peter (1981) have demonstrated that constant of the T. ni JHBP using a charcoal-dex- on day 5 at the last larval stadium of M. sexta tran adsorption technique and a hydroxylapatit e the Km of the esterase activity for racemic JH III protein binding technique yielded very similar (2E, 6E) was 1.4x10 -7 M. In addition, the K m values. In addition, the Ka measured at 30 ~ was for racemic JH I was 5.0 x 10- v M. These results similar to that obtained at 0.5 ~ The K d for the are in agreement with data in this report on 7". ni, dissociation of JH I from the M. sexta hemolymph where the K m on racemic JH I is approximately binding protein was also shown to be essentially 3 x as large a value as that for racemic JH III. constant between 4 ~ to 37 ~ (R.C. Peterson, In addition, these data are supported by Sparks Ph,D. dissertation), if the equilibrium dissociation (unpublished) who found that nonlabeled JH III constant K a does indeed remain relatively constant is a better competitive inhibitor of JH I or III hy- with increasing temperature, both the on-rate k~ drolysis than are JH's I or If. In both species, JH I and off-rate k d must increase proportionally. This was cleaved at a higher rate than was JH III, and situation is in marked contrast to steroid binding thus the differences in the rate of hydrolysis of globulins in mammals, where it has been demon- the two homologues must be dependent upon some strated that dissociation of complexes such as pro- factor other than that described by the apparent gerstone-progesterone binding globulin occurs Kin. Studies on deacylation of acylchymotrypsin much more rapidly at elevated temperatures have shown that the rate of the unimolecular reac- (Stroupe and Westphal t975), which alters K d. It tion k~at is dependent upon the size of the group should also be pointed out that all kinetic and equi- attached to the awl moiety (Dupaix et al. 1970). librium values reported here were obtained using Since the catalytic mechanism of JHE is probably racemic 2E, 6E, lO-cis juvenile hormones which similar to that of many serine proteases (Hammock were 1:1 mixes of 10R, llS (natural isomer) and et al. 1982), it is possible that JH I may be hydro- 10S, llR at the epoxide moiety. On analogy with lyzed more rapidly than JH III because of the more other species the binding affinities reported here rapid hydrolysis of the JH I acyl enzyme. With may be higher for the natural isomer of JH I substrate concentrations approaching or exceeding (Schooley et al. 1978; Klages et al. 1980). the K,, the deacylation step, and not the acytation The off-rate constant, k d of 0.308 min-1 at binding step, probably determines the overall reac- 0.5 ~ for the JHBP of T. ni, is a larger value than tion rate. At physiological concentrations of the that found for JH 1 receptors in the cytosoi of Dro- hormone which are actually lower than the molar sophila melanogaster Kr cells (k d = 1.34 x concentration of the JHE, the reaction will be es- 10- 2 rain- ~ at 23 ~ Chang et al. 1980) or in Dro- sentially first order in substrate and the acylation sophila hydei epidermis (k d =6.5 x 10 .4 min-1 at step may very well be rate-limiting. Since K m may 4 ~ Klages et al. 1980). These data suggest a t,/2 not equal K,~ for serine esterases and proteases value of 2.1 rain for the JH-JHBP complex in the (Bender et al. 1967), it will be important to deter- presence of excess JH which represents a much K.D. Wing ctal.: pl's and binding affinities ofJH esterases and binding proteins 221 faster dissociation than that observed for the re- JH titers seen in the Lepidoptera even in the ab- ceptors. Since attempts to determine k d by dilution sence of continued JH biosynthesis. However, the methods in the absence of excess JH resulted in dissociation of JH from JHBP appears fast enough slower dissociation (data not shown), interpreta- to support the rapid metabolism of JH observed. tion of off rate data must be cautious. Thus, both the on- and off-rates appear to be Attempts to determine an on-rate k a kinetically extremely rapid, and if we assume that these rates using JH I concentrations from 10 -6 M to 10 -9 M are higher at physiological temperatures, then we resulted in almost immediate binding at 0.5~ may envision a binding protein which very rapidly (Wing 1981).The calculated k, for the JH-JHBP picks up JH from the vicinity of the corpora allata complex of 4.5 x 105 M- 1 rain- ~ is lower than that during stages of active synthesis, or from the tis- found for Drosophila melanogaster K c celt cytosolic sues when the animal needs to rid itself of free receptors (k a = 1.29 x 106 M- 1 min- 1) and Dro- JH by increasing its degradation by JHE's. By the sophila hydei epidermal receptors (k, = 2 x same token, the hemolymph carrier readily would 10 6 M-1 min-1). It is apparent from these data release the hormone into solution to be picked up that the lower affinity of the JHBP compared to and retained by high affinity tissue receptors when the receptors is due to a mugh higher off rate for these are available. Riddiford and Mitsui (1978) the JHBP. have reported a high affinity JH binding activity The log P values for JH I and III (3.01, 3.15) in nuclei of M. sexta epidermal cells with K d = 2 x agree closely with values described earlier for ju- 10-8 M. Affinities of mammalian steroid transport venoids by Mumby and Hammock (1979) and proteins have also been found to be lower than clearly indicate that the JH's tend to partition into receptor affinities, and in particular the dissocia- lipid phases. As indicated earlier (Hammock et al. tion rate constant k a is usually much slower in 1975; Nowock et al. 1976) the rapid, nonspecific the case of a receptor (Westphal 1980). Thus, hor- uptake of JH by fat body followed nonsaturable mone complexes of the transport proteins dissoci- first order kinetics and was greatly retarded in the ate at a rate rapid enough to make free, biologi- presence of JHBP. Clearly the molarities and Kd'S cally active hormones available to the target cell. of the JHBP's in the Lepidoptera examined are When receptors are not present at high levels, sufficient to retain and distribute rapidly JH in or when it is developmentally critical to eliminate the aqueous compartment. Since most tissues also all JH, high concentrations of the hemolymph car- have been shown to contain JHBP's, it is likely rier may aid in removing unwanted JH from target that JH is largely within the aqueous pool through- tissues to be metabolized by hemolymph JHE's, out the insect. However, it should be recalled that whichseem to peak at stages when the JH titer K d values describe the equilibrium between the is decreasing. Thus, the JHBP which is present at JHBP's and the aqueous environment while in the micromolar concentrations probably is acting as in vivo situation the equilibrium may largely be an efficient extraction mechanism for pulling JH between JHBP and lipid depots. JH dissociates into the hemolymph, where it may be hydrolyzed very rapidly from the T. ni JHBP in the presence by the esterase. Since catabolism by the enzyme of excess JH with a tl/2 of about 2.1 min at 0.5 ~ C. is irreversible, the in vivo equilibrium should be Although metabolism clouds the picture, Nowock favorably shifted toward degradation when cor- et al. (1976) demonstrated that the ti/2 of efflux pora allata synthesis rates and tissue receptor titers from M. sexta fat body is much greater than are low. Precisely timed peaks of tissue and hemo- 30 min and using a compound of similar polarity lymph JHE in concert with the JHBP may be nec- it has been shown that even efflux from vesicles essary to eliminate all free JH to insure proper has a t j~ 2 of greater than 30 min (Zakim and Ves- development. In fact, inhibiting JHE in last stadi- sey 1977). In fact, in the absence of binding pro- um T. ni at critical stages by applying large doses teins lipophilic molecules may travel through a cell of EPPAT led to either a delay in pupation or without significant entry into the aqueous com- to the formation of malformed pupae (Sparks and partment by moving at the aqueous interface of Hammock 1981). Thus, a dynamic JH equilibrium cellular membranes. Thus, without JHBP present exists among the corpora allata, target tissue recep- the efflux of JH from lipophilic compartments into tors, and the esterase, with the hemolymph carrier the aqueous compartments, cells and the hemo- protein acting as an intermediary among the three. lymph where JHE activity is high is too slow to The levels of hormone synthesis, receptor and JHE support the hypothesis that JH catabolism by JHE titer, change during development depending on alone is responsible for the rapid decline in in vivo whether or not high hormone levels are required, 222 K.D. Wing et al. : pl's and binding atIinities of Jt-1 estcrases and binding proteins and all of these factors act in concert with the juvenile hormone stability and distribution in Manduca JHBP to express JH action. sexta fat body and imaginal disks in vitro. Mol Cell Endoc- rinot 3:167-184 Hammock BD, Sparks TC, Mumby SM (1977) Selective inhibi- Acknowledgements. The authors wish to thank Drs. T.C. Baker tion of JH esterase activity from cockroach hemoIymph. and M. Rust of the University of California at Riverside, and Pestic Biochem Physiol 7:517-530 Drs. C. Judson, S.S. Duffey and M. Birch of the University Hammock BD, Wing KD, McLaughlin J, Lovell VM, Sparks of California at Davis for use of their equipment and insect TC (1982) Trifluoromethylketones as possible transition colonies. This research was supported in part by NIEHS Grant state analog inhibitors of juvenile hormone esterase. Pestle No. 5-ROI-ESO2710-03. B.D. Hammock was supported by Bioehem Physiol 17: 76-88 NIEHS Research Career Development Award 5-KO4- Johnson G B (1973) Enzyme polymorphism and biosystematics: ESOO 107-04. the hypothesis of selective neutrality. Annu Rev Ecol Syste- mat 4:93-116 Jones D, Jones G, Hammock BD (1981) Growth parameters References associated with endocrine events in the larval Trichoplusia ni (Hiibner) and timing of these events with developmental Bender ML, Khzdy FJ, Wedler FC (1967) e-Chymotrypsin: markers. J Insect Physiol 27:779-788 enzyme concentration and kinetics. J Chem Educ 44:84-88 Jones D, Jones G, Wing KD, Rudnicka M, Hammock BD Bradford MM (1976) A rapid and sensitive method for the (1982) Juvenile hormone esterases of Lepidoptera. I. Acti- quantitation of microgram quantities of protein utilizing vity in the hemolymph during the last larval instar of 11 spe- the principle of protein-dye binding. Anal Biochem cies. J Comp Physiol 148:1-10 72: 248-254 Ktages G, Emmerich H, Peter MG (I980) High-affinity binding Chang ES, Coudron TA, Bruce M J, Sage BA, O'Connor JD, sites for juvenile hormone 1 in the larval integument of Dro- Law JH (1980) Juvenile hormone-binding protein from the sophila hydei. Nature 286:282-285 cytosol of Drosophila K c cells. Proc Natl Acad Sci USA Korenman SG (1969) Comparative binding affinity of estrogens 77:4657-4661 and its relation to estrogenic potency. Steroids 13:163-177 Coudron TA, Dunn PE, Seballos HL, Wharen RE, Sanburg Kramer K J, Childs CN (1977) Interaction of juvenile hormone LL, Law JH (1981) Preparation of homogeneous juvenile with carrier proteins and hydrolases from insect haemo- hormone specific esterase from the haemolymph of the to- lymph. Insect Biochem 7 : 397-403 bacco hornworm, Manduca sexta. Insect Biochem Kramer K J, Law JH (1980) Synthesis and transport of juvenile 11:453-461 hormone in insects. Ace Chem Res 13:297-303 deKort CAD, Granger NA (1981) Regulation of the juvenile Kramer K J, Dunn PE, Peterson RC, Law JH (1976) Interaction hormone titer. Annu Rev Entomol 26:1-28 of juvenile hormone with binding in insect hemolymph. In: Dupaix A, Bechet J3, Roucous C (1970) Separation of polar, Gilbert LI (ed) The juvenile hormones. Plenum Press, New steric and specific effects in the c~-chymotrypsin-catalyzed York, pp 327-341 hydrolysis of acylsubstituted p-nitrophenyl esters. Biochem Lineweaver H, Burk D (1934) The determination of enzyme Biophys Res Commun 41:464-470 dissociation constants. J Am Chem Soc 56:658-666 Ferkovich SM, Silhaeek DL, Rutter RR (1975) Juvenile hor- Mumby SM, Hammock BD (1979) Stability of epoxide-con- mone binding proteins in the haemolymph of the Indian taining juvenoids to dilute aqueous acid. J Agric Food Meal Moth. Insect Biochem 5:141-150 Chem 27:1223-1228 Fujita T, Isawa J, Hansch C (1964) A new substituent constant, Nowock J, Hammock BD, Gilbert LI (1976) The binding pro- q, derived from partition coefficients. J Am Chem Soc tein as a modulator of juvenile hormone stability and up- 86:5175 take. In: Gilbert LI (ed) The juvenile hormones. Plenum Gilbert LI, Goodman W, Granger N (1978) Regulation of juve- Press, New York, pp 354-373 nile hormone fiter in Lepidoptera. In: Gaillard PJ, Boer Peter MG (t981) Stereochemistry of juvenile hormone hydroly- ttM (eds) Comparative endocrinology. Elsevier/North-Hol- sis in insect hemolymph. In: Sehnat F, Zabza A, Mann land Biomedical Press, Amsterdam, pp 471-486 JJ, Cymborowski B (eds) Regulation of insect development Goodman W, Gilbert L[ (1978) The hemolymph titer of juve- and behaviour, International Conference. Karpacz, Poland, nile hormone binding protein and binding sites during the June 23-28 1980, pp 237-244 fourth larval instar of Manduca sexta. Gen Comp Endo- Powell JR (1975) Protein variation in natural populations of crinol 35 : 27-34 animals. In: Dobzhansky T, Hecht MK Steere WC (eds) Goodman W, Bollenbacher WE, Zvenko H, Gilbert LI (1976) Evolutionary biology. Plenum Corp, New York, pp 79-119 A competitive protein binding assay for juvenile hormone. Reddy G, Hwang-Hsu K, Kumaran AK (1979) Factors in- In : Gilbert L1 (ed) The juvenile hormones. Plenum Press, fluencing juvenile hormone esterase activity in the wax New York, pp 75-95 moth, Galleria mellonella. J Insect Physiol 25:65-71 Goodman W, Schooley DA, Gilbert LI (1978) Specificity of Riddiford LM, Mitsui T (1978) Loss of cellular receptors for the juvenile hormone binding protein: The geometrical juvenile hormone during the change in committment of the isomers of J H I. Proc Nail Acad Sci USA 75:185-t89 epidermis of the tobacco hornworm, Manduca sexta. In: Hammock BD, Quistad GB (1981) Metabolism and mode of Gaitlard PJ, Boer HH (eds) Comparative endocrinology. action of juvenile hormone, juvenoids and other insect Elsevier/North-Holland Biomcd Press, Amsterdam, p 519 growth regulators. In: Hutson DH, Roberts TR (eds) Pro- Roe RM, Hammond AM, Sparks TC (1983) Characterization gress in pesticide biochemistry, vol 1. John Wiley, New of the plasma juvenile hormone esterase in synchronous last York, pp 1-82 stadium female larvae of the sugarcane borer, Diatraea sac- Hammock BD, Sparks TC (1977) A rapid assay for insect juve- charalis (F). Insect Biochem t 3:163-170 nile hormone esterase activity. Anal Biochem 83 : 573-579 Rudnicka M, Hammock BD (1981) Approaches to the purifica- Hammock BD, Nowock J, Goodman W, Stamoudis V, Gilbert tion of the juvenile hormone esterase from the cabage LI (1975) The influence of hemolymph-binding protein on looper, Trichoplusia hi. Insect Biochem 11:437-444 K.D. Wing ct al.: pl:s and binding affinities of Jtt csterascs and binding protcins 223

Rudnicka M, Sehnal F, Jarolim V, Kochman M (1979) Hydro- Sparks I'C, Hammock BD, Riddiford LM (1983) Thc haemo- lysis and binding of the juvenile hormone in the haemo- lymph juvenile hormone esterase of Manduca sexfa (L.): lymph of Gallerla meltonella. Insect Biochem 9:569-575 Inhibition and regulation. J Insect Physiol (in press) Sanburg LL, Kramer KJ, K~zdy FJ, Law JH (1975) Juvenile Spector T (I978) Refinement of the Coomassie Blue method hormone-specific estcrases in the haemotymph of the to- of protein quantitation. Anal Biochem 86:142-146 bacco hornworn, Manduca sexta. J Insect Physiol Stroupe SD, Westphal U (1975) Stcroid-protein interactions 21 : 873-887 XXXIII. Stopped-flow fluorescence studies of the interac- Scatchard G (1949) The attractions of proteins for small mole- tion between steroid hormones and progesterone-binding cules and ions. Ann NY Acad Sci 51:660 672 globulin. J Biol Chem 250:8735-8739 Schooley DA, Bergot BJ, Goodman W, Gilbert LI (1978) Syn- Vince RK, Gilbert LI (1977) Juvenile hormone esterasc activity thesis of both optical isomers of insect juvenile hormone in precisely timed last instar larvae and pharatc pupae of I11 and their affinity for the juvenile hormone-specific bind- Manduca sexta. Insect Biochem 7 : 115-120 ing protein of Manduca sexta. Biochim Biophys Res Com- Wagner RP, Sclander RK (1974) lsozymes in insects and their mun 81:743--749 significance. Annu Rev Entomol 19 : 117-138 Shorey HH, Hale RL (1965) Mass rearing of the larvae of Westphal U (1980) Mechanism of steroid binding to transport nine noctuid species on a simple artificial medium. J Econ proteins. In: Benazzani E, DiCarlo F, Mainwaring WlP Entomol 58 : 522-524 (eds) Pharmacological modulation of steroid action. Raven Sparks TC, Hammock BD (1979a) A comparison of the in- Press, New York, pp 33-47 duced and naturally occurring juvenile hormone esterases Wing KD (1981) Juvenile hormone esterases of Trichoplusia from the last instar larvae of Trichoplusia hi. Insect Biochem ni and other Lepidoptera: characteristics, site of synthesis, 9:411-421 interaction with other proteins and inhibition. PhD disserta- Sparks TC, Hammock BD (1979b) Induction and regulation tion, University of California, Riverside of juvenile hormone esterases during the last larval instar Wing KD, Sparks TC, Lovell VM, Levinson SO, ttammock of the cabbage looper, Trichoplusia hi. J Insect Physiol BD (1981) The distribution of juvenile hormone esterase 25 : 551-560 and its interrelationship with other proteins influencing ju- Sparks TC, Hammock BD (1980) Comparative inhibition of venile hormone metabolism in the cabbage looper, Trichop- the juvenile hormone eslerases from Trichoplusia hi, Tene- lusia ni. Insect Biochem 11:473--485 brio molitor and Musca domestica. Pestle Biochem Physiol Winter A, Ek K, Andersson VB (1977) Analytical electrofocus- 14: 290-302 ing in thin layers of polyacrylamide gels. LKB Application Sparks TC, Willis WS, Shorey Htt, Hammock BD (1979) Hae- Note 250 molymph juvenile hormone esterase activity in synchronous Zakim D, Vessey DA (1977) Membrane-bound estrone as sub- last instar larvae of the cabbage looper, Trichoplusia hi. stratc for microsomal UDP-glucuronyltransferase. J Biol J Insect Physiol 25:125-132 Chem 252:7534-7537