Comparison of tyrosinase activity in the integument of xanthic and albino goldfish, Carassius auratus L.

Comp. Biochem. Physiol., 1978, Fol. 60B, pp. 81 to 85. Per qamon Press. Printed in Great Britain

C O M P A R I S O N OF TYROSINASE ACTIVITY IN THE I N T E G U M E N T OF XANTHIC A N D ALBINO GOLDFISH, CARASSIUS AURATUS L.* JOEL ABRAMOWITZand WALTER CHAVIN Department of Biology, Wayne State University, Detroit, MI 48202, U.S.A. (Received 24 June 1977)

Abstract--1. A tyrosinase-positive form of oculocutaneous albinism in the goldfish is described. 2. Both tyrosinase and peroxidase activity are present in the integuments of xanthic and albino goldfish. 3. The lack of integumental melanin pigmentation in both xanthic and albino goldfish is due, in part, to the presence of tyrosinase inhibitors. 4. The tyrosinase inhibitors present in the integument of xanthic and albino goldfish have different properties. 5. Triton X-100 treatment solubilizes xanthic and albino goldfish integumental tyrosinase.

INTRODUCTION In goldfish, albinism is a double recessive trait due to two independently assorting autosomal genes (Yamamoto, 1973). The presence of ocular tyrosinase in a form of oculocutaneous albinism in the goldfish has been described (Abramowitz et al., 1978). The xanthic goldfish normally lacks integumental melanin pigmentation but contains integumental tyrosinase so that melanization may be induced in vivo (Chavin, 1956) and in vitro (Chen et al., 1974). The present study was undertaken to investigate the biochemical mechanism responsible for the amelanotic phenotype in the integuments of xanthic and albino goldfish, Carassius auratus L.

tained radioactivity, the heat inactivated samples, including the membrane filters, were hydrolyzed (6 N HCI, 100°C, 24 hr), diluted to l N HCl with glass-distilled water and recollected as previously described (Chen & Chavin, 1965). Net tyrosinase activity was determined by subtracting the values of DDC-treated enzyme preparation from the values of the untreated enzyme preparation. In order to solubilize the particulate tyrosinase fraction, the pellet was resuspended in 1~ Triton X+100 (v/v) in glass-distilled water and incubated (4°C) with constant stirring. After 18 or 42 hr, the enzyme preparations were centrifuged as above and tyrosinase activities determined. Where applicable, the data are represented as the mean _ the standard error of the mean. Data were analyzed by the group comparison test (Woolf, 1968). Values were considered to be statistically different when P < 0.01.

MATERIALS AND METHODS

RESULTS

Fifteen normal xanthic (I.]-5.3g) and a female albino (l16g) goldfish, Carassius auratus L., were used in this study. Fish were maintained under constant conditions of photoperiod (12hr light, 12hr dark), temperature (25°C) and diet (Moore-Clark trout chow) for at least 8 weeks prior to use. Integumental homogenates were prepared as previously described (Chen& Chavin, 1965) and fractionated into soluble and particulate fractions (105,000g, 0-4°C, 72min). Tyrosinase activity was determined utilizing the radiometric assay of Chen& Chavin (1965), modified so that the specific activity of the uniformly labeled L-[1+C] tyrosine was 0.658 mCi/mM and the incubation time was increased to 24 hr. All assays were performed in triplicate. The control incubations consisted of (a) heat inactivated enzyme (5 or 90rain in boiling water) and (b) active enzyme with catalase (0.1 mg/ml, beef liver, 14400 units/ rag; Sigma) in the presence and absence of 6 mM diethyldithioearbamate (DDC). Additional controls consisted of enzyme preparations preincubated with 6ram DDC (60min, 25°C)followed by dialysis against water (3 x 1000 vol, 30min each, 4°C) to remove residual DDC prior to assay for tyrosinase activity. After quantitation of c o n * Contribution No. 367, Department of Biology, Wayne State University. 81 C+B.P. 6 0 / I n - F

Integumental tyrosinase activity was higher in the albino than in the xanthic goldfish (Table 1). The enzyme was associated with the particulate and soluble fractions in both integumental types. Fractionation of the xanthic goldfish integumental homogenate significantly increased tyrosinase activity in the particulate (545%) and the soluble (124%) fractions, compared to enzymic activity originally present in the homogenate. Fractionation of the albino integumental homogenate resulted in a similar pattern of increased tyrosinase activity; particulate tyrosinase activity increased 142% while soluble tyrosinase activity increased 72% above that present in the albino homogenate. It is clear that fractionation of the homogenates increases the total enzymatic activity. In the xanthic integumental particulate fraction, Triton X-100 decreased enzymatic activity at 18 hr but increased enzymatic activity at 42 hr (Table 2). Upon separation of the 18-hr particulate fraction into solubilized tyrosinase and the remaining insoluble fraction, the total enzymatic activity was increased 3-fold. Compared to the particulate fraction, the tyro-. sinase activity of the insoluble fraction was 165% higher, while the soluble fraction was 55~ lower.

82

JOEL ABRAMOWITZand WALTER CHAVIN

Table 1. Integumental tyrosinase activity in xanthic and albino goldfish Fraction* Phenotype

Homogenate

Particulate

Soluble

Xanthic Albino

14.8 + 0.3I" 43.6 _+ 1.0

95.5 _ 0.9 105.6 + 8.0

33.1 _ 0.4 75.2 _ 3.5

However, the activity of the catalase-treated albino homogenate and xanthic particulate fractions was greater than their respective DDC-treated enzyme preparations. DDC alone and DDC with catalase inhibited enzymatic activity to approximately the same degree. Pretreatment with DDC followed by dialysis, significantly increased tyrosine incorporation, compared to incubation in the presence of DDC in all the enzyme preparations.

* Separated at 105,000g, 72 min, 0-4°C. ?pmoles tyrosine converted to melanin/#g protein nitrogen. Values given are mean values + S.E.M. After 42 br of Triton X-100 treatment, the enzymatic activity in the insoluble fraction was 83~o higher and the solubilized fraction was 44~o lower than that present in the particulate fraction. The total enzymatic activity was increased 2.4-fold by centrifugal separation. The tyrosinase activity of the albino integumental particulate fraction was depressed at 18 hr of Triton X-100 treatment but returned to the initial level at 42 hr (Table 2). However, the tyrosinase activity patterns post-separation differed from those of similarly treated xanthic integument particulate enzyme preparations. Separation of the 18-hr albino particulate fraction resulted in a 2.8 fold increase in total enzymatic activity, the insoluble fraction being increased 56~o above that present in the particulate fraction. No significant difference was present in the solubilized enzyme activity compared to that of the particulate fraction. At 42 hr, the enzymatic activities of the insoluble and solubilized fractions were not significantly different from the activity of the particulate fraction. However, the total enzymatic activity was increased 1.9 fold. Heat-inactivation of the xanthic integumental particulate fraction decreased enzymatic activity (Table 3). However, heat inactivation did not decrease the "enzymatic" activity of the homogenates or other fractions of both types of animals. In fact, heat-inactivation increased the radioactivity of the xanthic homogenate and albino-soluble fractions treated for 90rain, and of the xanthic-soluble fractions treated for 5 min and for 90min (Table 3). Acid hydrolysis of the heat-inactivated preparations resulted in a 85.8-97.5~o reduction in contained tyrosine radiolabel. Compared to the untreated enzyme preparations, catalase depressed the quantity of tyrosine incorporated into melanin in the albino homogenate and xanthic particulate fractions, but did not alter tyrosinase activity in the other enzyme preparations (Table 3).

DISCUSSION

Integumental melanin pigmentation is absent in both xanthic and albino goldfish. However, xanthic goldfish embryos contain integumental melanocytes, whereas albino goldfish embryos do not (Yamamoto, 1973). Further, melanization may be induced in xanthic goldfish in vivo (Chavin, 1956) and in vitro (Chen et at., 1974) after appropriate stimulation. Thus, it is not surprising that adult xanthic goldfish possess integumental tyrosinase activity. Tyrosinase-positive oculocutaneous albinism, as observed in the present study, is well documented, since tyrosinase activity has also been demonstrated in albino teleosts (Hama, 1969; Abramowitz et al., 1976, 1978), frogs (Smith-Gill et al., 1972), mice (Hearing, 1973a; Pomerantz & Li, 1974), rats (Chen & Chavin, 1967; Gaudin & Fellman, 1967; Voulot, 1972; Voulot & Laviolette, 1976), hamsters (Pomerantz & Li, 1974; Voulot & Laviolette, 1976), rabbits (Voulot & Laviolette, 1976) and humans (Hu et al., 1961; Kulgeman & Van Scott, 1961; Witkop et at., 1971; King & Witkop, 1976), The enzyme is found in the particulate and soluble fractions of xanthic and albino goldfish integument. A similar distribution of tyrosinase activity has been demonstrated in the skin of black moor, grey, xanthic and white goldfish (Chen & Chavin, 1966), as well as the eyes of black and albino mice (Hearing, 1973a) and the eyes of xanthic and albino goldfish (Abramowitz et al., 1976, 1978). The absence of melanin formation in albinism has been attributed, in part, to tyrosinase inhibitors (Hu et al., 1961; Kulgeman & Van Scott, 1961; Smith-Gill et al., 1972; Voulot, 1972; Hearing, 1973a; Pomerantz & Li, 1974; Abramowitz et al., 1976, 1978). Such inhibitors appear to be present in the particulate fractions of the xanthic and albino goldfish integuments. Separation of the homogenate into particulate and soluble fractions increases the tyrosinase activities of both fractions. The total enzymatic activity in these fractions is greater than that of the homogenate from which they are derived. In addition, the soluble fraction enzymatic activity is greater than that of the

Table 2, Solubilization of xanthic and albino goldfish integumental particulate tyrosinase by 1% Triton X-100 Xanthic

Albino

Time

Particulate fraction

Insoluble* fraction

Solubilized* fraction

Particulate fraction

Insoluble fraction

Solubilized fraction

0 18hr 42hr

161.2 -t- 0.97 86.8 +_ 10.8 177.8 + 2.1

-229.9 +_ 24.3 325.6 + 3.5

-39.3 +__1.3 99.2 + 19.5

176.5 Jr 8.0 106.0 + ll.l 178.3 ___ 10.1

-165.2 ± 7.1 198.1 + 7.1

-128.3 ± 10.5 141.8 ± 6.5

* Separated at 105,000g, 72 rain, 0-4°C. I"pmoles tyrosine converted to melanin/#g protein nitrogen. Values given are mean values + S.E.M.

Tyrosinase activity in amelanotic goldfish

83

Table 3, Characterization of melanogenic activity in xanthic and albino goldfish integument i

p

,

,

,

Xanthic Treatment

None Boiled 5min Hydrolyzed:[: Boiled 90min Hydrolyzed Catalase

Albino

Homogenate

Particulate*

Soluble*

39.8 _ 0.3t 39.9 -I- 1.0 4.0 + 0.3 48.3 + 1.5 1.8 _ 0.1 36.9 _ 1.0

161.2 + 0.9 131.1 __+4.3 3.3 _ 0.6 120.7 _ 3.5 3.0 _ 0.5 134.6 + 5.1

60.8 _ 84.9 _ 3.3 + 84.4 + 12.0 _ 56.5 _

0.4 1.9 0.6 2.0 1.6 1.3

Homogenate 87.9 _ 87.5 _ 7.9 + 87.3 + 2.9 _ 71.4 +

1.0 2.9 1.9 3.8 0.3 1.2

Particulate

Soluble

176.5 + 8.0 145.1 _ 1.5 3.9 _ 0.2 153.2 + 6.5 4.6 + 0.4 169.8 + 11.8

122.0 __+3.5 129.2 + 6.4 15.4 _ 1.3 146.0 + 1.9 16.6 + 2.3 110.7 + 0.8

70.9 + 2.5 70.3 + 4.7 143.1 + 3.6

46.8 _ 1.0 45.2 _ 0.4 148.0 _ 2.3

(0.1 mg/ml)

DDC (6mM) Catalase + DDC DDC-dialysis§

25.0 + 0.7 23.3 _ 0.6 46.2 + 0.7

61.7 + 3.1 61.0 + 5.8 93.0 + 10.5

27.7 ___0.3 27.0 + 0.4 62.7 ___1.5

44.3 ___ 1.1 40.5 _ 0.9 75.8 __+1.1

* Separated at 105,000g, 72min, 0--4°C. "I"pmoles tyrosine incorporated/pg protein nitrogen. Values given are mean values + S.E.M. :~6 N HCI, 100°C, 24 hr. § Dialyzed against water (3 x 1000 vol, 30 min each, 4°C). homogenate, suggesting that an inhibitor is present in the particulate fraction. A similar distribution of tyrosinase inhibitors has been demonstrated in the eyes of xanthic and albino goldfish (Abramowitz et al., 1976, 1978). Increased enzymatic activity also occurs in the xanthic and albino goldfish integumental particulate fractions, compared to their respective homogenates. This may result from dilution of the inhibitor, as the particulate fraction i s prepared by resuspending the pellet in buffer. Thus, the lack of melanin pigmentation in both xanthic and albino goldfish may result, in part, from the presence of tyrosinase inhibitors. Triton X-100 treatment of xanthic and albino integumental particulate fractions reveals differences and similarities in the nature of the inhibitor(s) present in these fractions. Both particulate fractions treated with Triton X-100 demonstrated reduced enzymatic activity at 18 hr with a subsequent increase in activity at 42 hr. The decreased tyrosinase activity observed at 18hr may result from release or activation of the inhibitor(s) in these fractions. A similar pattern of reduced enzymatic activity with Triton X-100 treatment occurs in albino goldfish ocular particulate tyrosinase activity (Abramowitz et al., 1978). However, extended Triton X-100 treatment of goldfish ocular particulate tyrosinase continues to depress enzymatic activity (Abramowitz et al., 1978), while increased tyrosinase activity is observed with xanthic and albino goldfish integumental particulate tyrosinase. The 42-hr increase in goldfish integumental tyrosinase activity may result from inhibitor inactivation upon storage. Inactivation of tyrosinase inhibitors upon storage has been demonstrated in hagfish, garpike, Australian lungfish ( C h e n & Chavin, 1968), slow loris ( C h e n & Chavin, 1973) and human melanoma tyrosinase preparations ( C h e n & Chavin, 1975). Centrifugation of the Triton X-100 treated particulate fractions in the present study increased the tyrosinase activities of the resultant insoluble fractions, indicating the presence of an inhibitor in these fractions for the reasons suggested above. The solubilization of particulate xanthic and albino integumental tyrosinase may result from dissociation of enzymecontaining membranous structures as suggested pre-

viously (Abramowitz et al., 1978). The lower enzymatic activity observed in the xanthic solubilized fraction compared to the particulate fraction may result, in part, from the solubilization of the inhibitor. However, other factors may also affect the total detectable tyrosinase activity. For example, xanthic particulate integumental tyrosinase may be more strongly bound in the melanosome than albino particulate integumental tyrosinase since centrifugal separation of Triton X-100 treated albino particulate tyrosinase results in the same activity in the solubilized and particulate fractions. This also may explain the lower enzymatic activity of the solubilized fraction compared to its particulate fraction in the xanthic goldfish. Thus, the nature of the inhibitors and mechanism of tyrosinase inhibition present in the xanthic and albino goldfish integument appears to be different. Recently, the roles of tyrosinase and peroxidase in melanogenesis have elicited considerable interest. DDC treatment and its subsequent removal prior to evaluation of tyrosinase activity is used to differentiate tyrosinase- and peroxidase-mediated melanin formation (Okun et al., 1970, 1973, 1976; Hearing, 1973a, b; Hearing & Ekel, 1975; Mufson, 1975; Eppig & Hearing, 1976). In the present study, this approach indicates the presence of peroxidase in the skin of xanthic and albino goldfish, confirming the histochemical findings of Stone & Chavin (1976). However, peroxidase activity is localized in the erythrocytes and the white blood cells, not in melanocytes (Stone & Chavin, 1976). Although catalase depressed the quantity of tyrosine incorporated into melanin in the albino homogenate and the xanthic particulate fraction compared to the untreated enzyme preparation (indicating the presence of peroxidase) the activity of these catalase treated enzyme preparations was greater than the activity of their respective DDC treated enzyme preparations. Further, catalase did not affect the conversion of tyrosine to melanin in the other xanthic and albino integumental preparations, thereby revealing the presence of tyrosinase mediated melanin formation. Unaltered tyrosinase activity with catalase treatment has been reported using murine melanoma tyrosinase (Mufson, 1975), h u m a n ' integumental tyrosinase (Pomerantz & A n c e s , 1975)

84

JOEL ABRAMOWITZand WALTER CHAVIN

and xanthic and albino ocular tyrosinase (Abramowitz et al., 1978), while Hearing (1973b) demonstrated stimulation of murine ocular tyrosinase by catalase. Recently, the validity of heat inactivation control in tyrosinase assays has been questioned (Hearing & Ekel, 1975; Edelstein et al., 1975; Abramowitz et al., 1978). In the present study, heat inactivation increased the radioactivity of the samples above that present in the DDC or DDC-catalase treated samples. Similar findings have been reported for murine melanoma tyrosinase (Hearing & Ekel, 1975) and goldfish ocular tyrosinase (Abramowitz et al., 1978). This increase in radioactivity appears to result from nonspecific binding of tyrosine to denatured protein since acid hydrolysis substantially reduces sample radioactivity. The residual radioactivity in the acid hydrolyzed samples appears to result from humin formation, as this process has been shown to cause the incorporation of [14C] tyrosine into insoluble black radioactive material, even in the absence of proteinaceous material (Chavin, 1964). Thus, as proposed by Abramowitz et al. (1978), use of DDC as a control yields a more accurate assessment of melanogenic activity since (i) treatment of the various enzyme preparations with DDC consistently resulted in lower radioactivity than heat inactivation, (ii) the radioactivity of the heat-inactivated preparations results from nonspecific binding, and (iii) DDC inhibits the enzymes responsible for melanin synthesis. If DDC controls are used in conjunction with catalase controls for elimination of peroxidase activity, the presence of tyrosinase activity may be unequivocally identified. The lack of integumental melanin pigmentation in xanthic and albino goldfish results, in part, from the presence of tyrosinase inhibitors. These inhibitors appear to be different in the xanthic and albino goldfish. In addition to the presence of tyrosinase inhibitors, albinism in the goldfish also shows a defect in melanosome formation (Abramowitz et al., 1976, 1978). Thus, in the goldfish the albino phenotype is a multiple defect with alterations at the molecular and structural levels.

Skin (Edited by MONTAGNAW. & HU F.), Vol. 8, pp. 253-268. Pergamon Press, Oxford. CHEN Y. M. & CHAVINW. (1968) Activation of skin tyrosinase. Experientia 24, 550--552. CHENY. M. & CHAVtNW. (1973) Integumental distribution of tyrosinase activity in the slow loris, Nycticebus coucang, and the presence of integumental tyrosinase inhibitor(s). Experientia 29, 649-651. CHENY. M. & CHAVlNW. (1975) Melanogenesis in human melanomas. Cancer Res. 35, 606-612. CHEN S-T., WAHN H., TURNER W. A., TAYLOR J. D. & TCHEN T. T. (1974) MSH, cyclic AMP, and melanocyte differentiation. Recent Prog. Horm. Res. 30, 319--345. EDELSTEINL. M., CARIGLIAN., OKUN M. R., PATELR. P. & SMUCKERD. (1975) Inability of murine melanoma melanosomal "tyrosinase" (L-dopa oxidase) to oxidize tyrosine to melanin in polyacrylamide gel systems. J. invest. Derm. 64, 364--370. EPP1G J, J. JR & HEARINGV. J. (1976) The bifunctional role of mammalian, avian and amphibian tyrosinases in melanogenesis. In Pigment Cell, Vol. 3. Unique Properties of Melanocytes (Edited by RILEY V.), pp. 82-88. Karger, Basel. GAUDIN D. & FELLMANJ. H. (1967) The biosynthesis of dopa in albino skin. Biochim. biophys. Acta 141, 64-70. HAMA T. (1969) Mode d'existence de la tyrosinase dans l'albinos d'Oryzias latipes. C. r. S~anc. Soc. Biol. 163, 236-239. HEARING V. J. (1973a) Tyrosinase activity in subcellular fractions of black and albino mice. Nature New Biol. 245, 81-82. HEPatING V. J. (1973b) Mammalian melanogenesis: tyrosinase versus peroxidase involvement, and activation mechanisms, Archs Biochem. Biophys. 158, 720-725. HEAmNGV. J. & EKELLT. M. (1975) Involvement of tyrosinase in melanin formation in murine melanoma. J. invest. Derm. 64, 80-85. Hu F., FOSNAUGHR. P. & LESNEVP. F. (1961) Studies on albinism. Arch Derm. 83, 723-729. KING R. A. & WITKOPC. J., JR (1976) Hairbulb tyrosinase activity in oculocutaneous albinism. Nature, Lond. 263, 69-71. KULGEIdANT. P. & VAN SCOTT E. J. (1961) Tyrosina(se activity in melanocytes of human albinos, d. invest. Derm. 37, 73-76. MUFSON R. A. (1975) The tyrosinase activity of melanosomes from the Harding-Passey melanoma: The absence of a peroxidase component in vitro. Arch Biochem. Biophys. 167, 738-743. OKUN M. R, EDELSTEIN L. M., OR N., HAMADA G., DONREFERENCES NELANB. & LEVERW. F. (1970) Histochemical differentiation of peroxidase-mediated from tyrosinase-mediated ABRAMOWITZJ., TURNER W. A., JR, CHAVIN W. & TAYLOR melanin formation in mammalian tissues. Histochemie J. D. (1978) Tyrosinase positive oculocutaneous albinism 23, 295-309. in the goldfish, Carassius auratus L.-- an ultrastructural OKUNM. R., EDELSTEINL. M., PATELR. P. & DONNELLAN and biochemical study of the eye. Cell Tiss. Res. In press. B. (1973) Revised concept of mammalian melanogenesis: ABRAMOWITZJ., TURNER W. A., JR, TAYLOR J. D. & CHAThe possible synergistic functions of aerobic dopa oxiWN W. (1976) Ulstrastructural and biochemical evaludase and peroxidase. A review. Yale J. Biol. Med. 46, ation of the pigment epithelia in the xanthic and albino 535-540. goldfish. Am. Zool. 16, 233. OKUN M. R., PATEL R. P., DONNELLAN B., EDELSTEIN CHAWN W. (1956) Pituitary-adrenal control of melanizaL. M. & CAmOLIAN. (1976) Recent experiments on the tion in xanthic goldfish, Carassius auratus L. J. exp. role of aerobic dopa oxidase (tyrosinase) and peroxidase Zool. 133, 1-45. in mammalian melanogenesis. In Pigment Cell, Vol. CHAWN W. (1964) Factors in the assay of melanin. Am. 3, Unique Properties of Melanocytes (Edited by RILEY Zool. 4, 413. V.), pp. 89-97. Karger, Basel. CHEN Y. M. & CHAVIN W. (1965) Radiometric assay of PO~RANTZ S. H. & ANCESI. G. (1975) Tyrosinase activity tyrosinase and theoretical considerations of melanin forin human skin. Influence of race and age in newborns. mation. Analyt. Biochem. 13, 234-258. J. clin. Invest. 55, 1127-1131. CHEW Y. M. & CrIAVlN W. (1966) Tyrosinase activity in POMERANTZ S. H. & LI J. P-c. (1974) Tyrosinase in the goldfish skin. Proc; Soc. exp. Biol. Med. 121, 497-50L skin of albino hamsters and mice. Nature, Lond; 252, CHENY. M. & CoJ,vi~qW. (1967) Comparative biochemical 241-243. aspects of integumental and tumor tyrosinase activity SmTH-Gn.L S. J., RiCHAgDSC. M. & NACE G. W. (1972) in vertebrate melanogenesis. In Advances in Biology of Genetic and metabolic bases of two "albino" phenotypes

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