Characterization of homogeneous juvenile hormone esterase from larvae of Trichoplusia ni

Insect Biochem. Vol. 17, No. 2, pp. 373-382, 1987 Printed in Great Britain

0020-1790/87 $3.00+0.00 PergamonJournals Ltd

CHARACTERIZATION OF HOMOGENEOUS JUVENILE HORMONE ESTERASE FROM LARVAE OF TRICHOPLUSIA N I MARIA RUDNICKA* and DAVY JONESt Department of Entomology, University of Kentucky, Lexington, KY 40456-0091, U.S.A. (Received 4 October 1985; revised and accepted 29 April 1986)

Abstract--Juvenile hormone (JH) esterase from larvae of Trichoplusia ni was purified at least 4000-fold to homogeneity by means of polyethylene glycol precipitation followed by hydroxylapatite chromatography, chromatofocusing and electrophoresis. JH esterase electrophoreticaUy homogeneous on sodium dodecyl-sulphate polyacrylamidegels revealed a singlesubunit with an apparent mol. wt of 66,000. The purified enzyme was heterogeneous with respect to charge, as approx. 90% focused at a pH of 5.5 and the remainder at 5.3 on IEF gels. JH esterase in crude haemolymph possessed two apparent Kmvalues toward JH II (2.5 x I0 -s and 3.4 x 10 -7 M), whereas the purified enzyme exhibited only the high Km (3.8 × 10 -7 M ) .

Key Word Index: Juvenile hormone esterase purification, kinetic analysis, Trichoplusia ni

INTRODUCTION

MATERIALS AND METHODS

Juvenile hormone esterase (JHE) has been demonstrated to be of key importance in regulation of insect metamorphosis (Jones, 1985a; Jones and Sreekrishna, 1984; Jones, 1985b; Sparks and Hammock, 1980; Sparks, 1984). Much of the current research on JHE in insects has been conducted on larvae of Trichoplusia hi, and thus T. ni is serving as a model insect for the study of JHE (Hammock et al., 1981, 1984; Hammock, 1985). The JHE activity in T. ni has been used as a model active site for development of a broad spectrum of potential inhibitors (Abdel-aal et al., 1984; Abdel-aal and Hammock, 1985; Hammock et al., 1982; Prestwich et ai., 1984; Sparks and Rose, 1983). JHE from T. ni has also been the object of the kinetic analysis of enzyme-substrate interaction (Abdel-aal and Hammock, 1985; Sparks and Rose, 1983; Wing et al., 1984) and has likewise been used to study regulation of JHE activity (Click et al., 1985, 1986; Jones et al., 1981b; Jones and Hammock, 1983; Jones, 1985b; Sparks and Hammock, 1979; Sparks, 1984). The study of regulation, kinetics and inhibition of T. ni JH esterase would be substantially expediated if pure enzyme were available. Two attempts to purify JHE from T. ni have been published, with resulting purifications factors of x 175 and x 430 (Rudnicka and Hammock, 1981; Yuhas et al., 1983). We report in this paper a JH esterase purification procedure which has yielded at least 4000-fold purified enzyme, homogeneous by electrophoretic criteria. We also report some molecular and kinetic properties of the purified enzyme.

Insects Larvae of T. ni were reared on a pinto bean diet at 28 + 2°C 14 hr: 10hr light:dark as described by Shorey and Hale (1965) and Jones (1986). Larvae were synchronized as to developmental state (Jones et al., 1981a) and selected for bleeding on the day prior to wandering. Larvae were bled through the last abdominal proleg between 5 and 11 hr after lights on. Collected haemolymph was kept at -60°C until use.

JH esterase assay

*Present address: Department of Pathology, School of Medicine, University of Southern California, Los Angeles, CA 90007, U.S.A. 1"To whom correspondence should be addressed.

A partition assay (Hammock and Sparks, 1977) was routinely used to measure JH esterase activity against JH II as a substrate. Activities in the crude haemolymph, sample aliquots at each stage of purification and other analyses were assayed at final concentration of 5 x 10-6 M JH II. For kinetic analysis the range of substrate concentrations was

Chemicals JH II was obtained from Calbiochem while [C-10-3H]JH II was purchased from New England Nuclear (15.5 Ci/mmol). Labelled and unlabelled JH II were combined so as to give a stock concentration of 5 x 10-4, with ca 12,000dpm//zl. Polyethylene glycol (PEG, mol. wt 8000) was obtained from Aldrich, hydroxylap~ttite(HA-Ultrogel) was obtained from and prepared as described by LKB, Coomassie Fast Blue RR salt from Sigma and protein determination reagent from BioRad. Molecular weight markers obtained from Pharmacia were aldolase (160,000), fl-galactosidase (116,000), phosphorylase (97,400), BSA (68,000), ovalbumin (45,000), carbonic anhydrase (29,000) and cytochrome C (12,400). Chromatofocusing polybuffer 74, polybuffer exchanger 94, and Pharmalyte 4-6.5 were purchased from Pharmacia. Protein determination Protein concentration was determined colormetrically with the BioRad Coomassie Brilliant Blue G-250 procedure described by the supplier. Protein concentration was also or alternatively determined spectrophotometricaUy assuming 1 U of absorbance at 280nm as equalling Img of protein/ml.

373 I.B. 17/2--H

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MARIA RUDNICKA and DAVY JONES

from 2 x 10-s to 9 x 10 -6 M. Interpretation of apparent kinetic constants when two are present can be misleading unless the contribution of each to the velocity in the Km of the other is accommodated. Thus, we report kinetic constants corrected for the contribution of the other (Spears et al., 1971).

Purification procedure All steps of purification were performed at 5-8°C. Step L Polyethylene glycol (PEG) precipitation. PEG was prepared as a 50% solution (w/v) in 10 mM sodium phosphate buffer, pH 6.9. Frozen haemolymph was gently thawed and immediately centrifuged for 5 min at 7000 g. The 94ml supernatant was diluted 1:0.5 with the above buffer and held on ice. The 50% PEG solution was added dropwise to the diluted haemolymph, with slow magnetic stirring to raise the concentration of PEG to 8.5%. After 15 min of additional stirring, the suspensions was centrifuged for 15 min at 15,000g. The PEG concentration in the supernatant was adjusted to 26% by adding 50% PEG as above and stirred for 30 min. The material was centrifuged for 20 min at 21,000g and the resulting pellet dissolved in 32 ml of the same phosphate buffer and dialyzed extensively overnight against the same buffer. Step 2. Hydroxylapatite (HA) chromatography. The hydroxylapatite column (l.5cm i.d. x 60cm) filled with 80ml of HA gel was equilibrated with 10mM sodium phosphate buffer, pH 6.9. The dialyzed JH esterase sample was applied to the top of the column, and non-adsorbing protein washed out with equilibrating buffer until absorbance (280 nm) was below 0.1. Adsorbed proteins, including JH esterase, were eluted with a linear gradient of sodium phosphate, from 10 to 250 mM (pH 6.9), at a flow rate of 25-30 ml/hr. The 4 ml fractions were assayed for protein and JHE activity and those fractions containing enzymatic activity higher than 2.5 mol/min per ml were pooled. The resulting 124 ml sample was equilibrated with 10 mM phosphate buffer and concentrated to 6 ml using an Amicon cell equipped with YM10 membrane. Step 3. Chromatofocusing. Immediately before loading the sample onto the 10ml polyexchanger 94 column (0.7 cm i.d. x 25cm) it was equilibrated against histidine-HCl buffer, pH 6.2. The 5.9 ml sample was then applied to the column, previously equilibrated with the histidine-HCl buffer. The column was then washed with three column volumes of the same buffer and eluted with polybuffer 74-HC1, pH 4.9 at a flow rate of 10-12 ml/hr. The 1.4 ml fractions were collected into 100 #1 of 0.5 M dibasic phosphate to minimize the exposure of the enzyme to acidic conditions. Fractions containing over 16 nmol/min per ml of JHE activity were pooled (19.5 ml), and concentrated by ultrafiltration to 1.8 ml in histidine-HC1 buffer, pH 7.0. Step 4. Rechromatofocusing. The JHE sample from the previous step was equilibrated with 25 mM histidine-HCl buffer, pH 6.1 (Amicon system) and loaded onto a 7 ml chromatofocusing column (0.7cm i.d. x l 7 c m ) . After washing the column with 5 ml of the above buffer, 1 ml fractions were eluted at 6 ml/hr as above except that the amount of 0.5 M dibasic phosphate in the tubes was 75/~1. Pooled JHE activity (13.8 ml) was concentrated and equilibrated with Tris-HCl buffer, pH 7.2. Step 5. Semipreparative native gradient electrophoresis. JH esterase obtained following rechromatofocusing was applied to 4-15% gradient PAGE slab gels (1.5 mm). The vertical gels were run on a Hoefer electrophoresis unit. The 3.75% acrylamide stacking gels, cast in 62 mM Tris-HC1 (pH 6.7), were layered onto gradient resolving gels cast in 375 mM Tris-H2SO4, pH 8.9. Finally, an 8% acrylamide gel section in stacking buffer was cast for comb insertion. Electrophoresis was run in an 8°C chamber at 200 V constant voltage for 8 hr and then 250 V for approx. 9 hr, by which time the myoglobin marker was 3-4 cm from the bottom of the gel. One 0.5 cm lane was cut longitudinally along the

edge and then stained with Coomassie Blue and the rest of the gel was sliced laterally into 0.25 cm sections for elution in 3ml of 10mM phosphate buffer at 4°C for 6hr. The eluting buffer was removed, assayed for JHE activity and 3 ml of fresh buffer added for 3 hr more of elution. The first eluates containing activity were coneentated to 200/~1, pooled with the same fractions obtained from the second elution and concentrated again to 100/~1. This material was then washed three times with 1 ml of phosphate buffer using the Centricon System 10 (Amicon).

Analytical methods SDS-PAGE electrophoresis. Electrophoresis under denaturing conditions in acrylamide was used for first or second dimension (2-D) slab gels. In all cases the 10% resolving gel in 375 mM Tris-HCl, pH 8.8, and the 3.75% stacking gel in 125mM Tris-HC1, pH6.8 were run as described by the manufacturer of the electrophoresis unit (Hoefer). In 2-D gels, the resolving gel was cast to 1 cm from the top of the casting plates, and the stacking gel layered on top of it. Then, an isoelectric focusing tube gel was incubated with equilibrating buffer (Pharmacia), placed on top of the stacking gel and covered with 1% agarose. The second dimensional separation carried out at 200 V until the dye front reached the resolving gel and then 100 V for 5-6 hr. The voltage was increased to 200 V for the final 30-60 min, upon which time the dye front reached the bottom of the gel. The proteins were then visualized by staining with Coomassie Blue. Isoelectric focusing (IEF) in tube gels. Narrow range 4-6.5 Pharmalyte was used to cast 6% acrylamide gels in tubes (Winter et al., 1977). The gels (six tubes) were prefocused for 30-90min at 300V in 25mM histidine (cathodic solution) and 20 mM glutamic acid-10 mM phosphoric acid (anodic solution). After application of the samples, the cathodic solution was replenished and voltage changed as follows: 50 V/tube for 30 min, 75 V/tube for 30min, ll0V/tube for 7-8 hr and 150V/tube for the final hour. The gels were removed and then either stained for protein or 1-naphthyl acetate esterase activity, or applied to second dimension SDS--PAGE gels, or assayed for JHE activity. In order to localize this enzyme activity the IEF gels were cut into 1/3 cm, sections and the JHE activity eluted into 10mM phosphate buffer, pH 7.0. Visualization of 1-naphthyl acetate ( I-NA ) esterase. Tube gels from IEF were rinsed with 10 mM phosphate buffer, pH 6.9 and then incubated for 15-20min in the dark in substrate solution (ethanolic stock solution of 50 mM 1-NA diluted 10-fold with the above buffer) at room temperature. Then, the gels were rinsed with buffer and transferred into 0.2% Fast Blue RR in cold buffer. The color would develop within 5-15 min, after which the gels were fixed in isopropanol-acetic acid-water (25 : 10: 65). Gel filtration chromatography. JH esterase samples obtained before and after gradient PAGE were subjected to gel filtration chromatography on Sephacryl S-300. The column (1 cm i.d. × 82cm) was equilibrated with 25mM Tris-HCl buffer, pH 7.6, containing 100 mM NaCI. Chromatography was done with the same buffer at a flow rate of 8 ml/hr. The 1 ml fractions were collected and assayed for JH esterase activity. The column was previously calibrated with the following molecular weight markers:. aldolase (160,000), BSA (68,000), ovalbumin (45,000) and carbonic anhydrase (29,000). The protein profiles of these markers were monitored at 280 nm. The apparent molecular weight of JH esterase was calculated from the standard curve on the basis of its Rf. RESULTS

Enzyme purification PEG precipitation. In the example of purification given in this paper, removal o f proteins precipitating

375

JH esterase from larvae of T. ni Table 1. Purificationof the JHE from the haemolymph of final larval instar T. ni Total Specific Percentage Volume Protein activity activity original Step

(ml)

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Haemolymph 94 6617* 3591 0.54 100 -PEG precipitation9-26% 34 95.2 2495 26.0 70 48 Hydroxylapatite 124 23.2 1339 57.7 37 107 Chromatofocusing 19.5 5.42 1218 225 34 416 Reehromatofocusing 13.8 2.65 1148 432 32 800 Gradient eleetrophoresis 0.16 0.073* 156 2130 4t 3944 *Determinedby BioRadprocedure. Remainderdeterminedby measurementat 280 nm, assumingone absorbanceunit equals I mg/ml. BSA was used as the standard for both methods of determination. tThe enzymaticactivityis verylabileat this point, and in our experiencethe activitycan drop severalfold in the time this samplewas held prior to final JHE assay.

at 8.5% PEG and those not precipitating at 26% PEG resulted in a 48-fold purification of JHE, with 70% recovery (Table l). The composition of the protein bands in the 8.5-26% PEG fraction after gradient PAGE is shown in Fig. 3a. Hydroxylapatite chromatography. The second step allowed good separation of the peak of eluting JHE activity from most of the other proteins present (Fig. 1), and if total recovery of all JHE activity were not critical, taking only the JHE fractions on the left side of the peak would result in considerable additional purification. However, because we desired to retain as much JHE as possible for the next step, all fractions containi:-g JHE activity were retained. Consequently, the fold purification achieved during the second step ( x 2.2) was not as high as could have been achieved at this point. The change in protein content of the approx. 100-fold purified material at this step is shown by analytical gradient PAGE in Fig. 3b. Chromatofocusing and rechromatofocusing. JH esterase from the HA column was then purified another 4-fold by the use of chromatofocusing. The yield on this step represented near quantitative recovery of the enzyme. As seen in Fig. 2, the peak of JHE activity was on the leading edge on the peak of overall proteins eluted. At this stage of purification, analytical gradient PAGE showed elimination of a number of proteins and enrichment of several, including one comigrating with JHE activity (Fig. 3c). However, most of the staining material belonged to several proteins moving much faster on gradient electro-

phoresis than JHE. Because the relative placement of the JHE activity peak and the protein peaks varied slightly from preparation to preparation, the sample was rechromatofocused to improve the purification factor. This approach yielded a preparation of JHE now 800-fold purified, with near total recovery of the rechromatofocused JHE. Gradient electrophoresis purification. In the final step of purification, the 800-fold purified JHE was subjected to native gradient electrophoresis. The gels were run for an extended time to allow clearer separation of the bands. As shown in Fig. 4, the preparative gel discretely resolved a number of bands, including one comigrating with JHE activity. This band of JHE activity was eluted from the preparative gel and subjected to analytical procedures to test purity, molecular weight and charge. Molecular weight determination. Analysis on S D S - P A G E (Fig. 6) showed a single band with a mol. wt of 66,000, near the BSA standard (68,000). This molecular weight estimate is similar to that obtained by means of gel filtration chromatography using Sephacryl S-300 (not shown). Purity of the JH esterase. The high specific activity of the purified sample (2.13/~mol JH II hydrolyzed/min per mg protein) reflected the approx. 4000-fold purification achieved (Table 1). In our experience, the JHE activity following the electrophoretic separation declines rapidly. Thus, the sample must be assayed very quickly or else the specific activity will not be as high as it should be for the

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amount of JHE present. This situation causes the estimates of yield and fold purification to be underestimated. The time lag experienced by the example preparation discussed above prior to the JHE assay was one that would cause a several fold loss in detectable JHE activity. For this reason, we feel that the yield may actually be greater than 4% and the fold purification is even higher than x 4000. Isoelectric focusing in tube gels separated two bands of protein, vizualized after Coomassie Blue staining, with pI values of 5.5 and 5.3 (Fig. 5a). When a tube gel run in parallel at the same time as the one used for protein staining was instead stained for 1-NA esterase activity, it showed two clearly separated bands (Fig. 5b). The two bands of protein and the two bands of I-NAE activity exactly coincided, and the most strongly staining band in each was the more basic band (pI = 5.5). When another tube gel run in parallel was sliced, eluted and assayed for JHE activity, a strong major peak was observed which coincided wi{h the most intensely staining protein and I-NA esterase band (Fig. 5c). A second, minor peak (approx. 5% of total JHE activity) was detected which coincided with the more weakly staining, acidic protein and 1-NA esterase bands with a pI of 5.3. When IEF tube gels of purified JHE were applied to S D S - P A G E gels as a second dimension, the two protein bands on the tube gels maintained their integrity and both migrated to the same point, corresponding to a molecular weight of approx. 66,000 (Fig. 6). When the purified material was analyzed by native PAGE in tube gels, and stained with Coomassie Blue, a single protein band was observed (not shown). Kinetic analysis o f purified JHE. The kinetic interaction of the JHE activity in diluted haemolymph and of JHE after the purification procedure with JH II was examined. A Lineweaver-Burk plot for the activity in diluted haemolymph (Fig. 7a) showed a major inflection point, with substrate concentrations below 10 -7 M indicating a corrected K m of 3.4 x 10 -8 M (Vm~=22.3umol JH II hydrolyzed/min per ml haemolymph), while concentrations above 10-7M

indicated a corrected Km of 2.5 x 10 -7 M (Vm~ = 30.6nmol JH II/min per ml haemolymph). These data suggest the presence of two forms of JHE active sites in whole haemolymph. When haemolymph JHE was subjected to purification prior to kinetic analysis only a single apparent Km (average of 3.8 x 10 -7 M) was found at both the high and low substrate concentration regions (Fig. 7b). These results are consistent with recent data that, following partial purification by isoelectric focusing, the Km in the 10 -8 M region is lost (Jones et al., 1986b). DISCUSSION

The data presented here demonstrate the purification of JHE from T. ni to levels not previously obtained, permitting characterization of several kinetic and molecular aspects of the enzyme. The results reported here also have implications for a number of aspects of the current model for T. ni JHE. Purification procedure In recent years a number of attempts have been made to purify JHE from T. ni (Rudnicka and Hammock, 1981; Yuhas et al., 1983), Manduca sexta (Coudron et al., 1981; Abdel-aal and Hammock, 1985) and Galleria mellonella (Rudnicka and Kochman, 1984). Although more research has been published on T. ni JHE, in general the purification factors achieved ( x 175 and x 436) were much less than that for the other three insects ( x 800, × 1700 and x 2630). The procedure used here has achieved at least x 4000 purification of T. ni JHE. The procedure differs from the previous approaches in its combined use of isoelectric point (chromatofocusing) and native electrophoretic mobility as purification steps. The fold purification obtained was higher than the reported x 2630 purification of JHE from G. mellonella (Rudnicka and Kochman, 1984). The higher recovery in other studies on T. ni and M. sexta may be due to the lower fold purification achieved (Rudnicka and Hammock, 1981; Yuhas et

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Fig. 4. Protein separation and JHE activity following 4-15% gradient polyacrylamide electrophoresis. Above, Coomassie Blue stain of separation of the proteins obtained after the fourth step of purification. The protein bands shown were the only staining material on the gel. Below, the profile of eluted activity from 0.25 cm sliced fractions of the gradient gel. The activity of JHE is expressed as nmol JH II hydrolyzed/min per ml of fraction eluate. For the analytical step, we verified that JHE activity was associated only with the region of the gel stained for protein. For the purification step, JHE was eluted from the part of the gel containing JHE activity shown in the figure.

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Fig. 5. Analytical isoelectric focusing of purified JHE after the fifth step of purification. (a) Staining of gel with Coomassie Blue for protein. (b) Staining of gel for l-naphthyl acetate esterase activity. Three micrograms of protein were applied to each tube. (c) Profile of JHE activity expressed as nmol of JH II hydrolyzed/min per ml of gel fraction eluted from a tube gel run in parallel to those in (a) and (b). The two peaks of JHE activity coincide exactly with the two protein bands staining with Coomassie Blue and with the two I-NA esterase bands.

Fig. 6. (a) Analytical SDS polyacrylamide (10%) gel electrophoresis of the JHE sample after the fifth step of purification. (b) Two dimensional analysis of purified JHE. The pH 4-6.5 IEF tube gel was performed as a first dimension separation, and was then transferred to a 10% polyacrylamide SDS gel for the second dimension separation. Molecular weight markers for SDS-PAGE were fl-galactosidase (116,000), phosphorylase (97,400), BSA (68,000), ovalbumin (45,000) and carbonic anhydrase (29,000). 378

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Fig. 7. Kinetic analysis for (a) JHE activity in diluted haemolymph and (b) purified JHE, at substrate concentrations ranging from 8 x 10 -9 to 9 x 10 -6. Velocity in (a) is expressed as nmol JH II hydrolyzed/min per ml haemolymph, while that in (b) is expressed as JH II hydrolyzed/min per mg of purified JHE concentrate. The percent hydrolysis was minimized by appropriate dilution, and when greater than 5% it was corrected for by the excellent procedure described by Segel (1975).

al., 1983; Abdel-aal and Hammock, 1985) or assumptions of full recovery after the first step (Coudron et al., 1981). For situations where purity is of highest

importance, the approach used here will be useful in studies on T. ni JHE and perhaps for other insects. Multiplicity o f J H E activities

When conducting studies on the nature of an enzyme active site, or development of inhibitors for affinity columns or other purposes, it is very important to know the nature of the JHE activity being studied. Because previous isoelectric focusing of T. ni haemolymph proteins resolved, or were interpreted to resolve, only a single peak of JHE activity, kinetic and inhibitor studies have been conducted on the conclusion that a single JHE is present in crude or partially purified samples (Hammock et aL, 1981, 1984; Sparks and Rose, 1983). Recently, Jones et al. (1986b) found that careful application of isoelectric focusing methodology clearly resolved two peaks of JHE activity in T. ni haemolymph (pI 5.3 and 5.5).

It is thus very important to know whether or not these two activities represent charge modifications of the same protein, isozymes or two non-homologous proteins hydrolyzing the same substrate. The present study on purified JHE, which was electrophoretically homogeneous, also found two peaks of JHE activity on IEF gels, at pI 5.3 and 5.5. Although there are some nonhomologous enzymes possessing similar enzymatic activities which pass through rigorous purification steps together, it is more common that slight charge heterogenity on otherwise biochemically similar protein occurs. We favor the second interpretation for T. ni JHE. It is still not known whether the charge differences impart important roles in activity in vivo. Kinetics o f J H E

There has been confusion over the proper value of kinetic constants of T. ni JHE toward its JH substrates. Some researchers have measured the apparent Km to be in the 10 -7 M range (Sparks and Rose, 1983; Wing et al., 1984; Yuhas et al., 1983),

380

MARIA RUDNICKA and DAVY JONES

while others have estimated the K m to be in the 10-SM range (Abdel-aal and Hammock, 1985). However, it has been shown that crude preparations of JHE from either normal or pseudoparasitized T. ni actually possess JHE activity with both K m values (Jones et al., 1986). This result has been confirmed by more extensive analysis of JHE from normal larvae (Jones et al., 1986a) and by the present study. Jones et al. (1986b) proposed that conditions or factors in the haemolymph which favor the existence of two K m values are removed in purified or semipurifled preparations. The data obtained here are consistent with that hypothesis. The diluted haemolymph used in the example purification possessed two apparent Km values, 6.5 x 10 -s and 1.4 x 10 -7 M. However, the purified material possessed only a K m of 3.8 × 10 -7 M, which is not significantly different from 1.4 x 10 -7 M (P > 0.05). This result also implies that, at least in purified preparations assayed in vitro, the existing charge heterogeneity does not result in two clear, discrete K m values. It has been shown that the JHE of Drosophila hydei occurs in large aggregates (Bisser and Emmerich, 1981), and it is well known in enzymology that, alternatively, cofactors can be lost during purification of the protein. These possibilities should be investigated with respect to T. ni JH esterase. The haemolymph JHE activity of a number of Lepidoptera possess two or more electrophoretic forms of JHE (Roe et al., 1983; Wing et al., 1984). However, those authors did not analyze their estimate of K m towards JH I and JH III statistically, and Lineweaver-Burk plots derived from crude haemolymph showed no inflection point.

Activity towards other substrates

There has been and still is much controversy over definition of "general esterase" and "JH specific esterase," and the activity of JHE toward 1-NA ("general substrate"; Sanburg et al., 1975; Hammock, 1985; Hammock et al., 1984). Coudron et al. (1981) did not detect significant activity towards I-NA by their JHE preparation from M. sexta. Partially purified JHE from T. ni possessed activity towards I-NA (Yuhas et al., 1983). In the present study, homogeneous JHE possessed clear activity towards 1-NA (Fig. 5). Although it has been shown that JHE contributes little to the total 1-NAE activity in T. ni haemolymph (Sparks and Hammock, 1979), there remains the question of whether or not putative but unidentified alternative substrates in T. ni haemolymph competatively disrupt the in vivo interaction of JHE and JH. With the availability of purified JHE the interaction of JHE and potential alternative substrates can be addressed. Molecular weight o f T. ni J H E

The molecular weight obtained in this study for T. ni JHE was near 66,000, according to two indepen-

dent methods of estimation. This estimate is very close to that estimated for M. sexta and G. mellonella (Sanburg et al., 1975; Coudron et al., 1981; McCaleb et al., 1980). However, these values are higher than the 52,000 and 48,000 estimates obtained previously

for T. ni (Sparks and Hammock, 1979; Yuhas et al., 1983, respectively). Model o f T. ni J H E

Early concepts on JHE in Lepidoptera were developed from the pioneering studies of Whitmore et al. (1972), Sanburg et al. (1975), Weirich et al. 0973), Vince and Gilbert (1977) and others. On the basis of these studies on sphingid and saturiniid species researchers developed a useful early model (Gilbert et al., 1980; Kramer and Law, 1980) of JHE activity which stated that JHE (l) appears twice in last instar larvae but does not appear in the preultimate instar, (2) originates from the fat body, (3) appears in pupae treated with exogenous JH, (4) is apparently distinct from alpha-naphthyl acetate esterase activity, and (5) kinetically apparently behaves as a single enzyme. Subsequent researchers addressed important but unresolved questions such as: (1) Can it actually be demonstrated that JHE is necessary for larval metamophosis? (2) Is JHE a single protein? (3) Is the oceurence of two last instar peaks in activity a valid model for other Lepidoptera? (4) Does endogenous JH regulate JHE? (5) Is neurohormonal regulation of JHE involved? (6) What are the levels and molecular mechanisms by which JHE is regulated? (7) What are the biochemical properties of homogenous JHE? During the last decade, considerable new and original information has been developed, primarily from T. hi, which provide answers to the above and other pertinent questions. The results of these and the present study permit the lepidopteran model on JHE to be refined. The JHE activity in T. ni haemolymph is primarily due to two electrophoretic forms, each having a mol. wt of 66,000 (Fig. 5). Two other minor forms (pI = 5.6, 5.1) are present (Jones et al., 1986a, 1986d). In vivo conditions are such that JHE activities with two Km values are present, one in the 10-SM range and the other in the 10 -7 M range (Fig. 7; Jones et al., 1986b). Thus, the JHE activity is electrophoretically not a single protein and functions kinetically in vivo as multiple active sites. The JHE of M. sexta has also been shown to consist of multiple electrophoretic forms (unpublished data) and functionally multiple active sites (Abdel-aal and Hammock, 1985). The combination of K m and Vmaxvalues obtained in the present study suggest that both forms of active sites meaningfully contribute to haemolymph JHE activity, and therefore both should be considered as parameters of an in vivo model of JH action and metabolism. Although the Nihjout and Williams (1974) report on JH blockage of PTTH release led to speculation on an in vivo role of JHE in larval development and metamorphosis, such notions remained speculation in the absence of an original demonstration. Using an inhibitor with appropriate controls, it was shown that an extra larval molt will occur in the absence of functional JHE and that this is a non-pharmacological effect (Jones, 1985b). It has also recently been shown that this accumulation of endogenous JH will postpone PTTH or ecdysone production, extending the feeding stage (Jones, 1985b; Sparks and Hammock, 1980; Sparks et al., 1984). Juvenile hormone esterase with the isoelectric points and Km values of the final instar activity has also been recently reported from each

381

JH esterase from larvae of T. ni

larval instar (Jones and Click, 1987), in contrast to the initial model exposed by researchers in the field that JHE was an enzyme found only at and after larval metamorphosis. The occurrence of this preultimate instar activity in other Lepidoptera (Jones and Click, 1987) suggests it as a new aspect of the lepidopteran JHE model, and the occurrence of two JHE peaks during the final instar has also been validated as generally applicable to Lepidoptera (Jones et al., 1982). Results of topical applications of JH suggested the possibility of JH in regulation of JHE (Whitmore et al., 1972; Sparks and Hammock, 1979), and the necessary confirmation that endogenous JH induces JHE activity has recently appeared for both T. ni (Jones and Hammock, 1983) and M. sexta (Sparks et aL, 1984). A modification of the JHE model involves the demonstration of neurohormonal regulation of JHE activity (Jones et al., 1981b; McCaleb and Kumaran, 1980). The data in the present paper clarify whether or not the JHE model should distinguish JHE activity from alpha-naphthyl acetate esterase activity. JHE contributes little to total alpha-naphthyl acetate esterase activity, but JHE is not so substrate specific that such alternative ester substrates are not accepted and hydrolyzed by JHE active site(s). This finding has important implications for whether or not JHE is acylation rate-limited in vivo. Recent bioassay data place the haemolymph concentration of material with JH bioactivity as high as 10-SM JH II equivalents (Jones et al., 1986c). If this material which interacts with JH receptors in the bioassay also interacts with the JHE active site(s) (which this study shows will interact with non-JH substrates), then the in vivo concentration of material competing for the active site(s) is in the range of the lower K m of the haemolymph JHE activity. On the basis of recent estimates of JHE kinetic constants and the physicochemical measurement of the haemolymph JH concentration at 10 -9 M, it has been postulated that JHE in vivo functionally does not distinguish between JH homologs as substrate (Abdel-aal and Hammock, 1985). However, this postulate is valid only if the concentration of potential JH substrate is less than the region in which mixed order kinetics occurs (i.e. when the concentration less than range of the Km). Thus, attention must be given to the influence of deacylation-rate limited conditions on the ability of JHE to inactivate material which is biologically active at JH target sites. Clearly, our understanding of JHE has increased greatly during the past decade. Clearly, much work remains to be done in studying JHE regulation and in its characterization. With the availability of JHE purification techniques for antibody production (Jones et al., 1986a-d) and gene cloning, and with the recent refinement of in vitro techniques for study of JH and neurohormonal regulation of JHE (Click et aL, 1985, 1986), many of the remaining questions can be more easily addressed.

Acknowledgements--We wish to thank Dr Grace Jones for her assistance in this project and for reading the manuscript. This study was done in connection with a project of the Kentucky Agricultural Experiment Station (85-7-230) and was funded, in part, by NIH Grant GM 33995.

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