- Email: [email protected]
Electrochemical identification of dopachrome isomerase in Drosophila melanogaster
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 724-729
October 31, 1991
ELECTROCHEMICAL IDENTIFICATION OF DOPACHROME ISOMERASE IN DROSOPHILA MELANOGASTER J. Li and A. J. Nappi Department of Biology, Loyola University of Chicago, Chicago, IL 60626 R e c e i v e d September 13, 1991
SUMMARY. A principal reaction in the eumelanin biosynthetic pathway is the conversion of dopachrome (DC) to dihydroxyindole(s). Dopachrome isomerase (DI), the enzyme that catalyzes this reaction, was detected for the first time in larvae of D. melanogaster. Unlike the enzyme from B 16 mouse melanoma cells which converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the insect enzyme forms 5,6-dihydroxyindole (DHI). The activity of the insect DI was linear through 15 min incubation, and the amount of DHI produced was proportional to the amount of enzyme that was incorporated into the reaction mixtures. ~ 199~Ac~demiop..... ~n,
The enzyme tyrosinase plays a major role in the formation of eumelanin, catalyzing the initial hydroxylation of tyrosine to dopa, and the subsequent oxidation of dopa to dopaquinone. The reaction sequence that follows includes intramolecular cyclization and indolization of dopaquinone to form leucochrome, dopachrome (DC), 5,6-dihydroxyindole (DHI), and/or 5,6dihydroxyindole-2-carboxylic acid (DHICA), indole-5,6-quinone, and then eumelanin.
The
tyrosinase system of insects has long been accorded considerable interest because knowledge of the mechanism(s) of enzyme activation would be indispensable for a basic understanding of the factors regulating melanization and sclerotization reactions associated with cuticle formation, cuticular wound healing responses (1-3), and certain cellular immune reactions which sequester foreign organisms in pigmented capsules (4-10). For many years, tyrosinase was considered to be the only enzyme involved in melanogenesis. Recently, several factors aside from tyrosinase have been investigated extensively and found to be involved in the oxidative pathway of catecholamine metabolism (11-18). One of these factors was first identified in mammalian melanoma cells and called dopachrome conversion factor because of its ability to catalyze the decoloration or bleaching of DC (19,20). The enzyme, which has since been classified as dopachrome oxidoreductase (21), dopachrome tautomerase (22), and dopachrome isomerase (DI) Abbreviations: DC, dopachrome; DHI, 5,6-dlhydroxyindole; DHICA, 5,6-dihydroxyindole-2carboxylic acid; DI, dopachrome isomerase; MOPS, 3[N-Morpholino]propanesulfonic acid; PTU, 1-phenyl-2-thiourea. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction, in any form reserved.
724
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(23), catalyzes the conversion of DC to DHICA. The relative amounts of DHI and DHICA formed by enzymatic and nonenzymatic conversions of DC appear to be influenced by many factors, including metal ions (24-26).
Since these two dihydroxyindoles form reactive
intermediates that polymerize to form melanin, enzyme regulation at this stage in eumelanin synthesis may be as important as the initial tyrosinase-mediated oxidations. Recent studies by Sugumaran and Semenski (27) provide evidence that in Manduca sexta, the initial reaction catalyzed by the enzyme is the isomerization of iminochrome to quinone methide, and the aromatization of quinone methide appears to accompany its decarboxylation. It now appears that the DI from mammalian sources catalyzes the conversion of dopachrome to DHICA, whereas the enzyme from insects catalyzes the conversion of DC to DHI (14).
The purpose of this
investigation was to determine whether DI was present in larvae of D. melanogaster. This report summarizes our comparative investigations by high pressure liquid chromatography with electrochemical detection (HPLC-ED) of DI activity in Drosophila and in B16 melanoma cells. MATERIALS AND METHODS Chemicals and reagent. DHI was obtained from Regis Chemical Co. (Morton Grove, IL 60053). DHICA was synthesized by copper-catalyzed rearrangement of DC, and by ferricyanide oxidation of dopa (28). All other chemicals and reagents were from Sigma Chemical Co (St. Louis, MO). Buffer preparations. Contamination by heavy metals was avoided by treating the buffers that were used in the assays with Chelex 100 resin (Bio-Rad). Enzyme Preparations from Drosophila. The D. melanogaster that were used in this study were raised on standard cornmeal and yeast medium at 25°C. Fifteen third stage larvae (96 hr post oviposition) were removed from the medium, washed (twice) in MOPS buffer (pH 6.5), and then homogenized in 1 ml 50 mM MOPS buffer (pH 6.5) containing 1 mM EDTA and 0.2 mM PTU. The homogenate was centrifuged at 15000 x g and 4°C for 30 minutes. Proteins in the supernatant were extracted by ammonium sulfate (65 % saturation). Precipitation occurred at 4°C for 4 hr, and the sample was centrifuged (15000 x g) for 30 min. The pellet was re-suspended in 2 ml of 10 mM phosphate buffer (pH 6.8) and dialyzed against 4 changes of the same buffer. The sample was used as the enzyme source following dialysis. Enzyme Preparations from B16 cells. B16 mouse melanoma cells were cultured in Medium 199 supplemented with 10% fetal calf serum (v/v), at 37°C and 5% carbon dioxide in a humidified environment. The cells were detached from the culture flasks after 30 incubation in calcium and magnesium free HBSS. The cells were then centrifuged, re-suspended and washed (twice) in 50 mM MOPS buffer (pH 6.5). The cell pellets were then lysed on ice for 2 hr with 10 mM phosphate buffer (pH 6.8) containing 0.5% Triton X-100. The cell lysate was centrifuged at 15000 x g and 4°C for 30 minutes, and the supernatant was used as the crude enzyme preparation. Synthesis of Dopachrome. Dopachrome was prepared by mixing silver oxide (5 #g/ml) with 1 mM L-dopa prepared in distilled water. After 5 rain incubation the solution was filtered (twice) through Gelman 2/~m Acrodisc filters and stored on ice where it remained stable for at least 6 hr. Electrochemical analyses of the L-dopa concentrations before and after treatment with silver oxide indicated that this procedure resulted in a 62% conversion of L-dopa to dopachrome. Aliquots of the dopachrome solution were immediately used for the assays. Incubation Mixtures. Incubation mixtures were comprised of 100 txl dopachrome solution and 100 #1 enzyme preparation. For controls the enzyme preparation was either heat inactivated 725
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(100°C for 1 min), or it was replaced with an equal volume of buffer. PTU (80 nmol) was incorporated into the mixtures to inhibit phenol oxidase. At five min intervals 5#1 aliquots of the incubation mixture was withdrawn and analyzed by HPLC-ED to determine the formation of DHI and DHICA. Protein determinations were made using the Bradford microassay (29) with BSA as the standard. HPLC-ED Analyses. The HPLC-ED system consisted of a Gilson (Middleton, WI) Model 305 pump, a Model 231 sample injector fitted with a Rheodyne (Cotati, CA) Model 7010 injection valve, a 20 tzl sample loop, a Model 401 dilutor, and a Bioanalytical System (West Lafayette, IN) Model LC-4B amperometric detector fitted with a glassy carbon electrode. The working electrode was maintained at a potential of +750 mV versus a Ag/AgC1 reference electrode and a sensitivity of 100 to 500 nA full scale depending upon the amount of enzymatic activity. Separations were achieved at 40°C by a BAS Phase-II, 3-/zm ODS reverse phase column (3.2 mm I.D. x 10 cm). The mobile phase consisted of 0.1 M citrate buffer (pH 2.9) containing 5 % acetonitrile, 0.5 mM sodium octylsulfate, and 0.5 mM Na2EDTA at 40°C. The flow rate was 0.8 ml/min. Enzyme activity was based on the standard curve prepared by injecting increasing amounts of DI (0.01-0.3 nmol), and measuring the peak area. Standards were run before and after each set of assays and data were analyzed by linear regression. Specific enzyme activity is expressed as the amount of product (DHI) formed per min per mg protein. RESULTS Identification of DI Reaction Products.
To elucidate the nature of the DI-catalyzed reactions, the reaction products were analyzed by HPLC-ED. As shown in Fig. 1, dopa, DHI and DHICA were eluted at 1.8, 3.5 and 4.4 rain, respectively.
-DOPA
DOPA k.~
-DOPA
DHI
DIHCA
~,.
.4 DHICA
B A Figure 1. Representative chromatograms showing the separation and detection by HPLC-ED of dopa (1.8 min), DHI (3.5 min), and DHICA (4.4 rain). Reaction mixtures were comprised of 100/~1 of dopachrome solution end,an equal volume of (A) 10 mM phosphate buffer (pH 6.8) incubated for 40 min, (B) supernatant from B16 melanomacell lystate (100 ~g protein) incubated for 15 min, and (C) the supernatant from whole body homogenates of Drosophila (60/zg protein) incubated for 15 min. The potential was +750 mV, and the sensitivity was 500 nA full scale.
726
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
16
.,.j.~AI
~
8
,
4'
0
0
10
20 30 Timetmin)
40
Figure 2. Drosophila DI-catalyzed enhancement of DC conversion to DHI. Note the proportional increase in the amount of DHI formed when dopachrome solution (100 ~1) was incubated with an equal volume of enzyme preparation (30 /~g, open square; 60 /~g, solid square), 10 mM phosphate buffer (solid circle), and heat inactivated enzyme (open circle). Buffer Enhanced Conversion of Dopachrome to DHI. When DC was prepared in distilled water and stored on ice, it was stable for at least 6 hr. However, when incubated with MOPS or phosphate buffer, a small amount of DC was very gradually converted to DHI. In 40 min assays the total amount of DHI formed in buffer treated dopachrome solutions averaged 1.92 nmol.
A small amount of DHICA also formed non-
enzymatically in buffer treated DC solutions (Fig. 1). Conversion of Dopachrome to DHI by DI from
Drosophila.
Compared to buffer treated DC solutions, substantially larger amounts of DHI were produced when DC was incubated with enzyme preparations from
Drosophila.
When reaction
mixtures contained 30 pg enzyme protein the amount of DHI formed during 40 min incubations averaged 9 nmol (Fig. 2). When the amount of Drosophila enzyme protein incubated with DC was doubled, there was a proportional increase in the amount of DHI produced.
With heat
inactivated enzyme preparations the amount of DHI formed was virtually identical to that formed when buffer was used (Fig. 2). Under the described assay conditions DI activity was linear within 15 rain incubation, and the specific enzyme activity during this period of time was 11.5 nmol/min/mg protein.
Very low levels of DHICA formed in these incubation mixtures,
presumably from the nonenzymatic decarboxylation of DC since control incubations with buffer or heat inactivated enzymes produced nearly identical levels of DHICA. Conversion of Dopachrome to D H I C A by DI from B16 Melanoma Cells. For comparative purposes and to verify the applicability of the HPLC-ED method that we used to analyze DI activity in Drosophila, we incubated DC with an equal volume of supernatant 727
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from B16 melanoma cell lysate. Unlike the DI from Drosophila which formed DHI, the DI from B16 cells catalyzed the conversion of dopachrome to DHICA (Fig. 1). The small amount of DHI formed in these reaction mixtures was attributed to the non-enzymatic conversion of dopachrome to DHI, since virtually identical levels of this dihydroxyindole were produced in control incubations containing buffer or heat inactivated enzymes (not shown).
DISCUSSION In insects, the enzymatic regulation of melanogenesis is of considerable interest because the biosynthetic pathways are common to other processes including cellular immune responses, and cuticle formation and repair. While the tyrosinase-catalyzed reaction sequence leading from tyrosine to dopaquinone has been chemically well characterized, little is known of the subsequent reactions. Until recently, the reactions following the formation of dopaquinone were considered to occur spontaneously without regulatory control mechanisms. One reaction sequence distal to the synthesis of dopaquinone, i.e., the conversion of DC is known to be regulated by the enzyme DI. This report is the first to establish the presence of the enzyme DI in the supernatant of larval homogenates of Drosophila.
The DI of this insect catalyzed the decarboxylative
rearrangement of DC to form DHI.
There is presently no evidence that the Drosophila DI
catalyzed the conversion of DC to DHICA, as was shown to occur with DI from B16 melanoma cells. Although our study with DI from B16 cells confirms the observations made by other investigators that the mammalian enzyme potentiates the in vitro non-decarboxylative rearrangement of DC leading to the formation of DHICA rather than DHI (20, 21, 23, 24), the physiological significance of the process still remains unknown. Several studies have implicated DHICA as an important constituent of natural eumelanins, but the mechanism by which this indole is formed in vivo and is incorporated into the pigment polymer remains elusive. The presence of carboxylated indole units in natural melanins is probably due to the intervention in the biochemical pathway of metal ions which catalyze the formation of DHICA (25, 26). Additional studies are needed to clearly differentiate between two proposed mechanisms for the DI-catalyzed conversion of DC.
One mechanism considers DC to be initially oxidized or
decarboxylated to an indoline intermediate that then tautomerizes to DHICA or DHI, respectively. The second method proposes that DC undergoes an initial tautomerization to form quinone methide that either tautomerizes to DHICA
or undergoes
decarboxylative
tautomerization to yield DHI (27).
ACKNOWLEDGMENT We acknowledge support from the National Institutes of Health (AI 24199 ). 728
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Anderson, S. O. (1985) In Comprehensive Insect Physiology, Biochemistry and Pharmacology (G. A. Kerkut and L. Gilbert, Eds.), Vol. 3, pp. 59-74. Pergamon Press, New York. Brown, C.S. and Nestler, C. (1985) In Comprehensive Insect Physiology Biochemistry and Pharmacology (G. A. Kerkut and L. I. Gilbert, Eds.) Vol. 3, pp. 435-497. Pergamon Press, New York. Sugumaran, M. (1988) Adv. Insect Physiol. 21, 179-231. Nappi, A. J. (1975) In Invertebrate Immunity. Mechanisms of Invertebrate Vector-Parasite Relations (K. Maramorosch and R. Shope, Eds.), pp. 293-326. Academic Press, New York. Nappi, A. J., (1978) In Animal Models of Comparative and Developmental Aspects of Immunity and Disease ( E. Gershwin and E. L. Cooper, Eds.), p 15-24. Pergamon Press, New York. Gotz, P., and Boman H. G. (1985) In Comprehensive Insect Physiology Biochemistry and Pharmacology.(G. A. Kerkut G. A. and L. I. Gilbert, Eds.), pp. 453-485. Pergamon Press, Oxford. Nappi, A. J., and Carton Y. (1986) In Immunity in Invertebrates (M. Brehelin, Ed.), pp. 171-187. Springer-Verlag Berlin Heidelberg. Christensen, B. C., and Nappi A. J. (1988) Immune responses of arthropods. ISIAtlas of Science. 15-19. Christensen, B. C., and Tracy J. W. (1989) Amer. Zool. 29, 387-398. Sugumaran, M. (1990) In Defense Molecules. pp.47-62. Alan R. Liss, Inc. New York. Aso, Y. and Yamasaki, N. (1987) Seikagaku 59, 563. Saul S. J., and Sugumaran, M. (1988) FEBS Lett. 237, 155-158. Saul S. J. and Sugumaran M. (1989) FEBS Lett. 249, 155-158. Aso, Y., Immamura, Y. and Yamasaki, N. (1989) Insect Biochem. 19, 401-407. Aso, Y., Kramer, K. J., Hopkins, T. L. and Whetzel, S. Z. (1984) Insect Biochem. 14, 463-472. Andersen, K., Sun, S-C., Boman, H. G. and Steiner, H. (1989) Insect Biochem. 19, 629-637. Datta, T. K., Basu, P. S., Datta, P. K. and Banergee, A. (1989) Biochem. J. 260, 525529. Aso, Y., Nakashima, K., Yamasaki, N. (1990) Insect Biochem. 20, 685-689. Kroner, A. M. and Pawelek, J. 1982. J. Invest. Derm. 85, 229-231. Pawelek, J. M., Korner, A., Bergstrom, A. and Bologna, J. (1980) Nature 286, 617619. Barber, J. I., Yownsend, D., Olds, D. P. and King, R. A. (1984) J. Invest. Dermat. 83, 145-149. Aroca, P., Solano, F., Garcia-Borron, J. C. and Lozano, J. A. (1990) J. Biochem. Biophys. Meth. 21, 35-46. Pawelek, J. (1990) Biochem. Biophys. Res. Comm. 166, 1328-1333. Leonard, L. J., Townsend, D. and King, R. A. (1988) Biochemistry 27, 6156-6159. Palumbo, A., d'Ischia, M., Misuraca, G., Prota, G. (1987) Biochemica et Biophysica Acta 925, 203-209. Palumbo, A., d'Ischia, M., Misuraca, G., Prota, G. and Schultz, M. (1988) Biochimica et Biophysica Aeta 964, 193-199. Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078. Wakamatsu, K. and Ito, S. (1988). Anal. Biochem. 170, 335-340. Peterson, G. L. (1977) Anal. Biochem. 83, 346-356.
729
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 724-729
October 31, 1991
ELECTROCHEMICAL IDENTIFICATION OF DOPACHROME ISOMERASE IN DROSOPHILA MELANOGASTER J. Li and A. J. Nappi Department of Biology, Loyola University of Chicago, Chicago, IL 60626 R e c e i v e d September 13, 1991
SUMMARY. A principal reaction in the eumelanin biosynthetic pathway is the conversion of dopachrome (DC) to dihydroxyindole(s). Dopachrome isomerase (DI), the enzyme that catalyzes this reaction, was detected for the first time in larvae of D. melanogaster. Unlike the enzyme from B 16 mouse melanoma cells which converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the insect enzyme forms 5,6-dihydroxyindole (DHI). The activity of the insect DI was linear through 15 min incubation, and the amount of DHI produced was proportional to the amount of enzyme that was incorporated into the reaction mixtures. ~ 199~Ac~demiop..... ~n,
The enzyme tyrosinase plays a major role in the formation of eumelanin, catalyzing the initial hydroxylation of tyrosine to dopa, and the subsequent oxidation of dopa to dopaquinone. The reaction sequence that follows includes intramolecular cyclization and indolization of dopaquinone to form leucochrome, dopachrome (DC), 5,6-dihydroxyindole (DHI), and/or 5,6dihydroxyindole-2-carboxylic acid (DHICA), indole-5,6-quinone, and then eumelanin.
The
tyrosinase system of insects has long been accorded considerable interest because knowledge of the mechanism(s) of enzyme activation would be indispensable for a basic understanding of the factors regulating melanization and sclerotization reactions associated with cuticle formation, cuticular wound healing responses (1-3), and certain cellular immune reactions which sequester foreign organisms in pigmented capsules (4-10). For many years, tyrosinase was considered to be the only enzyme involved in melanogenesis. Recently, several factors aside from tyrosinase have been investigated extensively and found to be involved in the oxidative pathway of catecholamine metabolism (11-18). One of these factors was first identified in mammalian melanoma cells and called dopachrome conversion factor because of its ability to catalyze the decoloration or bleaching of DC (19,20). The enzyme, which has since been classified as dopachrome oxidoreductase (21), dopachrome tautomerase (22), and dopachrome isomerase (DI) Abbreviations: DC, dopachrome; DHI, 5,6-dlhydroxyindole; DHICA, 5,6-dihydroxyindole-2carboxylic acid; DI, dopachrome isomerase; MOPS, 3[N-Morpholino]propanesulfonic acid; PTU, 1-phenyl-2-thiourea. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction, in any form reserved.
724
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(23), catalyzes the conversion of DC to DHICA. The relative amounts of DHI and DHICA formed by enzymatic and nonenzymatic conversions of DC appear to be influenced by many factors, including metal ions (24-26).
Since these two dihydroxyindoles form reactive
intermediates that polymerize to form melanin, enzyme regulation at this stage in eumelanin synthesis may be as important as the initial tyrosinase-mediated oxidations. Recent studies by Sugumaran and Semenski (27) provide evidence that in Manduca sexta, the initial reaction catalyzed by the enzyme is the isomerization of iminochrome to quinone methide, and the aromatization of quinone methide appears to accompany its decarboxylation. It now appears that the DI from mammalian sources catalyzes the conversion of dopachrome to DHICA, whereas the enzyme from insects catalyzes the conversion of DC to DHI (14).
The purpose of this
investigation was to determine whether DI was present in larvae of D. melanogaster. This report summarizes our comparative investigations by high pressure liquid chromatography with electrochemical detection (HPLC-ED) of DI activity in Drosophila and in B16 melanoma cells. MATERIALS AND METHODS Chemicals and reagent. DHI was obtained from Regis Chemical Co. (Morton Grove, IL 60053). DHICA was synthesized by copper-catalyzed rearrangement of DC, and by ferricyanide oxidation of dopa (28). All other chemicals and reagents were from Sigma Chemical Co (St. Louis, MO). Buffer preparations. Contamination by heavy metals was avoided by treating the buffers that were used in the assays with Chelex 100 resin (Bio-Rad). Enzyme Preparations from Drosophila. The D. melanogaster that were used in this study were raised on standard cornmeal and yeast medium at 25°C. Fifteen third stage larvae (96 hr post oviposition) were removed from the medium, washed (twice) in MOPS buffer (pH 6.5), and then homogenized in 1 ml 50 mM MOPS buffer (pH 6.5) containing 1 mM EDTA and 0.2 mM PTU. The homogenate was centrifuged at 15000 x g and 4°C for 30 minutes. Proteins in the supernatant were extracted by ammonium sulfate (65 % saturation). Precipitation occurred at 4°C for 4 hr, and the sample was centrifuged (15000 x g) for 30 min. The pellet was re-suspended in 2 ml of 10 mM phosphate buffer (pH 6.8) and dialyzed against 4 changes of the same buffer. The sample was used as the enzyme source following dialysis. Enzyme Preparations from B16 cells. B16 mouse melanoma cells were cultured in Medium 199 supplemented with 10% fetal calf serum (v/v), at 37°C and 5% carbon dioxide in a humidified environment. The cells were detached from the culture flasks after 30 incubation in calcium and magnesium free HBSS. The cells were then centrifuged, re-suspended and washed (twice) in 50 mM MOPS buffer (pH 6.5). The cell pellets were then lysed on ice for 2 hr with 10 mM phosphate buffer (pH 6.8) containing 0.5% Triton X-100. The cell lysate was centrifuged at 15000 x g and 4°C for 30 minutes, and the supernatant was used as the crude enzyme preparation. Synthesis of Dopachrome. Dopachrome was prepared by mixing silver oxide (5 #g/ml) with 1 mM L-dopa prepared in distilled water. After 5 rain incubation the solution was filtered (twice) through Gelman 2/~m Acrodisc filters and stored on ice where it remained stable for at least 6 hr. Electrochemical analyses of the L-dopa concentrations before and after treatment with silver oxide indicated that this procedure resulted in a 62% conversion of L-dopa to dopachrome. Aliquots of the dopachrome solution were immediately used for the assays. Incubation Mixtures. Incubation mixtures were comprised of 100 txl dopachrome solution and 100 #1 enzyme preparation. For controls the enzyme preparation was either heat inactivated 725
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(100°C for 1 min), or it was replaced with an equal volume of buffer. PTU (80 nmol) was incorporated into the mixtures to inhibit phenol oxidase. At five min intervals 5#1 aliquots of the incubation mixture was withdrawn and analyzed by HPLC-ED to determine the formation of DHI and DHICA. Protein determinations were made using the Bradford microassay (29) with BSA as the standard. HPLC-ED Analyses. The HPLC-ED system consisted of a Gilson (Middleton, WI) Model 305 pump, a Model 231 sample injector fitted with a Rheodyne (Cotati, CA) Model 7010 injection valve, a 20 tzl sample loop, a Model 401 dilutor, and a Bioanalytical System (West Lafayette, IN) Model LC-4B amperometric detector fitted with a glassy carbon electrode. The working electrode was maintained at a potential of +750 mV versus a Ag/AgC1 reference electrode and a sensitivity of 100 to 500 nA full scale depending upon the amount of enzymatic activity. Separations were achieved at 40°C by a BAS Phase-II, 3-/zm ODS reverse phase column (3.2 mm I.D. x 10 cm). The mobile phase consisted of 0.1 M citrate buffer (pH 2.9) containing 5 % acetonitrile, 0.5 mM sodium octylsulfate, and 0.5 mM Na2EDTA at 40°C. The flow rate was 0.8 ml/min. Enzyme activity was based on the standard curve prepared by injecting increasing amounts of DI (0.01-0.3 nmol), and measuring the peak area. Standards were run before and after each set of assays and data were analyzed by linear regression. Specific enzyme activity is expressed as the amount of product (DHI) formed per min per mg protein. RESULTS Identification of DI Reaction Products.
To elucidate the nature of the DI-catalyzed reactions, the reaction products were analyzed by HPLC-ED. As shown in Fig. 1, dopa, DHI and DHICA were eluted at 1.8, 3.5 and 4.4 rain, respectively.
-DOPA
DOPA k.~
-DOPA
DHI
DIHCA
~,.
.4 DHICA
B A Figure 1. Representative chromatograms showing the separation and detection by HPLC-ED of dopa (1.8 min), DHI (3.5 min), and DHICA (4.4 rain). Reaction mixtures were comprised of 100/~1 of dopachrome solution end,an equal volume of (A) 10 mM phosphate buffer (pH 6.8) incubated for 40 min, (B) supernatant from B16 melanomacell lystate (100 ~g protein) incubated for 15 min, and (C) the supernatant from whole body homogenates of Drosophila (60/zg protein) incubated for 15 min. The potential was +750 mV, and the sensitivity was 500 nA full scale.
726
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
16
.,.j.~AI
~
8
,
4'
0
0
10
20 30 Timetmin)
40
Figure 2. Drosophila DI-catalyzed enhancement of DC conversion to DHI. Note the proportional increase in the amount of DHI formed when dopachrome solution (100 ~1) was incubated with an equal volume of enzyme preparation (30 /~g, open square; 60 /~g, solid square), 10 mM phosphate buffer (solid circle), and heat inactivated enzyme (open circle). Buffer Enhanced Conversion of Dopachrome to DHI. When DC was prepared in distilled water and stored on ice, it was stable for at least 6 hr. However, when incubated with MOPS or phosphate buffer, a small amount of DC was very gradually converted to DHI. In 40 min assays the total amount of DHI formed in buffer treated dopachrome solutions averaged 1.92 nmol.
A small amount of DHICA also formed non-
enzymatically in buffer treated DC solutions (Fig. 1). Conversion of Dopachrome to DHI by DI from
Drosophila.
Compared to buffer treated DC solutions, substantially larger amounts of DHI were produced when DC was incubated with enzyme preparations from
Drosophila.
When reaction
mixtures contained 30 pg enzyme protein the amount of DHI formed during 40 min incubations averaged 9 nmol (Fig. 2). When the amount of Drosophila enzyme protein incubated with DC was doubled, there was a proportional increase in the amount of DHI produced.
With heat
inactivated enzyme preparations the amount of DHI formed was virtually identical to that formed when buffer was used (Fig. 2). Under the described assay conditions DI activity was linear within 15 rain incubation, and the specific enzyme activity during this period of time was 11.5 nmol/min/mg protein.
Very low levels of DHICA formed in these incubation mixtures,
presumably from the nonenzymatic decarboxylation of DC since control incubations with buffer or heat inactivated enzymes produced nearly identical levels of DHICA. Conversion of Dopachrome to D H I C A by DI from B16 Melanoma Cells. For comparative purposes and to verify the applicability of the HPLC-ED method that we used to analyze DI activity in Drosophila, we incubated DC with an equal volume of supernatant 727
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from B16 melanoma cell lysate. Unlike the DI from Drosophila which formed DHI, the DI from B16 cells catalyzed the conversion of dopachrome to DHICA (Fig. 1). The small amount of DHI formed in these reaction mixtures was attributed to the non-enzymatic conversion of dopachrome to DHI, since virtually identical levels of this dihydroxyindole were produced in control incubations containing buffer or heat inactivated enzymes (not shown).
DISCUSSION In insects, the enzymatic regulation of melanogenesis is of considerable interest because the biosynthetic pathways are common to other processes including cellular immune responses, and cuticle formation and repair. While the tyrosinase-catalyzed reaction sequence leading from tyrosine to dopaquinone has been chemically well characterized, little is known of the subsequent reactions. Until recently, the reactions following the formation of dopaquinone were considered to occur spontaneously without regulatory control mechanisms. One reaction sequence distal to the synthesis of dopaquinone, i.e., the conversion of DC is known to be regulated by the enzyme DI. This report is the first to establish the presence of the enzyme DI in the supernatant of larval homogenates of Drosophila.
The DI of this insect catalyzed the decarboxylative
rearrangement of DC to form DHI.
There is presently no evidence that the Drosophila DI
catalyzed the conversion of DC to DHICA, as was shown to occur with DI from B16 melanoma cells. Although our study with DI from B16 cells confirms the observations made by other investigators that the mammalian enzyme potentiates the in vitro non-decarboxylative rearrangement of DC leading to the formation of DHICA rather than DHI (20, 21, 23, 24), the physiological significance of the process still remains unknown. Several studies have implicated DHICA as an important constituent of natural eumelanins, but the mechanism by which this indole is formed in vivo and is incorporated into the pigment polymer remains elusive. The presence of carboxylated indole units in natural melanins is probably due to the intervention in the biochemical pathway of metal ions which catalyze the formation of DHICA (25, 26). Additional studies are needed to clearly differentiate between two proposed mechanisms for the DI-catalyzed conversion of DC.
One mechanism considers DC to be initially oxidized or
decarboxylated to an indoline intermediate that then tautomerizes to DHICA or DHI, respectively. The second method proposes that DC undergoes an initial tautomerization to form quinone methide that either tautomerizes to DHICA
or undergoes
decarboxylative
tautomerization to yield DHI (27).
ACKNOWLEDGMENT We acknowledge support from the National Institutes of Health (AI 24199 ). 728
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Anderson, S. O. (1985) In Comprehensive Insect Physiology, Biochemistry and Pharmacology (G. A. Kerkut and L. Gilbert, Eds.), Vol. 3, pp. 59-74. Pergamon Press, New York. Brown, C.S. and Nestler, C. (1985) In Comprehensive Insect Physiology Biochemistry and Pharmacology (G. A. Kerkut and L. I. Gilbert, Eds.) Vol. 3, pp. 435-497. Pergamon Press, New York. Sugumaran, M. (1988) Adv. Insect Physiol. 21, 179-231. Nappi, A. J. (1975) In Invertebrate Immunity. Mechanisms of Invertebrate Vector-Parasite Relations (K. Maramorosch and R. Shope, Eds.), pp. 293-326. Academic Press, New York. Nappi, A. J., (1978) In Animal Models of Comparative and Developmental Aspects of Immunity and Disease ( E. Gershwin and E. L. Cooper, Eds.), p 15-24. Pergamon Press, New York. Gotz, P., and Boman H. G. (1985) In Comprehensive Insect Physiology Biochemistry and Pharmacology.(G. A. Kerkut G. A. and L. I. Gilbert, Eds.), pp. 453-485. Pergamon Press, Oxford. Nappi, A. J., and Carton Y. (1986) In Immunity in Invertebrates (M. Brehelin, Ed.), pp. 171-187. Springer-Verlag Berlin Heidelberg. Christensen, B. C., and Nappi A. J. (1988) Immune responses of arthropods. ISIAtlas of Science. 15-19. Christensen, B. C., and Tracy J. W. (1989) Amer. Zool. 29, 387-398. Sugumaran, M. (1990) In Defense Molecules. pp.47-62. Alan R. Liss, Inc. New York. Aso, Y. and Yamasaki, N. (1987) Seikagaku 59, 563. Saul S. J., and Sugumaran, M. (1988) FEBS Lett. 237, 155-158. Saul S. J. and Sugumaran M. (1989) FEBS Lett. 249, 155-158. Aso, Y., Immamura, Y. and Yamasaki, N. (1989) Insect Biochem. 19, 401-407. Aso, Y., Kramer, K. J., Hopkins, T. L. and Whetzel, S. Z. (1984) Insect Biochem. 14, 463-472. Andersen, K., Sun, S-C., Boman, H. G. and Steiner, H. (1989) Insect Biochem. 19, 629-637. Datta, T. K., Basu, P. S., Datta, P. K. and Banergee, A. (1989) Biochem. J. 260, 525529. Aso, Y., Nakashima, K., Yamasaki, N. (1990) Insect Biochem. 20, 685-689. Kroner, A. M. and Pawelek, J. 1982. J. Invest. Derm. 85, 229-231. Pawelek, J. M., Korner, A., Bergstrom, A. and Bologna, J. (1980) Nature 286, 617619. Barber, J. I., Yownsend, D., Olds, D. P. and King, R. A. (1984) J. Invest. Dermat. 83, 145-149. Aroca, P., Solano, F., Garcia-Borron, J. C. and Lozano, J. A. (1990) J. Biochem. Biophys. Meth. 21, 35-46. Pawelek, J. (1990) Biochem. Biophys. Res. Comm. 166, 1328-1333. Leonard, L. J., Townsend, D. and King, R. A. (1988) Biochemistry 27, 6156-6159. Palumbo, A., d'Ischia, M., Misuraca, G., Prota, G. (1987) Biochemica et Biophysica Acta 925, 203-209. Palumbo, A., d'Ischia, M., Misuraca, G., Prota, G. and Schultz, M. (1988) Biochimica et Biophysica Aeta 964, 193-199. Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078. Wakamatsu, K. and Ito, S. (1988). Anal. Biochem. 170, 335-340. Peterson, G. L. (1977) Anal. Biochem. 83, 346-356.
729