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Melanosomes from liver and skin of Rana esculenta L. A comparative chemical study
Comp. Biochem. Physiol. Vol. 90B, No. 2, pp. 397-400, 1988 Printed in Great Britain
0305-0491/88 $3.00+0.00 © 1988PergamonPress plc
MELANOSOMES FROM LIVER A N D SKIN OF RANA ESCULENTA L. A COMPARATIVE CHEMICAL S T U D Y SEBASTIANOSCIUTO,*~ ROSA CHILLEMI,* ANGELA PATTI,* GIOVANNI SICHEL~" and MARINA SCALIA$ *Dipartimento di Scienze Chimiche, University of Catania and $Istituto di Biologia generale, University of Catania, Italy (Tel.: 095 33-05-33) (Recewed 22 June 1987)
Abstract--1. Melanosomes from skin and liver ofRana esculenta L. have been isolated and some chemical properties of the relevant melanin and protein components were compared. 2. In both cases the pigments show spectroscopic (ESR) and chemical characteristics similar to those of eumelanins. The melanin content in skin melanosomes is higher than in the liver counterparts. 3. Amino acid patterns of the two protein components are different in their quantitative composition and both are characterized by high levels of glycine and proline. 4. The results as a whole indicate that skin and liver melanosomesfrom the same animal markedly differ in their chemical composition.
INTRODUCTION Our previous work dealt with the dark brown melanin contained in the Kupffer cells of the liver of Amphibia and Reptilia. We mainly devoted our attention on the liver melanin of Rana esculenta L. and compared some features of this pigment with those of melanin pigment which occurs in the skin of the same animal. In some of these investigations the two pigments showed curious dissimilarities in their features. Among them, in a study on the seasonal dependence of melanin content in frog liver (Cicero et al., 1977), it was shown that pigmentation increases in the cold periods of the year and decreases when it is warm. On the contrary, the melanin content in the skin of the same animal did not show significant variation during the year. Further, during an investigation on the ESR features of these pigments (Sichel et al., 1981), the spin density values found for isolated liver melanins were markedly lower than those obtained from the skin. Up to now, very few studies have been made on the chemical composition of liver and skin melanins of Rana esculenta; one explanation of the observed dissimilarities could lie in possible differences in the chemical structure of these pigments. On the other hand, one cannot exclude the possibility that the two pigments could have the same chemical structure but perform different metabolic functions, depending on their different location in the body. Moreover, the metabolic functions performed by these pigments could be strictly related to the chemical composition and ultrastructure of the melanosomes in which melanins are localized in both Kupffer cells and cutaneous melanocytes. But the chemical composition of these organelles is not yet known; the only :[:To whom all correspondence should be addressed. 397
investigation that has been carried out on the liver melanosomes of R. esculenta aimed at detecting any tyrosinase activity in their protein matrix (Cicero et aL, 1985), Therefore, it seemed interesting to undertake a comparative chemical study on isolated melanosomes from both Kupffer cells and cutaneous melanocytes. We report here the results of our investigation.
MATERIALS AND METHODS Isolation of melanosomes Wild specimens of Rana esculenta were caught near Catania, transferred to the laboratory and immediately killed by decapitation. Liver and skin were excised and separately processed. Livers were directly homogenized (1/5, v/v) in 0.25 M sucrose using a Polytron homogenizer; skins were scraped to remove pigmented cells and the latter were homogenized (1/10, v/v) in 0.1 M EDTA and 0.067M phosphate buffer at pH 7.4, using a Thomas Teflon-glass homogenizer. The homogenates were then centrifuged at 200g for 15min and the supernatants were subjected to 5000g for 15 min in order to separate the melanosomial fractions. These fractions were purified at 26,900g for 1 hr in a discontinuous gradient of sucrose (1-2 M). The purified melanosomial pellet was suspended in 10 mM sodium deoxycholate and I0 mM sodium laurilsulphate and treated by sonication. The resulting suspension was centrifuged at 50008 for 15min and the pellet was repeatedly washed, suspended and again centrifuged at 26,900g for 1 hr in a discontinuous gradient of sucrose (1-2 M). Melanosomes were lyophilized and weighed or directly used for electron microscopy and EPR analysis. Electron microscopy Samples of isolated melanosomes were dried by the critical point method, with CO2 as the transition fluid and then coated with a thin layer of gold-palladium and sputtered using an Emscope SM 300. The samples were examined with a Cambridge Stereo Scan 150 MK 2 scanning electron microscope (Fig. 1).
398
SEBAST1ANO SCIUTO et al.
a
Fig. 1. Electron micrographs of melanosomes isolated from liver (a) and skin (b) of Rana esculenta. Final magnification 40,000 × .
E S R measurement
For ESR measurements, melanosome samples were suspended in 3 mM ZnSO4. ESR absorption derivative X-band spectra were recorded on a Bruker ER 200 D spectrometer operating at 100 KHz modulation frequency and about 3.2 Gauss modulation amplitude. The g-value of resonance line was determined by intercalibration with an intercomparison standard of polycrystallyne DPPH (g = 2.0036). The obtained spectra are shown in Fig. 2 and the g values were g = 2.006 _+ 0.001 and g = 2.004 + 0.001 for skin and liver melanosomes, respectively. Isolation and chemical degradations o f melanins
To isolate the liver and skin melanins, the corresponding melanosomes were treated with HC1 6N in a sealed tube under N 2 for 20 hr at 120°C. After cooling, this suspension was centrifuged and melanin washed until washings were neutral and then lyophilized. The amount of melanin recovered was 49.3 and 50.4% of the total weight for liver and skin, respectively. Samples of melanin were dried in vacuo over P2Os for 48 hr at room temperature and analyzed for C,N,S by Elemental Analyzer (Carlo Erba). Alkaline fusion of melanins was carried out as previously reported by Piattelli et al. (1963); 5,6-dihydroxyindole was identified by paper chromatography using aqueous Fast Red B Salt (Fluka) (1-amino,2-methoxy,4-nitrobenzene diazotated) as chromogenic agent. Pigments were oxidized with 3% K M n O 4 according to Piattelli et al. (1962) and degradation products were analyzed by TLC SiO2
(benzene-acetic acid 50:50) using Fast Red B Salt as spraying agent. Treatment of melanin samples with 57% HI (Novellino et al., 1981) did not give detectable amounts of 3-hydroxy,4-aminophenylalanine and fl-6-(4-hydroxybenzothiazolyl)-alanine. All chromatographic analyses were carried out in comparison with authentic samples. The results of chemical degradations and elemental analyses are reported in Table 1. Hydrolysis o f protein component of melanosomes and amino acids analysis
Samples of melanosomes were hydrolyzed with HC1 6N (0.2 ml/mg of melanosomes) under N 2 for 20 hr at 120°C in the presence o f fl-mercaptoethanol (50/H/I ml HC1 6N) as reducing agent. Melanin was removed by centrifugation
a
i
.b
Table I. Result of chemical degradations and elemental analyses of melanins isolated from liver and skin melanosomes of Rana esculenta Liver Skin 5,6-dihydroxyindole* pyrrole-2,3-dicarboxylic acidt pyrrole-2,3,5-tricarboxylic acidt pyrrole-2,3,4,5-tetracarboxylic acid~" 3-hydroxy-4-amino-phenylalanine:~ f1-6-(4-hydroxy-benzothiazolyl)-alanine:~ Elemental analysis C% H% N% S%
+ + + + -
+ + + + -
54.59 4.28 6.17
47.11 2.97 5.87
1.90
1.60
*Alkaline fusion; ?permanganic oxidation; ~HI degradation.
I
3230
3240
3260
3260
3270
Fig. 2. ESR spectra of whole melanosomes from liver (a) and skin (b) of Rana esculenta.
Frog melanosomes and the hydrolyzate was taken to dryness and the residue dried under vacuum over KOH. HPLC amino acid analyses were performed by the pre-column derivatization method using o-phtalaldeide/fl-mercaptoethanol reagent. A Spectra Physics Model SP 8700 high performance liquid chromatograph equipped with a 10/~1 loop injector and fitted with a variable wavelength u.v. detector (u.v. 50 Varian) set at 330 nm was used with a Hypersil ODS column (HewlettPackard 100 x 4.6 mm i.d.). Amino acid derivatization and separation of amino acid derivatives by gradient elution with a binary solvent system were carried out according to Turnell and Cooper (1982). Amino acid derivatives were identified by their retention times relative to the reference peak produced by homoserine and quantified by comparing their peak areas with that of the internal standard. Amino acids were also analyzed by gas chromatographic method as their N-trifluoroacetyl n-butyl esters. Fused silica capillary column coated with OV-101 (30 m; i.d. 0.25mm; film 0.25pro) installed in a Fractovap 2900 Series gas chromatograph (Carlo Erba) equipped with a flame ionization detector and a splitter injector was used for analysis. Gas chromatographic conditions included: injector temperature 150°C, detector temperature 250°C, oven initial temperature 80°C, oven final temperature 230°C, programming rate 6°C/min, carrier gas (nitrogen) flow rate 1 ml/min. RESULTS AND DISCUSSION Melanosomes from the skin and liver of Rana esculenta were isolated by density gradient separation and their ultrastructural integrity and purity were directly evaluated by means of electron microscopy (Fig. 1). The liver melanosomes had a size (0.8/~m, max length) which was nearly two-fold that of the skin ones and both these organelles were ellipsoidal shaped. Whole melanosomes from either the liver or the skin gave single line ESR spectra as reported in Fig. 2. Both these spectra, which closely resemble those obtained by Sealy et aL (1982) from pure eumelanin under the same experimental conditions, are typical of semiquinones in dopamelanin and do not show any evidence of semiquinonimine species. These results indicated that the pigment in both the melanosomes was an eumelanin with little or no contribution of any phaeomelanin component. Elemental analysis and chemical degradations of the isolated melanins (Table 1) were in agreement with this result. In fact, the low sulphur content, the absence of 3-hydroxy-4-aminophenylalanineand fl-6(4-hydroxy-benzothiazolyl)-alanine among the degradation products following acid hydrolysis, in addition to the results of permanganic oxidation and alkali fusion, indicated that the melanins isolated from the liver or skin melanosomes had quite similar chemical features and that both these pigments had to be considered as dopamelanins. Obviously, these results do not indicate that the liver and skin melanins have the same chemical structure. The protein components of the isolated melanosomes were also studied with regards to their amino acid composition. Amino acid analyses of the hydrolyzates from melanosomes, carried out by gas chromatography and high performance liquid chromatography clearly gave different results between the skin and liver melanosomes (Table 2). Although both the amino acid patterns were characterized by high con-
399
Table 2. Amino acid compositionof the protein componentsof liver and skin melanosomesfrom Rana esculenta Amino acid Liver melanosomes Skin melanosomes ALA 6.649 6.571 ARG 5.169 4.802 ASN-ASP 9.782 7.933 CYSH 6.773 4.345 GLN-GLU l 1.043 8.51l GLY 12.625 30.045 HIS 2.809 1.578 ILE 3.688 2.436 LEU 5.376 3.652 LYS 3.156 4.200 MET 2.760 0.830 PHE 3.002 2.190 PRO 6.796 9.002 HY-PRO 0.910 3.402 SER 5.154 4,565 THR 4.653 2.084 TYR 3.357 1,630 VAL 6.297 2.493 The valuesare expressed as mol % of everyindividualaminoacid from the sum (100%) of all aminoacid recoveredafter hydrolysis.
tents of glycine and proline and the presence of the uncommon amino acid hydroxyproline, the amounts of these three amino acids in the skin melanosomes were remarkably higher than in the liver melanosomes. Abnormal levels of the amino acid cysteine, were also found in the hydrolyzates of both melanosomes, although higher values were found in those of the liver. Similar amino acid patterns had been found by Duchon et aL (1973) in melanosomes from "Stanford" melanomas and from ox choroid in a study on the chemical composition of ten kinds of various melanosomes, but the authors did not find hydroxyproline in any of the investigated melanosomes. This amino acid is typically found in collagen protein together with hydroxylysine; however, no trace of the latter amino acid was found in the hydrolyzates of the skin or liver melanosomes, and this, in addition to the results from the electron microscopy analysis, excluded a possible contamination by collagen protein. The unusual amino acid dihydroxyphenylalanine, sometimes found in trace amounts in various hydrolyzates of melanosomes (Duchon et al., 1973; Takahashi and Fitzpatrick, 1966), was not found in the samples we analyzed. The total amount of protein, calculated as the sum of amino acids recovered after acid hydrolysis, were 27 and 56% of the total weight of the melanosomes for the liver and the skin respectively. As the amount of recovered melanins was, in each case, near 50% of the whole melanosomes (see experimental), this strongly suggests that the liver melanosomes contain, in addition to melanins and protein matrix, some other substances as yet not identified. From all the data obtained from this comparative study, it seems clear that the melanosomes from cutaneous melanocytes and from Kupffer cells of R. esculenta differ remarkably in their size as well as in their chemical composition. As far as we know, this is the first report on such differences between melanosomes from pigmented cells which are located in different regions of the body of the same animal.
SEBASTIANO SCIUTO et al.
400
In the light of these results, the observed dissimilarities in some features of the liver and skin melanins mentioned in the Introduction, could be explained in terms of differences in the chemical composition and perhaps in the metabolic function of the relevant melanosomes. Acknowledgements--This work was partially supported by a grant from Ministero Pubblica Istruzione (Rome).
REFERENCES
Cicero R., Corsaro C., Sichel G. and Chillemi R. (1977) Correlazione fra variazioni di melanine epatiche e cutanee e variazioni stagionali. Boll. $oc. ital. Biol. sper. 53, 1772-1777. Cicero R., Maida I., Mallardi A. and Pintucci G. (1985) Melanin biosynthesis in the Kupffer cells of amphibia. Evidence for a tyrosinase system--VI. European Workshop on Melanin Pigmentation. Murcia, Spain. Cicero R., Sciuto S., Chillemi R. and Sichel G. (1982) Melanosynthesis in the Kupffer ceils of amphibia. Comp. Biochem. Physiol. 73A, 477-479. Duchon J., Borovansky J. and Hach P. (1973) Chemical composition of ten kinds of various melanosomes. Pigment cell 1, 165-170.
Novellino E., Ortonne J, P., Voulot C., Chioccara F., Misuraca G. and Prota G. (1981) Identification of cysteinyldopa-derived units in eumelanins from mammalian eyes. FEBS Lett. 125, 101-103. Piattelli M., Fattorusso E., Magno S. and Nicolaus R. A. (1962) The structure of melanins and melanogenesis--II. Sepiomelanin and synthetic pigments. Tetrahedron 18, 941-949. PiatteUi M., Fattorusso E., Magno S. and Nicolaus R. A. (1963) The structure of melanins and melanogenesis--IIl. The structure of sepiomelanin. Tetrahedron 19, 20612072. Sealy R. C., Hyde J. S., Felix C. C., Menon I. A., Prota G., Swartz H. M., Persad S. and Haberman H. F. (1982) Novel free radicals in synthetic and natural pheomelanins: distinction between dopa melanins and cysteinyldopa melanins by ESR spectroscopy. Proc. Natn. Acad. Sci. 79, 2885-2890. Sichel G., Brai M., Palminteri M. C. and Sciuto S. (1981) Seasonal dependence of ESR features of frog melanins. Comp. Biochem. Physiol. 7013, 611~513. Takahashi H. and Fitzpatrick T. B. (1966) Large amounts of deoxyphenylalanine in the hydrolysate of melanosomes from Harding-Passey mouse melanoma. Nature 209, 888-890. Turnell D. C. and Cooper J. D. H. (1982) Rapid assay for amino acids in serum or urine by pre-column derivatization and reversed-phase liquid chromatography. Clin. Chem. 28, 527-531.
0305-0491/88 $3.00+0.00 © 1988PergamonPress plc
MELANOSOMES FROM LIVER A N D SKIN OF RANA ESCULENTA L. A COMPARATIVE CHEMICAL S T U D Y SEBASTIANOSCIUTO,*~ ROSA CHILLEMI,* ANGELA PATTI,* GIOVANNI SICHEL~" and MARINA SCALIA$ *Dipartimento di Scienze Chimiche, University of Catania and $Istituto di Biologia generale, University of Catania, Italy (Tel.: 095 33-05-33) (Recewed 22 June 1987)
Abstract--1. Melanosomes from skin and liver ofRana esculenta L. have been isolated and some chemical properties of the relevant melanin and protein components were compared. 2. In both cases the pigments show spectroscopic (ESR) and chemical characteristics similar to those of eumelanins. The melanin content in skin melanosomes is higher than in the liver counterparts. 3. Amino acid patterns of the two protein components are different in their quantitative composition and both are characterized by high levels of glycine and proline. 4. The results as a whole indicate that skin and liver melanosomesfrom the same animal markedly differ in their chemical composition.
INTRODUCTION Our previous work dealt with the dark brown melanin contained in the Kupffer cells of the liver of Amphibia and Reptilia. We mainly devoted our attention on the liver melanin of Rana esculenta L. and compared some features of this pigment with those of melanin pigment which occurs in the skin of the same animal. In some of these investigations the two pigments showed curious dissimilarities in their features. Among them, in a study on the seasonal dependence of melanin content in frog liver (Cicero et al., 1977), it was shown that pigmentation increases in the cold periods of the year and decreases when it is warm. On the contrary, the melanin content in the skin of the same animal did not show significant variation during the year. Further, during an investigation on the ESR features of these pigments (Sichel et al., 1981), the spin density values found for isolated liver melanins were markedly lower than those obtained from the skin. Up to now, very few studies have been made on the chemical composition of liver and skin melanins of Rana esculenta; one explanation of the observed dissimilarities could lie in possible differences in the chemical structure of these pigments. On the other hand, one cannot exclude the possibility that the two pigments could have the same chemical structure but perform different metabolic functions, depending on their different location in the body. Moreover, the metabolic functions performed by these pigments could be strictly related to the chemical composition and ultrastructure of the melanosomes in which melanins are localized in both Kupffer cells and cutaneous melanocytes. But the chemical composition of these organelles is not yet known; the only :[:To whom all correspondence should be addressed. 397
investigation that has been carried out on the liver melanosomes of R. esculenta aimed at detecting any tyrosinase activity in their protein matrix (Cicero et aL, 1985), Therefore, it seemed interesting to undertake a comparative chemical study on isolated melanosomes from both Kupffer cells and cutaneous melanocytes. We report here the results of our investigation.
MATERIALS AND METHODS Isolation of melanosomes Wild specimens of Rana esculenta were caught near Catania, transferred to the laboratory and immediately killed by decapitation. Liver and skin were excised and separately processed. Livers were directly homogenized (1/5, v/v) in 0.25 M sucrose using a Polytron homogenizer; skins were scraped to remove pigmented cells and the latter were homogenized (1/10, v/v) in 0.1 M EDTA and 0.067M phosphate buffer at pH 7.4, using a Thomas Teflon-glass homogenizer. The homogenates were then centrifuged at 200g for 15min and the supernatants were subjected to 5000g for 15 min in order to separate the melanosomial fractions. These fractions were purified at 26,900g for 1 hr in a discontinuous gradient of sucrose (1-2 M). The purified melanosomial pellet was suspended in 10 mM sodium deoxycholate and I0 mM sodium laurilsulphate and treated by sonication. The resulting suspension was centrifuged at 50008 for 15min and the pellet was repeatedly washed, suspended and again centrifuged at 26,900g for 1 hr in a discontinuous gradient of sucrose (1-2 M). Melanosomes were lyophilized and weighed or directly used for electron microscopy and EPR analysis. Electron microscopy Samples of isolated melanosomes were dried by the critical point method, with CO2 as the transition fluid and then coated with a thin layer of gold-palladium and sputtered using an Emscope SM 300. The samples were examined with a Cambridge Stereo Scan 150 MK 2 scanning electron microscope (Fig. 1).
398
SEBAST1ANO SCIUTO et al.
a
Fig. 1. Electron micrographs of melanosomes isolated from liver (a) and skin (b) of Rana esculenta. Final magnification 40,000 × .
E S R measurement
For ESR measurements, melanosome samples were suspended in 3 mM ZnSO4. ESR absorption derivative X-band spectra were recorded on a Bruker ER 200 D spectrometer operating at 100 KHz modulation frequency and about 3.2 Gauss modulation amplitude. The g-value of resonance line was determined by intercalibration with an intercomparison standard of polycrystallyne DPPH (g = 2.0036). The obtained spectra are shown in Fig. 2 and the g values were g = 2.006 _+ 0.001 and g = 2.004 + 0.001 for skin and liver melanosomes, respectively. Isolation and chemical degradations o f melanins
To isolate the liver and skin melanins, the corresponding melanosomes were treated with HC1 6N in a sealed tube under N 2 for 20 hr at 120°C. After cooling, this suspension was centrifuged and melanin washed until washings were neutral and then lyophilized. The amount of melanin recovered was 49.3 and 50.4% of the total weight for liver and skin, respectively. Samples of melanin were dried in vacuo over P2Os for 48 hr at room temperature and analyzed for C,N,S by Elemental Analyzer (Carlo Erba). Alkaline fusion of melanins was carried out as previously reported by Piattelli et al. (1963); 5,6-dihydroxyindole was identified by paper chromatography using aqueous Fast Red B Salt (Fluka) (1-amino,2-methoxy,4-nitrobenzene diazotated) as chromogenic agent. Pigments were oxidized with 3% K M n O 4 according to Piattelli et al. (1962) and degradation products were analyzed by TLC SiO2
(benzene-acetic acid 50:50) using Fast Red B Salt as spraying agent. Treatment of melanin samples with 57% HI (Novellino et al., 1981) did not give detectable amounts of 3-hydroxy,4-aminophenylalanine and fl-6-(4-hydroxybenzothiazolyl)-alanine. All chromatographic analyses were carried out in comparison with authentic samples. The results of chemical degradations and elemental analyses are reported in Table 1. Hydrolysis o f protein component of melanosomes and amino acids analysis
Samples of melanosomes were hydrolyzed with HC1 6N (0.2 ml/mg of melanosomes) under N 2 for 20 hr at 120°C in the presence o f fl-mercaptoethanol (50/H/I ml HC1 6N) as reducing agent. Melanin was removed by centrifugation
a
i
.b
Table I. Result of chemical degradations and elemental analyses of melanins isolated from liver and skin melanosomes of Rana esculenta Liver Skin 5,6-dihydroxyindole* pyrrole-2,3-dicarboxylic acidt pyrrole-2,3,5-tricarboxylic acidt pyrrole-2,3,4,5-tetracarboxylic acid~" 3-hydroxy-4-amino-phenylalanine:~ f1-6-(4-hydroxy-benzothiazolyl)-alanine:~ Elemental analysis C% H% N% S%
+ + + + -
+ + + + -
54.59 4.28 6.17
47.11 2.97 5.87
1.90
1.60
*Alkaline fusion; ?permanganic oxidation; ~HI degradation.
I
3230
3240
3260
3260
3270
Fig. 2. ESR spectra of whole melanosomes from liver (a) and skin (b) of Rana esculenta.
Frog melanosomes and the hydrolyzate was taken to dryness and the residue dried under vacuum over KOH. HPLC amino acid analyses were performed by the pre-column derivatization method using o-phtalaldeide/fl-mercaptoethanol reagent. A Spectra Physics Model SP 8700 high performance liquid chromatograph equipped with a 10/~1 loop injector and fitted with a variable wavelength u.v. detector (u.v. 50 Varian) set at 330 nm was used with a Hypersil ODS column (HewlettPackard 100 x 4.6 mm i.d.). Amino acid derivatization and separation of amino acid derivatives by gradient elution with a binary solvent system were carried out according to Turnell and Cooper (1982). Amino acid derivatives were identified by their retention times relative to the reference peak produced by homoserine and quantified by comparing their peak areas with that of the internal standard. Amino acids were also analyzed by gas chromatographic method as their N-trifluoroacetyl n-butyl esters. Fused silica capillary column coated with OV-101 (30 m; i.d. 0.25mm; film 0.25pro) installed in a Fractovap 2900 Series gas chromatograph (Carlo Erba) equipped with a flame ionization detector and a splitter injector was used for analysis. Gas chromatographic conditions included: injector temperature 150°C, detector temperature 250°C, oven initial temperature 80°C, oven final temperature 230°C, programming rate 6°C/min, carrier gas (nitrogen) flow rate 1 ml/min. RESULTS AND DISCUSSION Melanosomes from the skin and liver of Rana esculenta were isolated by density gradient separation and their ultrastructural integrity and purity were directly evaluated by means of electron microscopy (Fig. 1). The liver melanosomes had a size (0.8/~m, max length) which was nearly two-fold that of the skin ones and both these organelles were ellipsoidal shaped. Whole melanosomes from either the liver or the skin gave single line ESR spectra as reported in Fig. 2. Both these spectra, which closely resemble those obtained by Sealy et aL (1982) from pure eumelanin under the same experimental conditions, are typical of semiquinones in dopamelanin and do not show any evidence of semiquinonimine species. These results indicated that the pigment in both the melanosomes was an eumelanin with little or no contribution of any phaeomelanin component. Elemental analysis and chemical degradations of the isolated melanins (Table 1) were in agreement with this result. In fact, the low sulphur content, the absence of 3-hydroxy-4-aminophenylalanineand fl-6(4-hydroxy-benzothiazolyl)-alanine among the degradation products following acid hydrolysis, in addition to the results of permanganic oxidation and alkali fusion, indicated that the melanins isolated from the liver or skin melanosomes had quite similar chemical features and that both these pigments had to be considered as dopamelanins. Obviously, these results do not indicate that the liver and skin melanins have the same chemical structure. The protein components of the isolated melanosomes were also studied with regards to their amino acid composition. Amino acid analyses of the hydrolyzates from melanosomes, carried out by gas chromatography and high performance liquid chromatography clearly gave different results between the skin and liver melanosomes (Table 2). Although both the amino acid patterns were characterized by high con-
399
Table 2. Amino acid compositionof the protein componentsof liver and skin melanosomesfrom Rana esculenta Amino acid Liver melanosomes Skin melanosomes ALA 6.649 6.571 ARG 5.169 4.802 ASN-ASP 9.782 7.933 CYSH 6.773 4.345 GLN-GLU l 1.043 8.51l GLY 12.625 30.045 HIS 2.809 1.578 ILE 3.688 2.436 LEU 5.376 3.652 LYS 3.156 4.200 MET 2.760 0.830 PHE 3.002 2.190 PRO 6.796 9.002 HY-PRO 0.910 3.402 SER 5.154 4,565 THR 4.653 2.084 TYR 3.357 1,630 VAL 6.297 2.493 The valuesare expressed as mol % of everyindividualaminoacid from the sum (100%) of all aminoacid recoveredafter hydrolysis.
tents of glycine and proline and the presence of the uncommon amino acid hydroxyproline, the amounts of these three amino acids in the skin melanosomes were remarkably higher than in the liver melanosomes. Abnormal levels of the amino acid cysteine, were also found in the hydrolyzates of both melanosomes, although higher values were found in those of the liver. Similar amino acid patterns had been found by Duchon et aL (1973) in melanosomes from "Stanford" melanomas and from ox choroid in a study on the chemical composition of ten kinds of various melanosomes, but the authors did not find hydroxyproline in any of the investigated melanosomes. This amino acid is typically found in collagen protein together with hydroxylysine; however, no trace of the latter amino acid was found in the hydrolyzates of the skin or liver melanosomes, and this, in addition to the results from the electron microscopy analysis, excluded a possible contamination by collagen protein. The unusual amino acid dihydroxyphenylalanine, sometimes found in trace amounts in various hydrolyzates of melanosomes (Duchon et al., 1973; Takahashi and Fitzpatrick, 1966), was not found in the samples we analyzed. The total amount of protein, calculated as the sum of amino acids recovered after acid hydrolysis, were 27 and 56% of the total weight of the melanosomes for the liver and the skin respectively. As the amount of recovered melanins was, in each case, near 50% of the whole melanosomes (see experimental), this strongly suggests that the liver melanosomes contain, in addition to melanins and protein matrix, some other substances as yet not identified. From all the data obtained from this comparative study, it seems clear that the melanosomes from cutaneous melanocytes and from Kupffer cells of R. esculenta differ remarkably in their size as well as in their chemical composition. As far as we know, this is the first report on such differences between melanosomes from pigmented cells which are located in different regions of the body of the same animal.
SEBASTIANO SCIUTO et al.
400
In the light of these results, the observed dissimilarities in some features of the liver and skin melanins mentioned in the Introduction, could be explained in terms of differences in the chemical composition and perhaps in the metabolic function of the relevant melanosomes. Acknowledgements--This work was partially supported by a grant from Ministero Pubblica Istruzione (Rome).
REFERENCES
Cicero R., Corsaro C., Sichel G. and Chillemi R. (1977) Correlazione fra variazioni di melanine epatiche e cutanee e variazioni stagionali. Boll. $oc. ital. Biol. sper. 53, 1772-1777. Cicero R., Maida I., Mallardi A. and Pintucci G. (1985) Melanin biosynthesis in the Kupffer cells of amphibia. Evidence for a tyrosinase system--VI. European Workshop on Melanin Pigmentation. Murcia, Spain. Cicero R., Sciuto S., Chillemi R. and Sichel G. (1982) Melanosynthesis in the Kupffer ceils of amphibia. Comp. Biochem. Physiol. 73A, 477-479. Duchon J., Borovansky J. and Hach P. (1973) Chemical composition of ten kinds of various melanosomes. Pigment cell 1, 165-170.
Novellino E., Ortonne J, P., Voulot C., Chioccara F., Misuraca G. and Prota G. (1981) Identification of cysteinyldopa-derived units in eumelanins from mammalian eyes. FEBS Lett. 125, 101-103. Piattelli M., Fattorusso E., Magno S. and Nicolaus R. A. (1962) The structure of melanins and melanogenesis--II. Sepiomelanin and synthetic pigments. Tetrahedron 18, 941-949. PiatteUi M., Fattorusso E., Magno S. and Nicolaus R. A. (1963) The structure of melanins and melanogenesis--IIl. The structure of sepiomelanin. Tetrahedron 19, 20612072. Sealy R. C., Hyde J. S., Felix C. C., Menon I. A., Prota G., Swartz H. M., Persad S. and Haberman H. F. (1982) Novel free radicals in synthetic and natural pheomelanins: distinction between dopa melanins and cysteinyldopa melanins by ESR spectroscopy. Proc. Natn. Acad. Sci. 79, 2885-2890. Sichel G., Brai M., Palminteri M. C. and Sciuto S. (1981) Seasonal dependence of ESR features of frog melanins. Comp. Biochem. Physiol. 7013, 611~513. Takahashi H. and Fitzpatrick T. B. (1966) Large amounts of deoxyphenylalanine in the hydrolysate of melanosomes from Harding-Passey mouse melanoma. Nature 209, 888-890. Turnell D. C. and Cooper J. D. H. (1982) Rapid assay for amino acids in serum or urine by pre-column derivatization and reversed-phase liquid chromatography. Clin. Chem. 28, 527-531.