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.