Isolation and characterization of hemolymph phenoloxidase from Heliothis virescens larvae

Comp. Biochem. PhysioL Vol. 102B, No. 4, pp. 891-896, 1992

0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd

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ISOLATION A N D CHARACTERIZATION OF HEMOLYMPH PHENOLOXIDASE FROM HELIOTHIS VIRESCENS LARVAE TIMOTHYD. LOCKEY and DONALDD. OURTH* Department of Biology, Memphis State University, Memphis, TN 38152, U.S.A. (Received 27 November 1991)

Abstract--1. Phenoloxidase was isolated from hemolymph of Heliothis virescens larvae and had a molecular weight of approximately 250 kDa. 2. The optimal pH of phenoloxidase activity was 9.0. 3. The optimal temperature for phenoloxidase activity was 45°C. 4. The enzyme kinetics of phenoloxidase had a Kra of 2.25 and Vm~xof 0.235 A A/min/mg. 5. The enzymatic activity of phenoloxidase was not affected by calcium or EGTA, but the activity was inhibited by both EDTA and SDS.

lymph PO. The kinetics of the enzymatic activity of PO have been determined for both the cuticle and hemolymph forms of PO and differ, even though both were isolated from M. sexta (Aso et aL, 1984; Aso et al., 1985; Morgan et al., 1990). The activation of proPO to PO requires different activators depending on the insect system being studied. The activation in most systems needs a protein activator and calcium to convert the inactive proPO to the active PO as in the crayfish (Ashida and Srderh/ill, 1984). Calmodulin, a protein activator of many enzymes that require the presence of calcium, has been detected in the fat body of Heliothis virescens (Lockey and Ourth, 1989). It also has been found that calmodulin is not involved in the enzymatic activity of phenoloxidase (Lockey and Ourth, 1992). Other conditions found to activate proPO include sodium dodecyl sulfate (SDS), the absence of calcium, lipopolysaccharide, trypsin and heat. Not only can these factors affect proPO activation but they can also affect the activity of PO. Phenoloxidase activity is affected by many different chemicals and physical conditions. In some PO systems, SDS is an activator of PO but in other systems the presence of SDS causes no increase in PO activity (Raghavan and Nadkarni, 1977). Differences in the characterization of PO can occur even within the same species. The stability of the enzyme in the presence of sodium and phosphate ions was determined for Lymantria dispar and Galleria mellonella (Dunphy, 1991). He found that G. mellonella was more sensitive to ion concentration than L. dispar. The stability of PO at a particular pH and the optimal pH for PO activity were different for each PO isolated from these two species. The characterization of PO was determined here for H. virescens using larval bemolymph. The molecular weight of PO was determined using high-pressure liquid chromatography (HPLC) gel filtration and SDS-PAGE. The optimal pH, optimal temperature and enzyme kinetics at which PO is active were determined. Along with the physical characteristics,

INTRODUCTION

In insects and invertebrates, the mechanism of nonself recognition may be controlled by the phenoloxidase (PO) pathway. The enzyme is involved in the encapsulation and melanization of infecting bacteria and fungi and sclerotization of the cuticle (Sfderh/ill and Smith, 1986). The foreign microorganism activates the phenoloxidase system causing deposition of melanin on the surface of the microorganism (Srderh/ill, 1982). Along with the deposition of melanin, the hemocytes enclose the microorganism by forming a layer of cells around the microorganism (Rather and Vinson, 1983). The exact mechanism by which the microorganism activates the PO system has not been determined but is thought to involve the lipopolysaccharide of the bacterial cell wall and the fl-l,3glucans of the fungal cell wall (Sfderh~ll and Smith, 1986). Phenoloxidase, in both the inactive prophenoloxidase (proPO) or active PO form can be demonstrated in hemolymph or hemocytes or both depending on the invertebrate species (Ratcliffe et al., 1984; Saul et al., 1987; Srderh/ill and Smith, 1983). The characterization of PO has been determined for various insects and invertebrates. The molecular weight of PO varies greatly with the insect from which the enzyme is isolated (Ashida, 1971; Gillespie et al., 1991; Morgan et al., 1990). Difficulty in ascertaining the molecular weight of PO pertains to whether it is found in an aggregated form that is resistant to chemical separation or if the enzyme is found in a native form composed of one subunit (Andersson et al., 1989; Gillespie et al., 1991). There is a difference in the physical and chemical characteristics of the cuticle and hemolymph forms of PO even within the same insect as found in Manduca sexta (Aso et al., 1985; Morgan et al., 1990; Thomas et al., 1989). The molecular weight of the cuticle PO tends to be of a higher molecular weight than the hemo*To whom correspondence should be addressed. 891

892

TIMOTHY D. LOCKEY and DONALD D. OURTH

chemical characteristics such as activators a n d inhibitors o f P O were also studied. D e t e r m i n i n g the characteristics o f P O will provide a better understanding of the optimal conditions needed by P O for m a x i m u m activity in H. virescens. These initial findings represent the first characterization o f P O in a Heliothis species. MATERIALS AND METHODS

Heliothis virescens eggs were obtained from the Bioenvironmental Insect Control Laboratory, U.S. Department of Agriculture, Stoneville, MS. The larvae were reared on artificial media in individual plastic cups. Each cup contained 10 ml of diet media (Ourth, 1988; Raulston and King, 1984). The cups were held at 24-25°C in a 12:12 (L: D) hour photoperiod. The larvae were 15 days old (5th instar) at the time of bleeding. The larvae were chilled for 5 min before bleeding. Hemolymph was collected by cutting a proleg and collecting the hemolymph into capillary tubes under ice-chilling conditions. The hemolymph from 106 larvae was pooled and centrifuged to remove cellular debris and then stored at -80°C. Phenoloxidase activity was measured using a microassay procedure. The assay was preformed in microtiter plates at room temperature. To each well, 50/21 of normal hemolymph were added to 100/21 of 0.01 M cacodylate buffer, pH 7.0. Then 50/21 of the substrate L-dopa (4 g/l; 25.4 mM) were added at timed intervals. Mushroom tyrosinase (1 mg/ I0 ml saline) (Sigma, St Louis, MO) was also assayed for PO activity and served as a positive control for the PO assay. The absorbance at 490 nm was determined after a 10 min incubation at room temperature. Polyacrylamide gel electrophoresis was carried out using a discontinuous system (Laemmli, 1970). A 5-20% acrylamide gradient resolving gel, pH 8.8 and 0.1% SDS with a 3% stacking gel of pH 6.8 and 0.1% SDS was used. The tank buffer was a Tris-glycine buffer, pH 8.3 with a final concentration of 0.1% SDS. Electrophoresis was performed at a constant voltage of 50 V for 18 hr. The gels were stained overnight in Coomassie Blue R250 and destained in a ratio of methanol: acetic acid: water (30:7.5:62.5). Samples of normal hemolymph (5/21) or 100/21 of 1 ml fractions from HPLC gel filtration of hemolymph were treated in sample buffer with 0.1% SDS and no 2-ME (non-reducing) or with 0.1% SDS and 2-ME (reducing). The reducing samples were boiled for 3 min and then allowed to cool before applying them to the SDS-PAGE gel. The isolation and molecular weight determination of PO was done by HPLC (ISCO, Lincoln, NE) using a Zorbax GF-250 (Dupont, Wilmington, DE) gel filtration column. The column was eluted with 0.2 M Na2HPO 4, pH 7.0 and had a flow rate of 1 ml/min. The fractions collected were assayed for PO activity by the method described above. The molecular weight of the peak fraction containing the PO activity was determined by comparing its HPLC retention time with Bio-Rad gel filtration standards (Bio-Rad, Richmond, CA) of IgG, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa and apoferritin, 443 kDa (Sigma, St Louis, MO). The three fractions containing the PO activity were pooled and again applied to the same HPLC column to isolate the peak fraction containing PO activity. For Western blot analysis, the proteins from the SDSPAGE gel described above were transferred to nitrocellulose (Towbin et al., 1979). The nitrocellulose was incubated for 1 hr in 10% Carnation non-fat dry milk in distilled water to block nonspecific protein binding sites. The primary antibody (rabbit-antimushroom tyrosinase) was diluted in 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.5% Tween 20 and incubated overnight at room temperature. The secondary antibody, goat-antirabbit conjugated with horseradish peroxidase, was also diluted in 5% non-fat

dry milk in TBS with 0.5% Tween 20 and incubated for 6 hr at room temperature. The bands were visualized by using Bio-Rad HRP color development reagent. Molecular weight standards were stained in 0.1% Amido black. The pH ranges used for PO enzymatic activity were from pH 2.0 to pH 11.0. The buffer for pH 2.0 5.5 was 0.05 M sodium citrate buffer, from pH 6.0 to 8.0 was 0.1 M phosphate buffer and from pH 8.5 to 11.0 was 0.05 M glycine-NaOH buffer. Phenoloxidase activity in the various buffers was determined as follows. Fifty microliters of saline-diluted hemolymph (1:25) were added to 100 p l of the appropriate pH buffer and then 50 #1 of L-dopa (4 g/i) were added at timed intervals. Absorbance was read at 490 nM after a 10 min incubation. Due to autooxidation of L-dopa at an alkaline pH, the substrate blank was subtracted from the hemolymph absorbances. To determine the optimal temperature for PO activity, 50/tl of the diluted hemolymph sample (1:25) were added to 100/21 of the 0.1 M cacodylate buffer, pH 7.0. This mixture was equilibrated to a proper temperature: 5°C, 21°C, 37°C, 45°C and 60°C for 15min. Then 50/21 of L-dopa (4 g/I) were added, and the reaction mixture was again returned to the appropriate temperature. The absorbance was determined at 490 nm after a 10 min incubation at the desired temperature. The PO activity was determined at different concentrations of the L-dopa substrate. The L-dopa concentration ranged from 0.6, 1.27, 2.54, 5.07, 10.14, 15.2, 20.3 and 25.4 mM. To 50/21 of the diluted hemolymph (1:25), 100/21 of 0.1 M cacodylate buffer, pH 7.0 were added, and then 50/21 of the appropriate L-dopa (4 g/l) substrate concentration were added to this mixture. The absorbance at 490 nm was determined after a 10-min incubation. Data were analyzed using the Lineweaver-Burk method to determine the Km and Vmax of PO activity. The effect of calcium, SDS, ethylene glycol-bis (/~-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and EDTA on PO activity were studied by incubating these separately with hemolymph, followed by determination of the PO activity. The diluted hemolymph (1:16) was preincubated with 200mM CaC12 or 1% SDS or 10mM EGTA. After preincubation for 30 min, the amount of PO activity was determined with the PO assay. After collection of hemolymph in 10 mM EDTA, no PO activity was detected even after the addition of calcium.

RESULTS

The molecular weight of P O from n o r m a l H. virescens h e m o l y m p h was determined to be 2 5 0 k D a by H P L C gel filtration (Fig. 1). W h e n the isolated P O was subjected to S D S - P A G E u n d e r n o n - r e d u c i n g conditions, one b a n d was seen after staining (Fig. 2). W h e n 2 - M E reducing conditions were employed, multiple b a n d s were detected. Western blot analysis was done to detect which b a n d s represented P O using rabbit-antityrosinase serum. I m m u n o b l o t analysis detected one m a j o r b a n d with a molecular weight of 219.1 k D A a n d one very m i n o r b a n d having a molecular weight o f 172.7 k D a u n d e r n o n r e d u c i n g conditions (Fig. 2). Therefore, the PO molecule is likely a m o n o m e r h a v i n g a molecular weight o f 250 k D a by H P L C analysis (Fig. 1). The optimal p H of P O activity was determined to be p H 9.0 with a small P O activity peak at p H 6.0 (Fig. 3). By assaying for P O activity at different temperatures, the optimal t e m p e r a t u r e of P O was determined to be 45°C (Fig. 4). In Fig. 5, the Vm~x for P O was s h o w n to be 0.235 (A A / m i n / m g ) , and the K m was 2.25 m M .

Heliothis phenoloxidase

893

6.0"

~Apoferridn

(443

Xd)

5.5

NOLOXIDASE

(250 Kd)

.< 5.0

Ovalbumin(44Kd) [.~

4.5 Myoglobin(17Kd) 4.0 8

9

1'0

11

HPLC RETENTION TIME (MINUTES) Fig. 1. Molecular weight of phenoloxidase using HPLC gel filtration on Zorbax GF-250 column. The retention time of phenoloxidase was compared with the retention times of four Bio-Rad molecular weight standards.

Phenoloxidase can be affected by different chemical inhibitors or activators. When H. virescens PO was incubated with the different activators and inhibitors, it was determined that 200mM CaCI 2 and 10mM EGTA had no effect on PO activity, as the amount of PO activity present was not significantly different from the control hemolymph (Table 1). Ten mM of

EDTA caused inactivation of PO activity, since no PO activity could be detected. The PO activity could not be restored by the addition of excess calcium into the reaction mixtures (Table 1). Sodium dodecyl sulfate also causes inactivation of PO, but the inactivation of PO is not as pronounced as seen with EDTA (Table 1).

A I

B 2

1

2

MW

MW 200

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Kd

20O Kd

-.w 1 1 6 97

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Fig. 2. A. SDS-PAGE of hemolymph phenoloxidase, 5 #1 of whole hemolymph were applied to the gel. B. Western blot analysis of hemolymph and detection with rabbit-antityrosinase antibody. Lane I: unreduced hemolymph. Lane 2: 2-ME reduced hemolymph.

894

TIMOTHY D. LOCKEr and DONALD D. OURTH Table 1. Phenoloxidase activity of hemolymph in the presence of chemical activators and inhibitors

0.30'

Phenoloxidase activity*

0.25 -

Hemolymph treatments

> [.

0.20 •

,~

0.15"

Hemolymph + 1% SDS Hemolymph + 10 mM EDTA Hemolymph + 10 mM EDTA + 10 mM CaC12 Hemolymph + 200 mM CaCI2 Hemolymph + 10 mM EGTA Hemolymph + 10 mM EGTA + 1 mM CaCI2 Hemolymph Control Mushroom Tyrosinase Control

0,10i

0.05"

Z ~.

0.00 2

3

4

5

6

7

0

9

10

I1

12

*One PO enzyme unit represents the amount of enzyme required to produce an absorbance of 0.001 O.D. at 490 nm. Data are for 10 min. Values are means of four replications.

p H

Fig. 3. Phenoloxidase activity vs pH. The pH ranges used were from pH 2.0 to pH I 1.0. The buffer for pH 2.0-5.5 was citrate buffer, from pH 6.0 to 8.0 was phosphate buffer and from pH 8.5 to 11.0 was glycine-NaOH buffer. Absorbance was read at 490 nm after a 10 rain incubation. Error bars represent __+SD of 3 replications.

DISCUSSION

Phenoloxidase is an enzyme that may be involved in nonself recognition by insects. Phenoloxidase is found in arthropods, insects and plants (Ashida and Dohke, 1980). Even though PO is found in many organisms, it may still have different molecular weights, optimal pH, temperature sensitivity and different PO activators and inhibitors depending on the organism (Thomas et al., 1989). The molecular weight of the PO enzyme of H. virescens was 250 kDa by HPLC gel filtration (Fig. 1), and this value was confirmed using SDS-PAGE and Western blotting (Fig. 2). The molecular weight of H. virescens PO is therefore similar to grasshopper PO which has a molecular weight of 212 kDa (Gillespie et al., 1991) and to tyrosinase from Xenopus skin of 229 kDa (Wittenberg and Triplett, 1985). But these molecular weight values for PO are different from the activated proPO found in the housefly which is 340 kDa (Tsukamoto et al., 1986). In H. virescens larvae, we have determined that the optimal pH for PO is pH 9.0 (Fig. 3). This is a different pH value to that found for

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20

40

60

90

80

370 0 0 1490 1470 1410 1480 1350

TEMPERATURE (CELSIUS)

Fig. 4. Phenoloxidase activity vs temperature. The optimal temperature of phenoloxidase activity was expressed as the phenoloxidase activity found after a preincubation period at the indicated temperature. Error bars represent + SD of 3 replications.

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(mM)

Fig. 5. Phenoloxidase activity vs L-dopa concentration. The phenoloxidase activity was determined at different concentrations of L-dopa substrate. The absorbance was determined at 490 nm after a 10 min incubation. Error bars represent + SD of 3 replications.

895

Heliothis phenoloxidase

cuticle PO of Manduca sexta which has an optimal pH of 6.0 (Aso et al., 1984). In the housefly, the PO was stable at a pH of 9.0 (Tsukamoto et al., 1986). The optimal temperature for H. virescens PO activity was 45°C (Fig. 4). Phenoloxidase from the cuticle of M. sexta was thermostable at a temperature of 45°C and labile at 75°C (Aso et al., 1984). The PO activity at the different concentrations of L-dopa substrate showed that the Vmaxwas 0.235 (A A/min/mg protein) and the Km was 2.25 mM for H. virescens (Fig. 5). The K m of 2.25 mM is similar to that seen in soluble tyrosinase II from the M. sexta integument which is 2.2 mM (Morgan et al., 1990). This would suggest that PO from H. virescens hemolymph is enzymatically similar to tyrosinase from M. sexta, even though the molecular weights of the two enzymes differ (270 kDa and 700 kDa, respectively). The optimal pH was also higher for H. virescens hemolymph (pH 9.0) when compared with M. sexta integument (pH 6.0) (Morgan et al., 1990). The Km of the hemolymph tyrosinase from M. sexta is 4.5 mM (Aso et al., 1984) which is twice the Km (2.25 mM) found for H. virescens (Fig. 5). Different activators and inhibitors of PO were analyzed for their effects on PO activity (Table 1). Sodium dodecyl sulfate has been shown to be an activator of proPO (Wittenburg and Triplett, 1985), but in Corcyra cephalonica SDS has no effect on PO activity (Raghavan and Nadkarni, 1977). In H. virescens, SDS caused a significant decrease in PO activity when the enzyme was assayed in the presence of SDS (Table 1). Calcium has been reported to be needed for proPO activity (Ashida and Srderhfill, 1984). In H. virescens larvae, PO activity was not affected by the presence or absence of calcium (Table 1). In the cockroach, hemolymph PO is not inhibited by EDTA at concentrations of 10 -3 M (Fisher and Brady, 1983). However, in the gypsy moth and greater wax moth, EDTA caused a reduction in PO activity (Dunphy, 1991). This inactivation could be reversed by the addition of calcium (Dunphy, 1991). In H. virescens, EDTA caused an irreversible inactivation of PO which could not be reversed by the addition of an excess amount of calcium (Table 1). The inactivation of H. virescens PO by EDTA, a divalent ion chelator, was not seen when the calciumspecific chelator EGTA was used. This may indicate that EDTA binds to the copper-containing PO and thereby inactivates H. virescens PO. The presence of EGTA did not have any effect on H. virescens PO activity (Table 1). In this study, the physical and chemical characteristics of hemolymph PO from H. virescens were determined. Morgan (1990) has found that the PO enzymes can be different even within the same species as seen in M. sexta. Although there are similar characteristics to other insect soluble hemolymph and cuticle phenoloxidases, no other insect PO enzyme was identical to, or closely related to, the PO of H. virescens based on our data. All of the insect phenoloxidases seem to have the same enzymatic activity including the oxidation of monophenols and diphenols. Our conclusion, then, is that the PO enzymes isolated from the different insect species, including H. virescens, are not identical in their physical and chemical properties. We have characterCBPB 102/4--Q

ized the hemolymph PO in H. virescens larvae with regard to its optimal conditions for enzymatic activity. Enzymatic characterization of PO is important in that it provides a better understanding of this enzyme which is needed for melanization and sclerotization reactions. In addition, melanization may be important in the insect defense response. Phenoloxidase, and its involvement in these important enzymatic reactions, is therefore necessary for insect survival. Acknowledgements--We thank Memphis State University

for a graduate research assistantship to T.D.L. and to Michael Patterson for technical assistance.

REFERENCES

Andersson K., Sun S. C., Boman H. G. and Steiner H. (1989) Purification of the prophenoloxidase from Hyalophora cecropia and four proteins involved in its activation. Insect Biochem. 19, 629-637. Ashida M. (1971) Purification and characterization of prephenoloxidase from hemolymph of the silkworm Bombyx mori. Archs Biochem. Biophys. 144, 749-762. Ashida M. and Dohke K. (1980) Activation of prophenoloxidase by the activating enzyme of the silkworm, Bombyx mori. lnsect Biochem. I0, 37-47. Ashida M. and Srderh/ill K. (1984) The prophenoloxidase activating system in crayfish. Comp. Biochem. Physiol. 77B, 21-26. Aso Y., Kramer K. J., Hopkins T. L. and Whetzel S. Z. (1984) Properties of tyrosinase and DOPA quinone immine conversion factor from pharate pupal cuticle of Manduca sexta (L.). Insect Biochem. 14, 463-472. Aso Y., Kramer K. J., Hopkins T. L. and Lookhart G. L. (1985) Characterization of haemolymph protyrosinase and a cuticular activator from Manduca sexta (L.). Insect Biochem. 15, 9-17. Dunphy G. B. (199I) Phenoloxidase activity in the serum of two species of insects, the gypsy moth, Lymantria dispar (Lymantriidae) and the greater wax moth, Galleria mellonella (Pyralidae). Comp. Biochem. Physiol. 98B, 535-538. Fisher C. W. and Brady U. E. (1983) Activation, properties and collection of haemolymph phenoloxidase of the American cockroach, Periplaneta americana. Comp. Biochem. Physiol. 75C, 111-114. Gillespie J. P., Bidochka M. J. and Khachatourians G. G. (1991) Separation and characterization of grasshopper hemolymph phenoloxidase by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Comp. Biochem. Physiol. 98C, 351-358. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lockey T. D. and Ourth D. D. (1989) Calmodulin activity in whole body and fat body tissue extracts of Heliothis virescens larvae. Biochem. biophys. Res. Commun. 158, 485-488. Lockey T. D. and Ourth D. D. (1992) Phenoloxidase activity independent of calmodulin and calcium in hemolymph of Heliothis virescens (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. (in press). Morgan T. D., Thomas B. R., Yonekura M., Czapla T. H., Kramer K. J. and Hopkins T. L. (1990) Soluble tyrosinases from pharate pupal integument of the tobacco hornworm, Manduca sexta (L.). Insect Biochem. 20, 251-260. Ourth D. D. (1988) Phenoloxidase activity, lack of bactericidal immunity, and oral susceptibility of tobacco budworm (Lepidoptera: Noctuidae) larvae to Serratia marcescens. J. Econ. Entomol. 81, 148-151.

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Raghavan K. G. and Nadkarni G. B. (1977) Control mechanisms of tanning in Corcyra cephalonica. J. Insect Physiol. 23, 765-771. Ratcliffe N. A., Leonard C. and Rawley A. F. (1984) Prophenoloxidase activation: nonself recognition and cell cooperation in insect immunity. Science 226, 557-559. Ratner S. and Vinson S. B. (1983) Encapsulation reactions in vitro by haemocytes of Heliothis virescens. J. Insect Physiol. 29, 855-863. Raulston J. R. and King E. G. (1984) Rearing the tobacco budworm, Heliothis virescens, and the corn earworm, Heliothis zea. In Advances and Challenges in Insect Rearing, pp. 167-175. Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA. Saul S. J., Bin L. and Sugumaran M. (1987) The majority of prophenoloxidase in the hemolymph of Manduca sexta is present in the plasma and not in the hemocytes. Devl comp. Immunol. 11, 479-485. Srderh/ill K. (1982) Prophenoloxidase activating system and melanization--a recognition mechanism of arthropods? a review. Devl comp. Immunol. 6, 601-611. Srderh/ill K. and Smith V. J. (1983) Separation of the haemocyte populations of Carcinus maenas and other

marine decapods, and prophenoloxidase distribution. Devl comp. lmmunol. 7, 229-239. Sfderh/ill K. and Smith V. J. (1986) Propbenoloxidaseactivating cascade as a recognition and defense system in arthropods. In Hemocytic and Humoral Immunity in Arthropods (Edited by Gupta A. P.), pp. 251-285. John Wiley and Sons, New York. Thomas B. R., Yonekura M., Morgan T. D., Czapla T. H., Hopkins T. L. and Kramer K. J. (1989) A trypsinsolubilized laccase from pharate pupal integument of the tobacco bornworm, Manduca sexta. Insect Biochem. 19, 611-622. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natn. Acad. Sci. U.S.A. 76, 4350-4354. Tsukamoto T., Ishiguro M. and Funatsu M. (1986) Isolation of latent phenoloxidase from prepupae of the housefly, Musca domestica. Insect Biochem. 16, 573-581. Wittenberg C. and Triplett E. L. (1985) A detergentactivated tyrosinase from Xenopus laevis I. Purification and partial characterization. J. biol. Chem. 260, 1253512541.