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Xanthurenic acid in the mutant strain scarlet of Drosophila melanogaster
Comp. Biochem. PhysioL Vol. 82B, No. 4, pp. 763-766, 1985
0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
Printed in Great Britain
XANTHURENIC ACID IN THE MUTANT STRAIN S C A R L E T OF D R O S O P H I L A M E L A N O G A S T E R YOSHISHIGE UMEBACHI,* AKIRA YOKOYAMA,* TAKANORI KAWASHIMA,* TOSHIKO DOI,* MITSUO WATANABE,~"MASAYOSHITABARAt and FUMIO IINUMAt *Department of Biology, Faculty of Science, Kanazawa University, Kanazawa, 920, Japan; and ?Faculty of Pharmaceutical Science, Teikyo University, Sagamiko, Kanagawa, 199-01, Japan (Received 3 May 1985)
Abstraet--l. Xanthurenic acid, 3-hydroxykynurenine, and kynurenine hydroxylase activity in bw, st and bwst of D. melanogaster were determined. 2. Both pupa and adult of bw, st and bwst showed much the same level of kynurenine hydroxylase activity. 3. The pupae of bw, st and bwst contained not a little 3-hydroxykynurenine. In st and bwst, the 3-hydroxykynurenine rapidly decreased at emergence, while in bw, the decrease was rather gradual. 4. In bw and st, a considerable amount of xanthurenic acid was synthesized during the pupal stage, while, in bwst, the synthesis was moderate. Moreover, in the adult stage, xanthurenic acid was much less in bwst than in bw and st.
INTRODUCTION
(Howells et al., 1977), (5) during the pupal stage, st contains much the same level of xanthurenic acid as in the wild type (Howells and Ryall, 1975), and (6) st contains not a little xanthurenic acid in the adult stage (Umebachi and Tsuchitani, 1955). As to bwst, however, no such detailed investigation has been made. The purpose of the present paper is to describe the changes of the levels of 3-hydroxykynurenine, xanthurenic acid, and kynurenine hydroxylase activity during the pupal and adult stages of bwst in comparison with bw and st.
Of the ommochrome-deficient mutants of D. melano gaster, it has well been established that vermilion and cinnabar lack the activity of tryptophan oxygenase (L-tryptophan: oxygen 2,3-oxidoreductase, EC 1.13.11.11) and kynurenine 3-hydroxylase (L-kynurenine, N A D P H : o x y g e n oxidoreductase, EC 1.14.13.9), respectively (Linzen, 1974; Dickinson and Sullivan, 1975). As to the m u t a n t scarlet (st), which also scarcely contains ommochrome and has a vermilion-type eye color, however, its genetic block has not completely been elucidated yet. The strongest possibility at present is that st is unable to transport kynurenine and 3-hydroxykynurenine into or unable to store them in the cells of Malpighian tubules of larvae and those of the eye disc of the developing adult (Beadle, 1937; Sullivan and Sullivan, 1975; Howells and Ryall, 1975; Howells et al., 1977). But, concerning tryptophan metabolism of st, there is a problem which has not been solved yet. It is the problem of xanthurenic acid formation. Umebachi and Tsuchitani (1955) reported for the first time the presence of xanthurenic acid in the adults of D. melanogaster. Strangely enough, it was noted that brown (bw) and st contained not a little xanthurenic acid, whereas the double recessive bwst contained little of it. Since then, the site and way of xanthurenic acid formation in st and the low level of xanthurenic acid in bwst have been interesting problems which remained to be solved. The properties of st reported up to the present concerning tryptophan metabolism may be summarized as follows: (1) st must be able to synthesize 3-hydroxykynurenine (Beadle, 1937), (2) st has the activity of kynurenine hydroxylase (Sullivan et al., 1973), (3) st contains not a little 3-hydroxykynurenine during the pupal stage, whereas adult has none or only a little of it (Howells and Ryall, 1975), (4) st excretes more 3-hydroxykynurenine than does the wild type strain
MATERIALS AND METHODS Materials D. melanogaster was raised at 25°C on the culture medium which contains malted rice (125 g), sugar (50 g), K2HPO4 (0.9g), agar (12.5 g), water (900ml), and raw yeast. The mutant strains used were bw, st and bwst. The eye color of bw, st and bwst is brown, vermilion, and white, respectively. The eye of bw contains xanthommatin but not drosopterins. The eye of st contains drosopterins but not xanthommatin (Nolte, 1954). The eye of bwst contains neither xanthommatin nor drosopterin. Eighty per cent ethanol extraction
Pupae or adults were homogenized in 80% ethanol and centrifuged at 3000 rpm for 5 rain at about 25°C or at 15,000 rpm (26,000g) for 30 min at 4°C. The supernatant was used for thin-layer chromatography, the method 1 of 3-hydroxykynurenine determination or the method 2 of xanthurenic acid determination. Hot-water extraction
Adults were homogenized in distilled water. The homogenate was warmed at 80°C for 10rain and centrifuged at 15,000 rpm (26,000g) for 30 rain at 4°C. The supernatant was filtered through the Toyo TM-2P membrane filter, and the filtrate was submitted to the method 1 of xanthurenic acid determination.
763
YOSHISHIGEUMEBACHIel al.
764
TCA extraction
Method 2. The above-mentioned 80% ethanol extract was evaporated to dryness in a rotary evaporator at 50~'C. The residue was dissolved in 120ml of water, and 30 ml of 1 N HCI was added. The resulting solution (150 ml) was applied to Dowex 50W X12 (H +) column (1 x 3cm). The column was washed with 50 ml of 0.2 N HC1 and then with 100 ml of 0 . 5 N HC1. Finally, xanthurenic acid was eluted with water (Satoh and Price, 1957). The first 12.5 ml of the water effluent was discarded and then the next 105 ml was collected. The xanthurenic acid fraction thus obtained (105 ml) was concentrated to 15 ml in a rotary evaporator at 6 0 C . Three ml of the sample thus obtained was added to a test tube containing 6 m l of 28% a m m o n i a water and 9 ml of 4 r a M Ca(NO3) 2 in 99.5% methanol. The mixture was shaken and kept at 80"C for 5 min. After cooling in running water, the fluorescence was measured with the abovementioned fluorophotometer. The excitation rays filter, screening filter of ultraviolet rays, and fluorescence-selective filter were AKA-UV-D2, AKA-UV-O2, and A K A - F L - B > respectively.
Pupae or adults were homogenized in 10% trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 20 rain at 4"C. The supernatant was filtered through the above-mentioned membrane filter, and the filtrate was used for the method 2 of 3-hydroxykynurenine determination.
Thin-layer chromatography One-dimensional thin-layer chromatography was carried out on the precoated cellulose plate (Merck No. 5716, 20 × 20cm). After development with a mixture of nbutanol, glacial acetic acid, and water (12 : 3 : 5), the plate was examined for fluorescent substance under ultraviolet rays, and then Ehrlich's diazo reaction (Umebachi and Tsuchitani, 1955) was performed.
Determination of 3-hydroxykynurenine This was performed with two kinds of methods. The first one was the method of Linzen (1963) and the second was the method of Inagami (1954). Method 1. To 4 ml of the above-mentioned 80,°/,) ethanol extract, 3.2 ml of 0.6 M potassium phosphate buffer (pH 6.8) and 0.8ml of 0.1 M potassium ferricyanide in 0.1 M potassium phosphate buffer (pH 6.8) were added. After standing in the cold room (about 4°C) for 7 min, 100 mg of ascorbic acid was added. Furthermore, after 3 ml of nbutanol and 0.8 ml of concentrated HC1 were added, the mixture was shaken and centrifuged at 3000 rpm for 5 min. Absorbance of the upper (n-butanol) layer was measured at 500 nm. In the blank, 4 ml of water was used instead of the sample. Method 2. Two ml of the above-mentioned T C A extract was mixed with 0.25 ml of 1% N a N O z, and the absorbance was measured at 400 nm. In the blank, 0.25 ml of water was used instead of the N a N O 2 solution.
Activity of kynurenine hydroxy/ase Pupae or adults were homogenized in 9 vol of 50raM Tris-HC1 buffer (pH 8.1) containing 0.36 M sucrose, 5 m M KCN, and 1 m M dithiothreitol in ice-cold condition. The homogenate was filtered through nylon cloth, and immediately after that, the filtrate was used as an enzyme preparation. The reaction mixture contained the following: (1) 0.5 ml of 4 m M N A D P H in the above-mentioned Tris-HCl buffer containing sucrose, KCN, and dithiothreitol, (2) 0.5 ml of 9 m M L-kynurenine sulphate in the same buffer, and (3) 0.5 ml of the above-mentioned enzyme solution. The blank mixture contained 0.5ml of the above-mentioned buffer instead of N A D P H solution. The incubation was performed at 37°C for 1 hr. The reaction was stopped by adding 1.5ml of 10% trichloroacetic acid. After being centrifuged at 3000rpm for 10min, the supernatant was filtered through the Toyo TM-2P membrane filter. The quantity of 3~hydroxykynurenine in the filtrate was determined by the above-mentioned Method 2.
Determination of xanthurenic acid This is also performed with two kinds of methods. Method 1. The above-mentioned hot-water extract was applied to Dowex 50W XI2 (H +) column (0.9 x 3 cm). The column was washed with 50 ml of 0.2 N HC1 and 100 ml of 0.5 N HC1. Finally, xanthurenic acid was eluted with water (Satoh and Price, 1958), and fractions of 5 ml were collected. To each fraction, one drop of 0.5 M potassium phosphate buffer (pH 7.4) and 5 ml of saturated N a O H solution were added. After standing for l hr, fluorescence of the solution was measured with a Shimazu-Kotaki U M microfluorophotometer. The excitation rays filter, screening filter of ultraviolet rays, and fluorescence-selective filter were A K A UV-D2, A K A - U V - O 2, and AKA-FL-B~, respectively.
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Kynurenine hydroxylase activity and 3-hydroxykynurenine level during pupal and adult stages T h e m u t a n t s bw, st a n d bwst all s h o w e d m u c h t h e s a m e level a n d t e n d e n c y o f t h e k y n u r e n i n e h y d r o x y lase activity (Fig. 1). I n all t h e cases, t h e activity i n c r e a s e s a f t e r p u p a t i o n , r e a c h e s t h e p e a k at t h e
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Fig. l. Kynurenine hydroxylase activities during the pupal and adult stages of bw, st and bwst. P, puparium formation; E, emergence.
Xanthurenic acid in D. melanogaster
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middle or late pupal stage, and then decreases to emergence. In the last larval stage also, the activity was found, though it was only on a low level. Changes of the 3-hydroxykynurenine level during the pupal and adult stages were determined by the Method 2. The results are given in Fig. 2, which shows that bw contains a high level of 3-hydroxykynurenine during both pupal and adult stages and that, in st and bwst, although a considerable amount of 3-hydroxykynurenine is synthesized during pupal stage, most of it is rapidly excreted at the time of or immediately after emergence. The amounts of 3-hydroxykynurenine as determined by the Method 1 in the samples of variouslyaged adults of bw, st, and bwst are given in Fig. 3, which shows that bw contains not a little 3-hydroxykynurenine and that, in st and bwst, the amount is only a small. 300
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Fig. 2. Levelsof 3-hydroxykynurenineduring the pupal and adult stages of bw (©), st (O), and bwst (/k). P, puparium formation; E, emergence.
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Developmental stoge Fig. 4. Levels of xanthurenic acid during the pupal and adult stages of bw (©), st (O), and bwst (/k). P, puparium formation; E, emergence. Each point represents the mean +_SE of three experiments.
Paper chromatographic studies showed that the 3-hydroxykynurenine present in the adult of bw was found in the head and Malpighian tubules, whereas, in the adults of st and bwst, 3-hydroxykynureninewas absent or only a trace in all the parts of body. Xanthurenic acid level during pupal and adult stages
This was determined by the Method 2. As given in Fig. 4, bw and st showed a high level of xanthurenic acid throughout the pupal and adult stages. Also bwst contained xanthurenic acid in the pupal stage, though the amount was only about 70~ of that of bw or st
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bw st bwst Fig. 3. The amounts of 3-hydroxykynurenine in the samples from variously-aged adults of bw, st and bwst. Mean + SE
for the number of experiments shown in parentheses.
bw st bwst Fig. 5. The amounts of xanthurenic acid in the samples from variously-aged adults of bw, st and bwst. Means _+SE for the number of experiments shown in parentheses.
YOSH1SH1GEUMEBACHIel al.
766
(Fig. 4), and one day after emergence, the xanthurenic acid decreased to about 50~ of bw or st. The amounts of xanthurenic acid in the samples from variously-aged adults of bw, st and bwst as determined by the Method 1 are given in Fig. 5, which shows that bw and st contain a considerable amount of xanthurenic acid, while bwst contains only a small amount of it. Interestingly, paper chromatographic studies showed that the xanthurenic acid found in the adult of bw was present in both head and Malpighian tubules, whereas, in st, xanthurenic acid was found only in the head and was absent, or if any present, only a little in Malpighian tubules. In bwst, xanthurenic acid was not detected on the paper chromatogram, indicating that the amount was only small. DISCUSSION In bw, the presence of the high level of both 3-hydroxykynurenine and xanthurenic acid throughout the pupal and adult stages is natural and is what is expected. As to st, not a little 3-hydroxykynurenine is present during the pupal stage, but almost all of it seems to be excreted at or immediately after emergence. This fact coincides with Howells and Ryall's report (1975). Moreover, Howells et al. (1977) have reported that st excretes more 3-hydroxykynurenine than does the wild type. In addition, Beadle (1937) and Sullivan and Sullivan (1975) have reported that the Malpighian tubules and eye of st can scarcely take up kynurenine. These facts give grounds for the hypothesis that st is unable to transport 3-hydroxykynurenine into or unable to store it in the cells of eye disc and Malpighian tubules. Wessing and Eichelberg (1968) have also performed paper chromatographic studies on the tryptophan metabolites in various eye color mutants of D. melanogaster and described that the Malpighian tubules of st lack 3-hydroxykynurenine. In contrast to 3-hydroxykynurenine, xanthurenic acid is present not only in the pupal stage of st but also in its adult. And yet all or almost all of the xanthurenic acid present in adult seem to be in the head. Where in the head and where in the cells is the xanthurenic acid synthesized? What relationship is there between the presence of xanthurenic acid in head and the above-mentioned hypothesis? This is an interesting problem for future research. Moreover, interestingly enough, despite the presence of a considerable amount of xanthurenic acid in the adult of both bw and st, the adult of bwst contains
only a low level of it. This fact cannot be satisfactorily explained at the present time. This may suggest the possible relationship between tryptophan and pteridine metabolism in the eye-pigment cells. This must also be the subject of future research. Anyway, xanthurenic acid in D. melanogaster presents interesting problems from the standpoint of eye pigment formation as well as from the viewpoint of comparative biochemistry of tryptophan metabolism.
REFERENCES
Beadle G. W. (1937) Development of eye colors in Drosophila: fat bodies and Malpighian tubes in relation of diffusible substances. Genetics 22, 587-611. Dickinson W. J. and Sullivan D. T. (1975) Gene Enzyme Systems in Drosophila, pp. 36-39, 41-42. Springer-Verlag, Berlin. Howells A. J. and Ryall R. L. (1975) A biochemical study of the scarlet eye-color mutant of Drosophila melanogaster. Biochem. Genet. 13, 273-282. Howells A. J., Summers K. M. and Ryall R. L. (1977) Developmental patterns of 3-hydroxykynurenine accumulation in white and various other eye color mutants of Drosophila melanogaster. Biochem. Genet. 15, 1049-1059. Inagami K. (1954) Chemical and genetical studies on the formation of the pigment in the silkworm. III. On the microanalysis of 3-hydroxykynurenine. J. seric. Sci., Tokyo 23, 299-303 (in Japanese). Linzen B. (1963) Eine spezifische quantitative Bestimmung des 3-Hydroxykynurenins. 3-Hydroxykynurenin and Xanthommatin in der Imaginalentwicklung von Calliphora. Hoppe-Seyler's Z. physiol. Chem. 333, 145 148. Linzen B. (1974) The tryptophan-ommochrome pathway in insects. In Advances in Insect Physiology Vol. 10 (Edited by Treherne et al.), pp. 117-246. Academic Press, London. Nolte D. J. (1954) The eye-pigmentary system of Drosophila. IV. The pigments of the vermilion group of mutants. J. Genet. 52, 111-126. Satoh K. and Price J. M. (1958) Fluorometric determination of kynurenic acid and xanthurenic acid in human urine. J. biol. Chem. 230, 781-789. Sullivan D. T., Kitos R. J. and Sullivan M. C. (1973) Developmental and genetic studies on kynurenine hydroxylase from Drosophila melanogaster. Genetics 75, 651-661. Sullivan D. T. and Sullivan M. C. (1975) Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster. Biochem. Genet. 13, 603-613. Umebachi Y. and Tsuchitani K. (1955) The presence of xanthurenic acid in the fruit-fly, Drosophila melanogaster. J. Biochem. (Tokyo) 42, 817-824. Wessing A. and Eichelberg D. (1968) Die ftuoreszierenden Stoffe aus den Malpighischen Gef/issen der Wildform und verschiedener Augenfarbenmutanten von Drosophila melanogaster. Z. Naturf. 23b, 376-386.
0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
Printed in Great Britain
XANTHURENIC ACID IN THE MUTANT STRAIN S C A R L E T OF D R O S O P H I L A M E L A N O G A S T E R YOSHISHIGE UMEBACHI,* AKIRA YOKOYAMA,* TAKANORI KAWASHIMA,* TOSHIKO DOI,* MITSUO WATANABE,~"MASAYOSHITABARAt and FUMIO IINUMAt *Department of Biology, Faculty of Science, Kanazawa University, Kanazawa, 920, Japan; and ?Faculty of Pharmaceutical Science, Teikyo University, Sagamiko, Kanagawa, 199-01, Japan (Received 3 May 1985)
Abstraet--l. Xanthurenic acid, 3-hydroxykynurenine, and kynurenine hydroxylase activity in bw, st and bwst of D. melanogaster were determined. 2. Both pupa and adult of bw, st and bwst showed much the same level of kynurenine hydroxylase activity. 3. The pupae of bw, st and bwst contained not a little 3-hydroxykynurenine. In st and bwst, the 3-hydroxykynurenine rapidly decreased at emergence, while in bw, the decrease was rather gradual. 4. In bw and st, a considerable amount of xanthurenic acid was synthesized during the pupal stage, while, in bwst, the synthesis was moderate. Moreover, in the adult stage, xanthurenic acid was much less in bwst than in bw and st.
INTRODUCTION
(Howells et al., 1977), (5) during the pupal stage, st contains much the same level of xanthurenic acid as in the wild type (Howells and Ryall, 1975), and (6) st contains not a little xanthurenic acid in the adult stage (Umebachi and Tsuchitani, 1955). As to bwst, however, no such detailed investigation has been made. The purpose of the present paper is to describe the changes of the levels of 3-hydroxykynurenine, xanthurenic acid, and kynurenine hydroxylase activity during the pupal and adult stages of bwst in comparison with bw and st.
Of the ommochrome-deficient mutants of D. melano gaster, it has well been established that vermilion and cinnabar lack the activity of tryptophan oxygenase (L-tryptophan: oxygen 2,3-oxidoreductase, EC 1.13.11.11) and kynurenine 3-hydroxylase (L-kynurenine, N A D P H : o x y g e n oxidoreductase, EC 1.14.13.9), respectively (Linzen, 1974; Dickinson and Sullivan, 1975). As to the m u t a n t scarlet (st), which also scarcely contains ommochrome and has a vermilion-type eye color, however, its genetic block has not completely been elucidated yet. The strongest possibility at present is that st is unable to transport kynurenine and 3-hydroxykynurenine into or unable to store them in the cells of Malpighian tubules of larvae and those of the eye disc of the developing adult (Beadle, 1937; Sullivan and Sullivan, 1975; Howells and Ryall, 1975; Howells et al., 1977). But, concerning tryptophan metabolism of st, there is a problem which has not been solved yet. It is the problem of xanthurenic acid formation. Umebachi and Tsuchitani (1955) reported for the first time the presence of xanthurenic acid in the adults of D. melanogaster. Strangely enough, it was noted that brown (bw) and st contained not a little xanthurenic acid, whereas the double recessive bwst contained little of it. Since then, the site and way of xanthurenic acid formation in st and the low level of xanthurenic acid in bwst have been interesting problems which remained to be solved. The properties of st reported up to the present concerning tryptophan metabolism may be summarized as follows: (1) st must be able to synthesize 3-hydroxykynurenine (Beadle, 1937), (2) st has the activity of kynurenine hydroxylase (Sullivan et al., 1973), (3) st contains not a little 3-hydroxykynurenine during the pupal stage, whereas adult has none or only a little of it (Howells and Ryall, 1975), (4) st excretes more 3-hydroxykynurenine than does the wild type strain
MATERIALS AND METHODS Materials D. melanogaster was raised at 25°C on the culture medium which contains malted rice (125 g), sugar (50 g), K2HPO4 (0.9g), agar (12.5 g), water (900ml), and raw yeast. The mutant strains used were bw, st and bwst. The eye color of bw, st and bwst is brown, vermilion, and white, respectively. The eye of bw contains xanthommatin but not drosopterins. The eye of st contains drosopterins but not xanthommatin (Nolte, 1954). The eye of bwst contains neither xanthommatin nor drosopterin. Eighty per cent ethanol extraction
Pupae or adults were homogenized in 80% ethanol and centrifuged at 3000 rpm for 5 rain at about 25°C or at 15,000 rpm (26,000g) for 30 min at 4°C. The supernatant was used for thin-layer chromatography, the method 1 of 3-hydroxykynurenine determination or the method 2 of xanthurenic acid determination. Hot-water extraction
Adults were homogenized in distilled water. The homogenate was warmed at 80°C for 10rain and centrifuged at 15,000 rpm (26,000g) for 30 rain at 4°C. The supernatant was filtered through the Toyo TM-2P membrane filter, and the filtrate was submitted to the method 1 of xanthurenic acid determination.
763
YOSHISHIGEUMEBACHIel al.
764
TCA extraction
Method 2. The above-mentioned 80% ethanol extract was evaporated to dryness in a rotary evaporator at 50~'C. The residue was dissolved in 120ml of water, and 30 ml of 1 N HCI was added. The resulting solution (150 ml) was applied to Dowex 50W X12 (H +) column (1 x 3cm). The column was washed with 50 ml of 0.2 N HC1 and then with 100 ml of 0 . 5 N HC1. Finally, xanthurenic acid was eluted with water (Satoh and Price, 1957). The first 12.5 ml of the water effluent was discarded and then the next 105 ml was collected. The xanthurenic acid fraction thus obtained (105 ml) was concentrated to 15 ml in a rotary evaporator at 6 0 C . Three ml of the sample thus obtained was added to a test tube containing 6 m l of 28% a m m o n i a water and 9 ml of 4 r a M Ca(NO3) 2 in 99.5% methanol. The mixture was shaken and kept at 80"C for 5 min. After cooling in running water, the fluorescence was measured with the abovementioned fluorophotometer. The excitation rays filter, screening filter of ultraviolet rays, and fluorescence-selective filter were AKA-UV-D2, AKA-UV-O2, and A K A - F L - B > respectively.
Pupae or adults were homogenized in 10% trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 20 rain at 4"C. The supernatant was filtered through the above-mentioned membrane filter, and the filtrate was used for the method 2 of 3-hydroxykynurenine determination.
Thin-layer chromatography One-dimensional thin-layer chromatography was carried out on the precoated cellulose plate (Merck No. 5716, 20 × 20cm). After development with a mixture of nbutanol, glacial acetic acid, and water (12 : 3 : 5), the plate was examined for fluorescent substance under ultraviolet rays, and then Ehrlich's diazo reaction (Umebachi and Tsuchitani, 1955) was performed.
Determination of 3-hydroxykynurenine This was performed with two kinds of methods. The first one was the method of Linzen (1963) and the second was the method of Inagami (1954). Method 1. To 4 ml of the above-mentioned 80,°/,) ethanol extract, 3.2 ml of 0.6 M potassium phosphate buffer (pH 6.8) and 0.8ml of 0.1 M potassium ferricyanide in 0.1 M potassium phosphate buffer (pH 6.8) were added. After standing in the cold room (about 4°C) for 7 min, 100 mg of ascorbic acid was added. Furthermore, after 3 ml of nbutanol and 0.8 ml of concentrated HC1 were added, the mixture was shaken and centrifuged at 3000 rpm for 5 min. Absorbance of the upper (n-butanol) layer was measured at 500 nm. In the blank, 4 ml of water was used instead of the sample. Method 2. Two ml of the above-mentioned T C A extract was mixed with 0.25 ml of 1% N a N O z, and the absorbance was measured at 400 nm. In the blank, 0.25 ml of water was used instead of the N a N O 2 solution.
Activity of kynurenine hydroxy/ase Pupae or adults were homogenized in 9 vol of 50raM Tris-HC1 buffer (pH 8.1) containing 0.36 M sucrose, 5 m M KCN, and 1 m M dithiothreitol in ice-cold condition. The homogenate was filtered through nylon cloth, and immediately after that, the filtrate was used as an enzyme preparation. The reaction mixture contained the following: (1) 0.5 ml of 4 m M N A D P H in the above-mentioned Tris-HCl buffer containing sucrose, KCN, and dithiothreitol, (2) 0.5 ml of 9 m M L-kynurenine sulphate in the same buffer, and (3) 0.5 ml of the above-mentioned enzyme solution. The blank mixture contained 0.5ml of the above-mentioned buffer instead of N A D P H solution. The incubation was performed at 37°C for 1 hr. The reaction was stopped by adding 1.5ml of 10% trichloroacetic acid. After being centrifuged at 3000rpm for 10min, the supernatant was filtered through the Toyo TM-2P membrane filter. The quantity of 3~hydroxykynurenine in the filtrate was determined by the above-mentioned Method 2.
Determination of xanthurenic acid This is also performed with two kinds of methods. Method 1. The above-mentioned hot-water extract was applied to Dowex 50W XI2 (H +) column (0.9 x 3 cm). The column was washed with 50 ml of 0.2 N HC1 and 100 ml of 0.5 N HC1. Finally, xanthurenic acid was eluted with water (Satoh and Price, 1958), and fractions of 5 ml were collected. To each fraction, one drop of 0.5 M potassium phosphate buffer (pH 7.4) and 5 ml of saturated N a O H solution were added. After standing for l hr, fluorescence of the solution was measured with a Shimazu-Kotaki U M microfluorophotometer. The excitation rays filter, screening filter of ultraviolet rays, and fluorescence-selective filter were A K A UV-D2, A K A - U V - O 2, and AKA-FL-B~, respectively.
~o
.
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Kynurenine hydroxylase activity and 3-hydroxykynurenine level during pupal and adult stages T h e m u t a n t s bw, st a n d bwst all s h o w e d m u c h t h e s a m e level a n d t e n d e n c y o f t h e k y n u r e n i n e h y d r o x y lase activity (Fig. 1). I n all t h e cases, t h e activity i n c r e a s e s a f t e r p u p a t i o n , r e a c h e s t h e p e a k at t h e
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Fig. l. Kynurenine hydroxylase activities during the pupal and adult stages of bw, st and bwst. P, puparium formation; E, emergence.
Xanthurenic acid in D. melanogaster
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middle or late pupal stage, and then decreases to emergence. In the last larval stage also, the activity was found, though it was only on a low level. Changes of the 3-hydroxykynurenine level during the pupal and adult stages were determined by the Method 2. The results are given in Fig. 2, which shows that bw contains a high level of 3-hydroxykynurenine during both pupal and adult stages and that, in st and bwst, although a considerable amount of 3-hydroxykynurenine is synthesized during pupal stage, most of it is rapidly excreted at the time of or immediately after emergence. The amounts of 3-hydroxykynurenine as determined by the Method 1 in the samples of variouslyaged adults of bw, st, and bwst are given in Fig. 3, which shows that bw contains not a little 3-hydroxykynurenine and that, in st and bwst, the amount is only a small. 300
I
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Fig. 2. Levelsof 3-hydroxykynurenineduring the pupal and adult stages of bw (©), st (O), and bwst (/k). P, puparium formation; E, emergence.
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1 (Days) Adult
Developmental stoge Fig. 4. Levels of xanthurenic acid during the pupal and adult stages of bw (©), st (O), and bwst (/k). P, puparium formation; E, emergence. Each point represents the mean +_SE of three experiments.
Paper chromatographic studies showed that the 3-hydroxykynurenine present in the adult of bw was found in the head and Malpighian tubules, whereas, in the adults of st and bwst, 3-hydroxykynureninewas absent or only a trace in all the parts of body. Xanthurenic acid level during pupal and adult stages
This was determined by the Method 2. As given in Fig. 4, bw and st showed a high level of xanthurenic acid throughout the pupal and adult stages. Also bwst contained xanthurenic acid in the pupal stage, though the amount was only about 70~ of that of bw or st
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for the number of experiments shown in parentheses.
bw st bwst Fig. 5. The amounts of xanthurenic acid in the samples from variously-aged adults of bw, st and bwst. Means _+SE for the number of experiments shown in parentheses.
YOSH1SH1GEUMEBACHIel al.
766
(Fig. 4), and one day after emergence, the xanthurenic acid decreased to about 50~ of bw or st. The amounts of xanthurenic acid in the samples from variously-aged adults of bw, st and bwst as determined by the Method 1 are given in Fig. 5, which shows that bw and st contain a considerable amount of xanthurenic acid, while bwst contains only a small amount of it. Interestingly, paper chromatographic studies showed that the xanthurenic acid found in the adult of bw was present in both head and Malpighian tubules, whereas, in st, xanthurenic acid was found only in the head and was absent, or if any present, only a little in Malpighian tubules. In bwst, xanthurenic acid was not detected on the paper chromatogram, indicating that the amount was only small. DISCUSSION In bw, the presence of the high level of both 3-hydroxykynurenine and xanthurenic acid throughout the pupal and adult stages is natural and is what is expected. As to st, not a little 3-hydroxykynurenine is present during the pupal stage, but almost all of it seems to be excreted at or immediately after emergence. This fact coincides with Howells and Ryall's report (1975). Moreover, Howells et al. (1977) have reported that st excretes more 3-hydroxykynurenine than does the wild type. In addition, Beadle (1937) and Sullivan and Sullivan (1975) have reported that the Malpighian tubules and eye of st can scarcely take up kynurenine. These facts give grounds for the hypothesis that st is unable to transport 3-hydroxykynurenine into or unable to store it in the cells of eye disc and Malpighian tubules. Wessing and Eichelberg (1968) have also performed paper chromatographic studies on the tryptophan metabolites in various eye color mutants of D. melanogaster and described that the Malpighian tubules of st lack 3-hydroxykynurenine. In contrast to 3-hydroxykynurenine, xanthurenic acid is present not only in the pupal stage of st but also in its adult. And yet all or almost all of the xanthurenic acid present in adult seem to be in the head. Where in the head and where in the cells is the xanthurenic acid synthesized? What relationship is there between the presence of xanthurenic acid in head and the above-mentioned hypothesis? This is an interesting problem for future research. Moreover, interestingly enough, despite the presence of a considerable amount of xanthurenic acid in the adult of both bw and st, the adult of bwst contains
only a low level of it. This fact cannot be satisfactorily explained at the present time. This may suggest the possible relationship between tryptophan and pteridine metabolism in the eye-pigment cells. This must also be the subject of future research. Anyway, xanthurenic acid in D. melanogaster presents interesting problems from the standpoint of eye pigment formation as well as from the viewpoint of comparative biochemistry of tryptophan metabolism.
REFERENCES
Beadle G. W. (1937) Development of eye colors in Drosophila: fat bodies and Malpighian tubes in relation of diffusible substances. Genetics 22, 587-611. Dickinson W. J. and Sullivan D. T. (1975) Gene Enzyme Systems in Drosophila, pp. 36-39, 41-42. Springer-Verlag, Berlin. Howells A. J. and Ryall R. L. (1975) A biochemical study of the scarlet eye-color mutant of Drosophila melanogaster. Biochem. Genet. 13, 273-282. Howells A. J., Summers K. M. and Ryall R. L. (1977) Developmental patterns of 3-hydroxykynurenine accumulation in white and various other eye color mutants of Drosophila melanogaster. Biochem. Genet. 15, 1049-1059. Inagami K. (1954) Chemical and genetical studies on the formation of the pigment in the silkworm. III. On the microanalysis of 3-hydroxykynurenine. J. seric. Sci., Tokyo 23, 299-303 (in Japanese). Linzen B. (1963) Eine spezifische quantitative Bestimmung des 3-Hydroxykynurenins. 3-Hydroxykynurenin and Xanthommatin in der Imaginalentwicklung von Calliphora. Hoppe-Seyler's Z. physiol. Chem. 333, 145 148. Linzen B. (1974) The tryptophan-ommochrome pathway in insects. In Advances in Insect Physiology Vol. 10 (Edited by Treherne et al.), pp. 117-246. Academic Press, London. Nolte D. J. (1954) The eye-pigmentary system of Drosophila. IV. The pigments of the vermilion group of mutants. J. Genet. 52, 111-126. Satoh K. and Price J. M. (1958) Fluorometric determination of kynurenic acid and xanthurenic acid in human urine. J. biol. Chem. 230, 781-789. Sullivan D. T., Kitos R. J. and Sullivan M. C. (1973) Developmental and genetic studies on kynurenine hydroxylase from Drosophila melanogaster. Genetics 75, 651-661. Sullivan D. T. and Sullivan M. C. (1975) Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster. Biochem. Genet. 13, 603-613. Umebachi Y. and Tsuchitani K. (1955) The presence of xanthurenic acid in the fruit-fly, Drosophila melanogaster. J. Biochem. (Tokyo) 42, 817-824. Wessing A. and Eichelberg D. (1968) Die ftuoreszierenden Stoffe aus den Malpighischen Gef/issen der Wildform und verschiedener Augenfarbenmutanten von Drosophila melanogaster. Z. Naturf. 23b, 376-386.