Introduction  latipes into the Netherlands from Japan where they have been maintained as an important species for

1. Introduction  latipes into the Netherlands from Japan where they have been maintained as an important species for

Body-color pigmentation is the most prominent

research.

phenomenon and pigment mutants have attracted One of co-authors (Takeuchi T.) collected in 1977 geneticists from the earliest studies within and the fading body-color phenotypic variant of O. latipes between species in killifish, Oryzias latipes [1]. In from natural population at Kochi prefecture, southern mammals body-color pigments were derived from the part island of Japan, then revealed it the melanin melanocyte cell in mammals, and are from pigment took place in wild type O. latipes. It changed white-colored leucophore, red-colored xanthophore, into something like orange-red appearance of “the and black-colored melanophore in fishes [2, 3]. Tomita collection” and again it restored its color of Pigmentation studies in O. latipes have a history the wild type, designated the new fading body-color reminiscent by Tomita [4], who systematically studied variant as BdlR strain (Fig. 1). In this article, the genetics of variants collected from spontaneous formation of pheomelanin pigment at the fin and natural populations or from cultured population stocks characterization of tyrosinase activity collected from named as “the Tomita collection” [1]. O. latipes is an

the fin fraction are discussed.

endemic species, except north edge island Hokkaido, in Japan. From the Yedo era (1603-1867) in Japan, O.

2. Materials and Methods

latipes has been a familiar fish and was depicted in the

2.1 Fish Strain

ukiyoe (woodblock print), during the same era, the well-known scientist, P.F.B. Siebold imported O.

Oryzias.latipes B-R-(K-11 strain was used as the control [5]) belonging to the Western Seto-Inland

Corresponding author: Nobuhiko Asada, PhD, proffesor, research field: population genetics. E-mail: population, and a fading body-color variant, BdlR asada@zool.ous.ac.jp.

Pheomelanin Formation and Low Tyrosinase Activity in Fading Body

Color Variant BdlR Strain Oryzias latipes

Fig. 1 Oryzias latipes. Above B-R- (K-11) strain (Western-Seto-Inland population, Okayama Prefecture, Japan, control) and below BdlR strain (Kochi Prefecture, Japan). Standard bar indicates 1 cm.

strain, wear reared at the room temperature (15 °C tube on ice, and then centrifuged as 16,000 rpm for -30 °C) after our previous study [5].

two minutes at 4 °C. The given extract, approximately

10 μL, served to measure tyrosinase activity after [5],

2.2 Chemicals with some following modifications. The research

L-hydroxyphenylalanine (L-tyrosine), mixture holded as 500 μL of 10 mM L-dopamine (or L-3,4-dihydroxyphenylalanine (L-dopa),

10 mM L-tyrosine, and L-dopa) in 200 mM L-3-4-dihydroxyphenylalanine (L-dopamine), and Tris-maleate buffer, pH 6.0 at room temperature, and

L-cysteine were purchased from Nakarai Tesque Inc.

20 μL of enzyme fraction. After incubation for five (Kyoto, Japan). Copper content was estimated using

minutes at 30 °C, the absorbance of dopachrome was commercial kit, Metalo Assay, MG Metallogenics,

measured at the wave length 475 nm using Funakoshi Co. Ltd. (Tokyo, Japan).

Spectrophotometer Hitachi U-2000 (Tokyo, Japan).

2.3 Measurement of Melanin-Pigment Content

2.5 Measurement of Copper Content

A single whole body of O. latipes was placed in a To determine copper molecule content in tyrosinase small volume of fresh water (10 mL) and a single

protein in O. latipes, the scale was declined using the scale was obtained after anesthetization on ice. Given

commercial kit after preparation of extract at 582 nm fraction was observed melanin pigments under a light

using Hitachi U-2000 (Tokyo, Japan). microscope (magnification, ×15). Coloration and

statistical significance of melanin pigments was

3. Results and Discussion

determined in accordance with the methodology of

3.1 Fading Body-Color in BdlR Strain Sokal and Rohlf [6].

Spontaneous variant, BdlR strain in O. latipes

2.4 Assay of Tyrosinase Activity appeared very strong phenotype in the body-color

A single tail-fin fraction was collected [5] as, and fading and very weaker melanin pigmentation than in the given fraction was homogenized in the Eppendorf

the wild type control, K-11 strain, especially at

Pheomelanin Formation and Low Tyrosinase Activity in Fading Body

Color Variant BdlR Strain Oryzias latipes

black-colored dorsal line and at tail fin (Fig. 1). three minutes (after mixture of extract-substrate was Difference of mean number on melanin pigment at the

plotted, the reaction was showed as sigmoid (Fig. 2). scale between wild type K-11 strain (37.4 ± 11.1 value,

In general, enzyme has an allosteric site different n = 10) and BdlR strain (17.4 ± 11.7 value, n = 10)

from its substrate, that is, two sites are estimated to was statistically significant (p < 0.001) suggested that

involve different from steric specificity. Allosteric site body color variation in BdlR strain due to the variation

can bind allosteric effector specific reversible. of melanin color and forming the fading pheomelanin.

Therefore, tyrosinase in O. latipes was suggestied to L-Cysteine is one of the amino acids playing

be an allosteric enzyme (protein) and estimated that constriction of the functional SH protein containing

this enzyme has two separated different steric sites, sulfur molecules. When a small powder of L-Cysteine

especially catalytic site, and has different steric was added to a single scale, the scale-color was

specificity and that tyrosinase protein can bind gradually blight like whole bogy especially in BdlR

allosteric effector specific and reversible, then strain as shown in Fig. 1. These enzymatic analyses,

metabolic activity was regulated high and low activity BdlR strain was estimated as one of the low tyrosinase

of enzyme. So, tyrosinase activity and protein in O. activity spontaneous variant caught in natural latipes is clearly revealed as the proten-substrate

populations. fitting Ping-Pong BiBi model known as acetyl CoA

3.2 Tyrosinase Activity and Enzyme Kinetics

carboxylase.

As showing the forgoing graph in BdlR strain (Fig. Tyrosinase activity was first detected from fin

2), final tyrosinase activity was lower than in control extract using L-tyrosine, L-dopa, and L-dopamine as

the enzyme substrate since BdlR strain showed fading body-color as mentioned above. The value of enzyme activity indicated as an absorbance at the wave length 475 nm was shown in K-11 strain (0.083 using L-dopamine as substrate) whereas quite low value was shown in BdlR strain (0.006 using L-dopamine as same substrate), meaning that BdlR strain was the first distinguished killifish diminishing and low tyrosinase activity variant corresponding to fading body color at whole body collected at nature.

Enzyme activity was estimated both as tyrosinase-type mono-phenol oxidase and dopa oxidase-type di-phenol oxidase whereas no evidence was detected oxigenase activity in this study. Substrate specificity was shown in both strains that the highest enzyme activity was detected used L-dopanine and minimum in L-tyrosine. From above evidence, body-color variation can be analyzed

molecular level.

Fig. 2 Time course of tyrosinase activity. Tyrosinase

Time cource of tyrosinase activity showed sigmoidal activity in tail-fin extract using 10 mM L-dopamine as an

enzyme substrate of Oryzias latipes. Horizontal and vertical

saturation curve when relative tyrosinase activity

axis indicate the time started mix of enzyme with the

(absorbance value) against time was plotted up to

substrate and tyrosinase activity at 25 °C.

Pheomelanin Formation and Low Tyrosinase Activity in Fading Body

Color Variant BdlR Strain Oryzias latipes

Fig. 3 Initial velocity. Estimation of initial velocity represented conjugation with the formula v=d[p]/dt: where v represented velocity and p represented product. Bar indicates an arithmetric line. Horizontal and vertical axis indicates time from mix substrate-enzyme with second.

3.3 Copper-Involving Structure at the Enzyme Active and substrate can bind each other, enzyme kinetics

K-11 strain, time necessary for both enzyme protein

Center

designated as initial velocity was calculate from both After our previous study in prophenol oxidase graph and the formula as v = d[p]/dt where v is a

(tyrosinase in vertebrates) protein in Drosophila velocity, p is a product. The mean v values were

melanogaster, The ribbon-model diagram of significantly lower in BdlR strain (0.0022, n = 5,

higher-order structure made by Circular Dichroism probability was not shown) than in control K-11 strain

(CD) spectra of prophenol oxidase protein revealed (0.0034, n = 5, probability was not shown) (Fig. 3).

that dicopper Cu (II) were separated as 2.9 A each These results showed from these values, fading

other and six His residues were surrounded body-color variant BdlR strain can mean both low

by α-helices at the catalytic site [7]. In O. latipes, activity and late velocity of tyrosinase activity and

cupper content was firstly detected from a single scale forming pheomelanin spontaneous variant.

fraction using the commercial kit, at first, almost the

Pheomelanin Formation and Low Tyrosinase Activity in Fading Body

Color Variant BdlR Strain Oryzias latipes

same copper contents was detected, but no significant Furutani-Saiki, M., Ozato, K., and Wakamatsu, Y. 2004. difference was detected into two strains (values were “The Tomita Collection of Medaka Pigmentation Mutants as a Resource for Understanding Neural Crest Cell

not shown). By the way, no copper was included in Development.” Mechanisms of Development 121:

the culture medium certified from the medium

841-859.

company. These results showed that tyrosinase [2] Takeuchi, T., and Manabe, E. 1981. “An Electron activity was involved and copper molecule at active

Microscope Study on the Development of Melanosome in site play important role for enzyme activity in O.

the Early Embryogenesis in the Medaka, Oryzias latipes.” latipes (Fig. 4). Fading body-color was based on

The Research Bulletin of Shujitsu Women’s College and Shujitsu Junior College 11: 61-87. (Japanese with

pheomelanin and variance at the copper-binding

English abstract)

catalytic site in BdlR. strain. [3] Takeuchi, T., and Manabe, E. 1984. “Genetical Study on