609 W 611. Pergamon Press. Printed in G~
VIELANIZATION IN HO] OF THE TOBACCO BU] TENS F. (LEPIDOPTERA
~.TES 9
JIDAE)
YES, A. JOHNSON AND M. S. SCHECI~
rol Laboratory, AEQI, Agricultural I Beltsviile, Md. 20705, U.S.A.
ice, USDA,
(Received 25 March 1974)
Abstract--1. Melanization is decreased or prevented in homog,genates of 1 worm, Heliothis virescens, by reagents that protect sulfhydryl groups, by phenylthiourea, by thiourea and by the cationic exchange resin, Dowex I" 50W 2. The results are consistent with the hypothesis that "brownintg enzymes' of sulfhydryl to disulfide. 3. Choice of the inhibitor to be used in vitro is dependent uupon the bi investigation.
INTRODUCTION
gRKENING of insect homogenates or hemodue to the activity of cresolases and related enzymes,'sis a problem for insect biochemists. This brown color is often associated with a change in the 3f pigments or in the activity of enzymes color of (Williamson, 1970; Hayes, 1972). Bakry (1966), El Azizz (1967) and Leesch (1970) investigated this n in some detail and attempted to relate the reaction ion of "browning" by the organic thiocynates inhibition to their activity as carbamate synergists. Also, Bakry (1966) examined the mechanism of several of the browning reactions. rk was to determine The purpose of the present work ;olase-type enzymes methods of inhibiting these cresolase-t, in crude homogenates during the preparation of other enzymes and the extractionn of of insect inse hasect pigments and to obtain insight into the te mechanism(s) of cresolase reactions. MATERIALS AND METHODS )DS ETHODS Eggs of the tobacco budworm, Heliothis virescens F., furnished by Southwestern Cotton Insects Investigations, Brownsville, Texas, or by Tobacco Insects Investigations, Oxford, North Carolina, were washed led in a 0-5~0 commercial hypochlorite solution and allowed owed to hatch. The first instar larvae were then placed on modified Vanderzant-Adkisson medium (1960) and nd the insects were ndle regimen of reared to the last instal" in a photoperiodi toperiodic either LD 16 : 8 or LD 10 : 14. The test test homogenate was trvae in the last instar prepared by homogenizing three larvae g total) in 5 ml of an (weighing between 600 and 900 nag 0"25 M sucrose solution through which water-pumped nitrogen gas had been bubbled for 15 min to remove air.
In the tests t¢ made !
obacco bud3 M urea, by by oxidation ~cesses under
he effects of various
classesof chemicals c o~ l, we added 0.1 ml of homogcnat ;enate to 0.1, 0-05 or 0-01 ml of an aqu( aeous solution testchemical. c of the test All materials were pipet petted or placed in the well wells of a porcelain spot plate and observed for browning for 4--28 hr. Control solutions consisted of plus 0.1 ml of 0.25 0.2~ M sucrose. 0-I ml of homogenate i Three such mixtures and the control were assayed a~, during a single work day. In In the the tests te made to determine the effect (of removal of cations Ire from the homogenate, we treated 5 ml of the homogenate with 12g of drained Dowex 50W-X8 (sodium form). fc The browning of the supe ~ernatant after the resin ,~in se settled was determined witha and without wi various additives. Also, in other similar tests made to determine the effects of the disruption of the tertiaDT structure of proteins on enzymes involved in melanization, we prepared 5 ml of homogenate in 8 M urea that was or ,genization by was not dcaerated 15 min before homog bubbling nitrogen gas through the solution. In the tests of the effect of a browning inhibitor on insect growth, we reared the insects (Ad Adkisson et aL, 1960) as before except that the medium contained cc 0.40, 0.50 or 1.0 g/l of phenylthiourea and made ma tests with hemolymph or with homogenates from sur~ surviving insects. RESULTS AND DISCUSSIOP ;ION Table 1 reports the effectiveness of ses several types of additives in inhibiting browning in the homogenate. 1 Mercaptoethanol, thioglycolic acid, ccysteine and dithiothreitol, all often used to prote( )rotect sulfhydryl This (mercapto-) groups, inhibited the reaction. lea indicates that if the sulfhydryl groups are a protected, lutathione was not the enzyme is not active. Glutathk effective in the concentrations tested, probably because of steric factors. If sulfhyclr3 ydryl goups are 609
HAYES, A. JOHNSONAND M. S. SCHECHT ewise inhibited activity also. ~"ineffectiveness mzoate. Moreagents such as
in homogenates ~orm
'1~ t~SLt~ld
)toethanol /colic acid hreitoH" te aione :tate romercuribenzoate ~alt) 'pus acetate 1 hydrosulfite ic acid ne um ferricyanide thiourea :a ddazole L/- J.l~llz~ 3doxyphenol l~. 3,4-Dioxyphenylalanine Melatonin
t effective tration*
t.:Ull~.t;lltl~tlOll"
0'05% 0.059/o 0.005% 0"00l~o Not effective at 0.15% Not effective at 0"5~o Not effective at 0'5% saturated 0"05~o 0.05%, 0.025~ 50% saturated 0"5% 5% saturated 0"0570§ Not effective at 0-57o Not effective at 0"5~o saturated Enhanced browning rate Not effective at 0"157oo
her 0"01, 0-05 or 0.1 ml of a solution of the test * Either materiald was added to 0.1 ml of homogenate in a spot When 0-01 or 0"05 ml was used, the volume of the plate. When xture was diluted to 0.2 ml with 0-25 M sucrose, test mixture •rc~l was wag prepared nrenared by hv adding addinp ng 0.1 ml of 0.2_5 0.25 M A control sucrose to 0.I ml of homogenate. I Added in a solution containin g 0•25 M sucrose and hiothreitol. reitol. 0.002 M CaCI~ in addition to the dithiothreit I: Bleached the sample. § Very slight browning noted after 24 hr. sodium hydrosulfite and ascorbicc acid and of nitrovning shows that, as gen degassing in preventing browning is well known, oxidation must occur to obtain melanization. The observations that protecting or blocking anization tion and that sulfhydryl groups inhibits melanizatioJ oxygen is required for browning; are consistent with sulfhydryl to disulthe hypothesis that oxidation of sulfh fide is required for activation of this enzyme system• ion of sulfhydryl to This suggestion, i,e. that oxidation nzyme activity, can disulfide is a prerequisite for enz reconcile the observation of Bayer (1954) who suggested that sulfhydryl groups are present in ertesz & Zito (1962) phenolases and the finding of Kertesz who reported that phenolase is not a sulfhydryl containing enzyme.
In our prepara ne (half saturated) prevented brownit ' by inhibiting the transfer of hydroge H during hydroxylation. The extreme 1 tenone to insects and fish and its lesser birds and mammals undoubtedly reflect y greater importance of aromatic hydro: eeer forms• Gutman et al. (1970) indica le sulfhydryl groups interact with ferric: finding that ferricyanide inhibits the br( e 1) provides further support for our c( it sulfhydryl groups m ust p c 1] must be free to reac ruing can occur• Of the materials ylthiourea was most effective in i inhibitk ~ation although thiourea was also acti Because benzimidazole and p-benz2 were inactive, we concluded that tll onded sulfur atom ~ined with an :leus were important combinec )revention of o e further concluded in preyer that aromatic chara arolr ts not effective• Homogenates lC )m the 24-day-old insects that tt had b n media containing 0•40 g of phenylthi tired more than 6 hr (overnigh to mela (overnight) genates from control insects turned tu brox • Hemolymph from free Lts which presumably control insects, i~ contained i while containec phenylthiourea, browned readily that from insects fed the mediurr :lium containing phenylthiourea remained unmelaniz melanized. Insects reared on the phenylthiourea medium pupated slowly--r ty--no pupation occurred in 19 d, days, and only 1 per cen cent in 24 days; among control insects i 19 per cent pup~)ated in 13 days and 54 per cer cent in 24 days• Also, weight gain was half that of the control• ,lthiourea at a concentration of 1 g/l. was toxic Phenylthi to the insects, presumably because it inhibited hydroxyk ,lations necessary for normal development. d( Sucrose-N 2 deaeration
r - - -
8M u r e a - n o t deaerated
- - ]
8M u r e a - N2 deaer~tion
7
IOO (I) 92 (2) ]
Relative t i m e "to brown
260 (2)
Fig. 1. Effect of 8 M urea and deaeration leration with nitrogen before homogenization on the browninlg reaction in tobacco budworm. crude homogenates of larvae of the toba¢ Melatonin, predictably, did not affect affec the rate of acc~ browning. This compound inhibits 9its accumulation of melanin in dogs (Rickards, 1965) an and aggregates pre-existing melanin in dermal mela melanophores of amphibians (Kaufman, 1962)• It therefore there acts on preformed pigments and not directly on pigment synthesis• Figure 1 indicates that melanization was delayed when homogenate was prepared in a solution
melanization in homogenates of Helioth~ that the urea
l
of sulfhydryl 3 the disulfides. mogenate was 50W-X8 in the e toward which
ADKISSONP. L., VA
ng of a homovae and treated
,e
Concentration Effect
~xyphenylalanine 0.1 saturated m alanine H 34 H 34 tylalanine
No browning Pink color in 4 min 45 rnin No browning No browning
0.1 saturated 0.5 saturated 0-25~ 0.05 mM No browning 0"25~o 0.05 rnM 0.1% 0.15 saturated No browning 0.5~ Bleached
virescens larvae in the last instar (1.8-2.7 g) were enized in 15 ml of 0.25 M sucrose that had been enated for 15 min with nitrogen.
the enzyme was most active. In our hands the addition m of Cu z+, N A D P H and phenylalanine as suggested ted by Kaufman (1962) or of tyrosine did not result in activation. We have apparently removed small cationic cofactors required for oxidation of phenyl~ [alanine and tyrosine. Such a treatment does, er, provide a practical means for prevention however of browning in in vitro studies.
ALLISONN. E. (l
S., BULL D. L. & tt germ medium for ,n. Ent. 53, 759-762. 3f action of organic 63-110. Ph.D. thesis,
rearing the pink be BARRYN. M. S. (1 thiocyanates as ins University of Califc e. )metric study of the B o ~ P. D. (1954) groups with organic reaction of protei mercurials. J. Am. 4331-4334. EL Azlz S. A. E. A. ~xidases and detoxieation of carbamate fi • 79-162. Ph.D. thesis, Universi of Califc University e. GtrrMAr4 M., MERSM IY J. • SINGER T. P. (1970) Action of hibitors on different forms oof the resp linked reduced nicotinamide-adenine tinamidi dehydrogenase. Biochemistr,y 9, 2678-2 HAYES D. K., WASH IECHTER M. S. (1972) Monoar Monoamine oxidas trvae of the European corn borer. bet J. econ -1232. of the phenylalanine KAUrMANS. (1962) C 7hem. 237, 2712-2713. hydroxylation cofa aolase. In Oxygenases KERTESZD. ]" & ZITO ] (Edited Edited 1by HAYAIS~ -354. Academic Press, New York. md biochemical basis LEESCHJ. G. (1970)" ® to mosquito larvae, for the sselective to~ Ph.D. thesis, University of c California, pp. 66--67. 66Riverside. RICKARDS D. A. (1965) The therapeutic effect e of melatonin on ol canine melanosis. J. Invest. Dermatol. 44, 13-16. Wlt~MSC WILLIAMSONR. L. & ScrmcrrrER M. S. (1970) (197C Microsomal epoxidal )oxidation of aldrin in lepidopterous larvae. lar~ Biochem. PharmacoL 19, 1719-1727. Key Word W~ Index--Melanization; Heliot~this virescens; rotenone; SH groups.
VIELANIZATION IN HO] OF THE TOBACCO BU] TENS F. (LEPIDOPTERA
~.TES 9
JIDAE)
YES, A. JOHNSON AND M. S. SCHECI~
rol Laboratory, AEQI, Agricultural I Beltsviile, Md. 20705, U.S.A.
ice, USDA,
(Received 25 March 1974)
Abstract--1. Melanization is decreased or prevented in homog,genates of 1 worm, Heliothis virescens, by reagents that protect sulfhydryl groups, by phenylthiourea, by thiourea and by the cationic exchange resin, Dowex I" 50W 2. The results are consistent with the hypothesis that "brownintg enzymes' of sulfhydryl to disulfide. 3. Choice of the inhibitor to be used in vitro is dependent uupon the bi investigation.
INTRODUCTION
gRKENING of insect homogenates or hemodue to the activity of cresolases and related enzymes,'sis a problem for insect biochemists. This brown color is often associated with a change in the 3f pigments or in the activity of enzymes color of (Williamson, 1970; Hayes, 1972). Bakry (1966), El Azizz (1967) and Leesch (1970) investigated this n in some detail and attempted to relate the reaction ion of "browning" by the organic thiocynates inhibition to their activity as carbamate synergists. Also, Bakry (1966) examined the mechanism of several of the browning reactions. rk was to determine The purpose of the present work ;olase-type enzymes methods of inhibiting these cresolase-t, in crude homogenates during the preparation of other enzymes and the extractionn of of insect inse hasect pigments and to obtain insight into the te mechanism(s) of cresolase reactions. MATERIALS AND METHODS )DS ETHODS Eggs of the tobacco budworm, Heliothis virescens F., furnished by Southwestern Cotton Insects Investigations, Brownsville, Texas, or by Tobacco Insects Investigations, Oxford, North Carolina, were washed led in a 0-5~0 commercial hypochlorite solution and allowed owed to hatch. The first instar larvae were then placed on modified Vanderzant-Adkisson medium (1960) and nd the insects were ndle regimen of reared to the last instal" in a photoperiodi toperiodic either LD 16 : 8 or LD 10 : 14. The test test homogenate was trvae in the last instar prepared by homogenizing three larvae g total) in 5 ml of an (weighing between 600 and 900 nag 0"25 M sucrose solution through which water-pumped nitrogen gas had been bubbled for 15 min to remove air.
In the tests t¢ made !
obacco bud3 M urea, by by oxidation ~cesses under
he effects of various
classesof chemicals c o~ l, we added 0.1 ml of homogcnat ;enate to 0.1, 0-05 or 0-01 ml of an aqu( aeous solution testchemical. c of the test All materials were pipet petted or placed in the well wells of a porcelain spot plate and observed for browning for 4--28 hr. Control solutions consisted of plus 0.1 ml of 0.25 0.2~ M sucrose. 0-I ml of homogenate i Three such mixtures and the control were assayed a~, during a single work day. In In the the tests te made to determine the effect (of removal of cations Ire from the homogenate, we treated 5 ml of the homogenate with 12g of drained Dowex 50W-X8 (sodium form). fc The browning of the supe ~ernatant after the resin ,~in se settled was determined witha and without wi various additives. Also, in other similar tests made to determine the effects of the disruption of the tertiaDT structure of proteins on enzymes involved in melanization, we prepared 5 ml of homogenate in 8 M urea that was or ,genization by was not dcaerated 15 min before homog bubbling nitrogen gas through the solution. In the tests of the effect of a browning inhibitor on insect growth, we reared the insects (Ad Adkisson et aL, 1960) as before except that the medium contained cc 0.40, 0.50 or 1.0 g/l of phenylthiourea and made ma tests with hemolymph or with homogenates from sur~ surviving insects. RESULTS AND DISCUSSIOP ;ION Table 1 reports the effectiveness of ses several types of additives in inhibiting browning in the homogenate. 1 Mercaptoethanol, thioglycolic acid, ccysteine and dithiothreitol, all often used to prote( )rotect sulfhydryl This (mercapto-) groups, inhibited the reaction. lea indicates that if the sulfhydryl groups are a protected, lutathione was not the enzyme is not active. Glutathk effective in the concentrations tested, probably because of steric factors. If sulfhyclr3 ydryl goups are 609
HAYES, A. JOHNSONAND M. S. SCHECHT ewise inhibited activity also. ~"ineffectiveness mzoate. Moreagents such as
in homogenates ~orm
'1~ t~SLt~ld
)toethanol /colic acid hreitoH" te aione :tate romercuribenzoate ~alt) 'pus acetate 1 hydrosulfite ic acid ne um ferricyanide thiourea :a ddazole L/- J.l~llz~ 3doxyphenol l~. 3,4-Dioxyphenylalanine Melatonin
t effective tration*
t.:Ull~.t;lltl~tlOll"
0'05% 0.059/o 0.005% 0"00l~o Not effective at 0.15% Not effective at 0"5~o Not effective at 0'5% saturated 0"05~o 0.05%, 0.025~ 50% saturated 0"5% 5% saturated 0"0570§ Not effective at 0-57o Not effective at 0"5~o saturated Enhanced browning rate Not effective at 0"157oo
her 0"01, 0-05 or 0.1 ml of a solution of the test * Either materiald was added to 0.1 ml of homogenate in a spot When 0-01 or 0"05 ml was used, the volume of the plate. When xture was diluted to 0.2 ml with 0-25 M sucrose, test mixture •rc~l was wag prepared nrenared by hv adding addinp ng 0.1 ml of 0.2_5 0.25 M A control sucrose to 0.I ml of homogenate. I Added in a solution containin g 0•25 M sucrose and hiothreitol. reitol. 0.002 M CaCI~ in addition to the dithiothreit I: Bleached the sample. § Very slight browning noted after 24 hr. sodium hydrosulfite and ascorbicc acid and of nitrovning shows that, as gen degassing in preventing browning is well known, oxidation must occur to obtain melanization. The observations that protecting or blocking anization tion and that sulfhydryl groups inhibits melanizatioJ oxygen is required for browning; are consistent with sulfhydryl to disulthe hypothesis that oxidation of sulfh fide is required for activation of this enzyme system• ion of sulfhydryl to This suggestion, i,e. that oxidation nzyme activity, can disulfide is a prerequisite for enz reconcile the observation of Bayer (1954) who suggested that sulfhydryl groups are present in ertesz & Zito (1962) phenolases and the finding of Kertesz who reported that phenolase is not a sulfhydryl containing enzyme.
In our prepara ne (half saturated) prevented brownit ' by inhibiting the transfer of hydroge H during hydroxylation. The extreme 1 tenone to insects and fish and its lesser birds and mammals undoubtedly reflect y greater importance of aromatic hydro: eeer forms• Gutman et al. (1970) indica le sulfhydryl groups interact with ferric: finding that ferricyanide inhibits the br( e 1) provides further support for our c( it sulfhydryl groups m ust p c 1] must be free to reac ruing can occur• Of the materials ylthiourea was most effective in i inhibitk ~ation although thiourea was also acti Because benzimidazole and p-benz2 were inactive, we concluded that tll onded sulfur atom ~ined with an :leus were important combinec )revention of o e further concluded in preyer that aromatic chara arolr ts not effective• Homogenates lC )m the 24-day-old insects that tt had b n media containing 0•40 g of phenylthi tired more than 6 hr (overnigh to mela (overnight) genates from control insects turned tu brox • Hemolymph from free Lts which presumably control insects, i~ contained i while containec phenylthiourea, browned readily that from insects fed the mediurr :lium containing phenylthiourea remained unmelaniz melanized. Insects reared on the phenylthiourea medium pupated slowly--r ty--no pupation occurred in 19 d, days, and only 1 per cen cent in 24 days; among control insects i 19 per cent pup~)ated in 13 days and 54 per cer cent in 24 days• Also, weight gain was half that of the control• ,lthiourea at a concentration of 1 g/l. was toxic Phenylthi to the insects, presumably because it inhibited hydroxyk ,lations necessary for normal development. d( Sucrose-N 2 deaeration
r - - -
8M u r e a - n o t deaerated
- - ]
8M u r e a - N2 deaer~tion
7
IOO (I) 92 (2) ]
Relative t i m e "to brown
260 (2)
Fig. 1. Effect of 8 M urea and deaeration leration with nitrogen before homogenization on the browninlg reaction in tobacco budworm. crude homogenates of larvae of the toba¢ Melatonin, predictably, did not affect affec the rate of acc~ browning. This compound inhibits 9its accumulation of melanin in dogs (Rickards, 1965) an and aggregates pre-existing melanin in dermal mela melanophores of amphibians (Kaufman, 1962)• It therefore there acts on preformed pigments and not directly on pigment synthesis• Figure 1 indicates that melanization was delayed when homogenate was prepared in a solution
melanization in homogenates of Helioth~ that the urea
l
of sulfhydryl 3 the disulfides. mogenate was 50W-X8 in the e toward which
ADKISSONP. L., VA
ng of a homovae and treated
,e
Concentration Effect
~xyphenylalanine 0.1 saturated m alanine H 34 H 34 tylalanine
No browning Pink color in 4 min 45 rnin No browning No browning
0.1 saturated 0.5 saturated 0-25~ 0.05 mM No browning 0"25~o 0.05 rnM 0.1% 0.15 saturated No browning 0.5~ Bleached
virescens larvae in the last instar (1.8-2.7 g) were enized in 15 ml of 0.25 M sucrose that had been enated for 15 min with nitrogen.
the enzyme was most active. In our hands the addition m of Cu z+, N A D P H and phenylalanine as suggested ted by Kaufman (1962) or of tyrosine did not result in activation. We have apparently removed small cationic cofactors required for oxidation of phenyl~ [alanine and tyrosine. Such a treatment does, er, provide a practical means for prevention however of browning in in vitro studies.
ALLISONN. E. (l
S., BULL D. L. & tt germ medium for ,n. Ent. 53, 759-762. 3f action of organic 63-110. Ph.D. thesis,
rearing the pink be BARRYN. M. S. (1 thiocyanates as ins University of Califc e. )metric study of the B o ~ P. D. (1954) groups with organic reaction of protei mercurials. J. Am. 4331-4334. EL Azlz S. A. E. A. ~xidases and detoxieation of carbamate fi • 79-162. Ph.D. thesis, Universi of Califc University e. GtrrMAr4 M., MERSM IY J. • SINGER T. P. (1970) Action of hibitors on different forms oof the resp linked reduced nicotinamide-adenine tinamidi dehydrogenase. Biochemistr,y 9, 2678-2 HAYES D. K., WASH IECHTER M. S. (1972) Monoar Monoamine oxidas trvae of the European corn borer. bet J. econ -1232. of the phenylalanine KAUrMANS. (1962) C 7hem. 237, 2712-2713. hydroxylation cofa aolase. In Oxygenases KERTESZD. ]" & ZITO ] (Edited Edited 1by HAYAIS~ -354. Academic Press, New York. md biochemical basis LEESCHJ. G. (1970)" ® to mosquito larvae, for the sselective to~ Ph.D. thesis, University of c California, pp. 66--67. 66Riverside. RICKARDS D. A. (1965) The therapeutic effect e of melatonin on ol canine melanosis. J. Invest. Dermatol. 44, 13-16. Wlt~MSC WILLIAMSONR. L. & ScrmcrrrER M. S. (1970) (197C Microsomal epoxidal )oxidation of aldrin in lepidopterous larvae. lar~ Biochem. PharmacoL 19, 1719-1727. Key Word W~ Index--Melanization; Heliot~this virescens; rotenone; SH groups.
