Báo cáo khoa học: Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus docx

Eur. J. Biochem. 270, 4026–4038 (2003) Ó FEBS 2003

doi:10.1046/j.1432-1033.2003.03795.x

Purification, characterization and molecular cloning of tyrosinase
from the cephalopod mollusk, Illex argentinus
Tetsushi Naraoka1,2, Hidemitsu Uchisawa1, Haruhide Mori2, Hajime Matsue3, Seiya Chiba2
and Atsuo Kimura2
1

Aomori Industrial Research Center, Aomori; 2Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Sapporo; 3Aomori University of Health and Welfare, Aomori, Japan

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase)
was isolated from the ink of the squid, Illex argentinus.
Squid tyrosinase, termed ST94, was found to occur as a
covalently linked homodimeric protein with a molecular
mass of 140.2 kDa containing two copper atoms per a
subunit. The tyrosinase activity of ST94 was enhanced by
proteolysis with trypsin to form a protein, termed ST94t,
with a molecular mass of 127.6 kDa. The amino acid
sequence of the subunit was deduced from N-terminal amino
acid sequencing and cDNA cloning, indicating that the
subunit of ST94 is synthesized as a premature protein with
625 amino acid residues and an 18-residue signal sequence
region is eliminated to form the mature subunit comprised of

607 amino acid residues with a deduced molecular mass of
68 993 Da. ST94 was revealed to contain two putative
copper-binding sites per a subunit, that showed sequence
similarities with those of hemocyanins from mollusks,

tyrosinases from microorganisms and vertebrates and the
hypothetical tyrosinase-related protein of Caenorhabditis
elegans. The squid tyrosinase was shown to catalyze the
oxidation of monophenols as well as o-diphenols and to
exhibit temperature-dependency of o-diphenolase activity
like a psychrophilic enzyme.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase)
is one of the copper-containing phenoloxidases that are
widely distributed in nature. The enzyme is known to be a
key enzyme in the melanogenic pathway that catalyzes the
initial rate-determining reaction, the oxygenation of monophenols to o-diphenols (monophenolase activity), as well as
the oxidation of o-diphenols to corresponding o-quinones
(o-diphenolase activity) [1,2]. Type 3 copper proteins,
including tyrosinases, arthropod phenoloxidases and hemocyanins, have been isolated from many organisms. The
evolutional relationships of the structures have also been
elucidated on the basis of the amino acid sequences
conserved around two copper-binding sites that form an

oxygen-binding active center [3–6]. These proteins are
classified into a superfamily and are of interest from the
viewpoint of their molecular evolution [7–9].
These copper proteins are particularly important in
arthropods. Arthropod phenoloxidases [10–13], which are
given the same EC number as tyrosinase, are known to be
involved in the host defense system termed the prophenoloxidase cascade as a terminally active molecule in the system
[14–17]. Hemocyanins are macromolecules that function
as oxygen carriers in the hemolymph of arthropods and
mollusks [7]. Hemocyanins are also found to exhibit
phenoloxidase activity [6,18,19], which is amplified after

certain treatments such as proteolysis or exposure to
detergents, or by interactions with specific proteins
[6,8,20–22]. This activation suggests that these hemocyanins
may have roles as phenoloxidases in some important
biological events.
Among the mollusks, the emission of ink for defense
against predators is a well-known characteristic behavior of
most cephalopods, which indicates their high capacity for
melanogenesis. We reported previously that a fraction
from the ink of the squid, Illex argentinus, in which the
illexin-peptidoglycan (IPG) possessing a novel mucopolysaccharide structure and tyrosinase were contained, showed
anti-tumor activity against Meth A fibrosarcoma in BALB/
c mice [23–26]. The anti-tumor activity was thought to be
expressed through immunostimulation because the fraction
had macrophage-stimulating activity [23]. Consistent with
these observations is the clinical use of hemocyanin from the
keyhole limpet (a marine gastropod, Megathura crenulata)
as an immunotherapeutic agent for the treatment of bladder
carcinoma [27], and other observations suggesting that

Correspondence to T. Naraoka, Aomori Industrial Research Center,
4-11-6 Daini-tonyamachi, Aomori 030–0113, Japan.
Fax: + 81 17 7399613, Tel.: + 81 17 7399676,
E-mail:
Abbreviations: DHPPA, 3,4-dihydroxyphenylpropionic acid; DOPA,
3,4-dihydroxyphenylalanine; IPG, illexin-peptidoglycan; KLH,
keyhole limpet hemocyanin; MBTH, 3-methyl-2-benzothiazolinone
hydrazone; PPAE, prophenoloxidase-activating enzyme;
pro-PPAE, zymogen of PPAE; ST94, tyrosinase from Illex argentinus;
ST94t, proteolyte of ST94 with trypsin.

Enzymes: tyrosinase (EC 1.14.18.1); trypsin (EC 3.4.21.4).
Note: After submission and during the review of this article, the cDNA
sequence of tyrosinase from Sepia officinalis has been opened in
DDBJ/EMBL/GeneBank databases on 5 July 2003 (Accession no.
AJ297474) by Lieb, B., Erteld, D., Poli, A., Palumbo, A. & Markl, J.
(Received 11 May 2003, revised 12 August 2003,
accepted 18 August 2003)

Keywords: Illex argentinus; tyrosinase; copper protein; melanogenesis; cephalopod.


Ó FEBS 2003

Tyrosinase from the mollusk, Illex argentinus (Eur. J. Biochem. 270) 4027

molluscan tyrosinases also are biologically and biomedically
significant. Tyrosinase activity has been demonstrated in the
inks of some other cephalopods [28]. Recently, there have
been substantial advances in elucidating the mechanism of
ink production in Sepia officinalis [29–33]. For cephalopod
tyrosinases, however, only limited information is available
on their characteristics. This seems to have been due to the
difficulty of isolating the tyrosinases that occur in ink, which
show extremely complex polymorphism (as observed at
least in ink of I. argentinus [26]). In other mollusks, although
tyrosinases have been isolated from a bivalve [34] and a
gastropod [35], there have been no reports on their amino
acid sequences.
In a previous paper, we reported a protein that occurred
in the ink of I. argentinus with weak tyrosinase activity,

which migrated as a 94-kDa protein on polyacrylamide gel
electrophoresis under native condition [26]. The protein,
termed ST94, was assumed to be a partially activated
tyrosinase, one of the abundant proteins in the ink. In this
paper, we describe the purification, proteolytic activation,
some enzymatic properties and molecular cloning of the
squid tyrosinase ST94. This is the first report on the primary
structure of a molluscan tyrosinase (see Footnote on
p. 4026), which contributes to evolutional studies on type
3 copper proteins.

Materials and methods

MALDI-TOF mass spectrometry experiments were performed on a Voyager-DE STR (Applied Biosystems, Foster
City, CA, USA) according to the manufacturer’s instructions. Synapinic acid dissolved in a 2 : 1 mixture of 0.1% (by
volume) aqueous trifluoroacetic acid and 0.1% (by volume)
trifluoroacetic acid containing acetonitrile was used as a
matrix for the analyses. Spectrometry was performed in
positive linear mode. Bovine serum albumin (BSA) and
horse heart myoglobin were used as mass number standards.
Amino acid sequences were analyzed by the gas-phase
Edman degradation method using a protein sequencer
PPSQ-10 (Shimadzu Corp., Kyoto, Japan), according to the
manufacturer’s instructions.
Protein content was determined by the method of Lowry
et al. [36] or by measuring absorbance at 205 nm [37] using
BSA as a standard. Uronic acid, hexose and methylpentose
were determined by the carbazole-sulfuric acid method [38],
the phenol–sulfuric acid method [39] and the cysteine–
sulfuric acid method [40], respectively.

Copper analysis was performed using an atomic absorption analyzer Z5010 with a graphite atomizer (Hitachi,
Tokyo, Japan). Protein was dissolved in 1 mM sodium
phosphate (pH 7.4), and analyzed by the standard addition
method using commercially available copper standard
solution. The copper content of the buffer used was checked
in advance and found to be 0.1 ngỈmL)1 close to the
detection limit.

Materials

Purification of tyrosinase ST94

L-3,4-Dihydroxyphenylalanine (L-DOPA), D-3,4-dihydroxy-

Ink sacs of I. argentinus were homogenized with four
volumes of acetone () 30 °C) using a Waring blender, then
filtered through a glass filter and the residue was dried
in vacuo. The defatted powder (100 g) was extracted with
1 L of 10 mM sodium phosphate buffer (pH 7.4) at 4 °C for
12 h with stirring and then centrifuged (10 000 g, 30 min) to
obtain the crude tyrosinase extract. Ammonium sulfate
concentration of the extract was brought to 30% saturation
with solid ammonium sulfate and allowed to stand at 4 °C
for 12 h. The turbid solution was centrifuged (10 000 g,
30 min) and the supernatant was brought to 60% saturation
concentration by addition of solid ammonium sulfate at
4 °C, and allowed to stand for 12 h. The resulting precipitate was collected by centrifugation (10 000 g, 30 min),
dissolved in a small volume of 10 mM sodium phosphate
(pH 7.4), and dialyzed against the same buffer (fraction
AS60, 300 mL).

The fraction AS60 (100 mL) was added to 20 mL of 3 M
ammonium sulfate containing 10 mM sodium phosphate
(pH 7.4) and applied to a column (2.6 · 28 cm) of PhenylSepharose CL-4B equilibrated with 0.5 M ammonium
sulfate in 10 mM sodium phosphate (pH 7.4). After washing
with the same buffer, the column was eluted with a linear
gradient of 0.5–0 M ammonium sulfate in 10 mM sodium
phosphate (pH 7.4) in a total volume of 1.5 L, and then
further eluted with 10 mM sodium phosphate (pH 7.4) at a
flow rate of 1 mLỈmin)1. Fractions of 10 mL were collected
and analyzed for uronic acid using the carbazole–sulfuric
acid method and for protein and pigment using the
absorbance at 280 nm. Tyrosinase activity was monitored
as follows. A 5-lL sample was added to a microplate,
200 lL of 5 mM L-DOPA in 0.1 M sodium phosphate buffer

phenylalanine (D-DOPA), dopamine, pyrocatechol, L-tyrosine, D-tyrosine, tyramine, 3,4-dihydroxyphenylpropionic
acid (DHPPA), 3-methyl-2-benzothiazolinone hydrazone
(MBTH), kojic acid, arbutin, phenylthiourea, tropolone,
mushroom tyrosinase and keyhole limpet hemocyanin
(KLH) were supplied from Sigma (St. Louis, MO, USA).
N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)treated bovine pancreatic trypsin was purchased from
Funakoshi Co. Ltd. (Tokyo, Japan). Phenyl-Sepharose
CL-4B, polyacrylamide gel plates for electrophoresis were
from Amersham Biosciences (Uppsala, Sweden). The other
chemicals were supplied by Wako Pure Chemical Industries,
Ltd. (Osaka, Japan).
Analytical methods
PAGE under native conditions (native-PAGE) and
denaturing conditions (SDS/PAGE), and two-dimensional
PAGE (2D-PAGE) were performed using a PhastSystem

(Amersham Biosciences). For denaturing with a reducing
reagent, the protein was treated in the sample buffer
containing 2.5% (m/v) SDS, 5% (v) 2-mercaptoethanol,
5% (v) glycerol and 62.5 mM Tris/HCl (pH 6.8) for
5 min at 95 °C. For SDS/PAGE under nonreducing
conditions, the protein was treated using the sample
buffer from which 2-mercaptoethanol was omitted. After
the run, the gel was stained for visualizing proteins with
Coomassie Brilliant Blue (CBB), or stained for detecting
of tyrosinase activity with 5 mM L-DOPA or 0.5 mM
L-tyrosine in 0.1 M sodium phosphate buffer (pH 6.8) at
room temperature.


4028 T. Naraoka et al. (Eur. J. Biochem. 270)

(pH 6.8) was added to the plate, and the absorbance of the
mixture was measured using a Labsystems microplate
reader with a 492-nm filter after incubation at 25 °C for
10 min. Fractions containing ST94 were detected by nativePAGE (10–15% gradient gel), then concentrated and
desalted by ultrafiltration using YM10 membrane (Amicon,
Beverly, MA, USA). ST94 was purified further using
an anion-exchange column (4.6 · 100 mm) of Poros
HQ/M (Applied Biosystems) with a BioCAD 700E HPLC
system (Applied Biosystems). For the elution (flow rate
10 mLỈ min)1), a linear gradient of NaCl, from 0 to 1.0 M
over 16.6 min in 50 mM Tris/HCl (pH 7.0) was applied
(fractions of 2 mL). ST94 was detected by native-PAGE
(10–15% gradient gel). Fractions containing ST94 (eluting
at 0.27 M NaCl) were concentrated and desalted by

ultrafiltration as described above, then recovered as a
solution of 1 mM sodium phosphate (pH 7.4).
Trypsin-treatment of ST94
ST94 dissolved in 10 mM Tris/HCl buffer (pH 8.0) at a final
concentration of 250 lgỈmL)1 was treated with TPCKtreated trypsin (2.5 lgỈmL)1) for 2 h at 25 °C. The resulting
proteolyte of ST94, termed ST94t, was purified by gel
permeation HPLC. Gel permeation HPLC was performed
with a L7100S HPLC system (Hitachi) using a column of
G3000SWXL (7.8 mm · 300 mm; TOSOH, Tokyo, Japan)
equilibrated with 0.2 M NaCl in 0.1 M sodium phosphate
(pH 7.0) (flow rate 0.5 mLỈmin)1). Fractions containing
ST94t were concentrated and desalted by ultrafiltration as
described above, then recovered as a solution of 1 mM
sodium phosphate (pH 7.4).
Assay of tyrosinase activity
Tyrosinase activity was assayed by the dopachrome method
[41] as follows. The standard reaction mixture contained
5 mM L-DOPA, 0.1 M sodium phosphate buffer (pH 6.8)
and the enzyme solution in a total volume of 3 mL. The
reaction took place in a cuvette with a path length of 1 cm
and the absorbance at 475 nm was monitored continuously
using a spectrophotometer U-3210 (Hitachi) at 25 °C. One
unit of tyrosinase was defined as the amount of enzyme
required to oxidize 1 lmol of L-DOPA per min under the
above conditions, which was calculated using the molar
extinction coefficient of dopachrome (3600 M)1Ỉcm)1).
The substrate specificity of tyrosinase was analyzed by the
MBTH method [42,43]. The assay was carried out in 3 mL
of reaction mixture containing an appropriate concentration of substrate, 5 mM MBTH, 2% (by volume) N,Ndimethylformamide and the enzyme solution in 50 mM
sodium phosphate buffer (pH 6.8). In the analyses for

monophenols, the corresponding o-diphenol at a final
concentration of 1 lM was added to the reaction mixture
to shorten the lag period. Formation of the MBTH-adduct
of o-quinone was followed at the isosbestic point wavelength
of each MBTH-adduct at 25 °C, and the steady-state rate
of oxidation of substrate was determined using the molar
extinction coefficient of MBTH-adduct at the isosbestic
point wavelength taken from the literature [43]. The Km and
Vmax values for different substrates were obtained from the
Hanes–Woolf equation.

Ó FEBS 2003

Extraction of RNA and first strand cDNA preparation
All DNA and RNA manipulations were carried out by
standard techniques except where otherwise noted [44].
PCR experiments were performed using a GeneAmp PCR
System 9700 (Applied Biosystems). Poly(A)+ RNA was
extracted from an ink sac (1.5 g) of I. argentinus using a
QuickPrep mRNA purification kit (Amersham Biosciences). A portion of the homogenate corresponding to
500 mg of ink sac was applied to an oligo(dT)-cellulose
spun column. Poly(A)+ RNA eluted from a column was
ethanol-precipitated, and redissolved in 50 lL of water.
First strand cDNAs for 5¢- and 3¢-RACE were prepared
from the ink sac poly(A)+ RNA using a SMART RACE
cDNA Amplification kit (Clontech, Palo Alto, CA, USA)
according to the manufacturer’s instructions. For each
cDNA preparation, 0.46 lg of poly(A)+ RNA was used.
After reverse-transcription, reaction mixtures (10 lL) were
diluted by addition of 20 lL of 1 mM EDTA containing

10 mM Tricine/KOH buffer (pH 8.5), heated at 72 °C for
7 min, then cooled on ice and used for further experiments.
Cloning and sequencing of tyrosinase ST94 cDNA
Degenerate RT-PCR was carried out to detect ST94 cDNA
using the first strand cDNA for 5¢-RACE as a template with
sense and antisense primers corresponding to a portion
of the N-terminal amino acid sequence of ST94
(MVDVSQSD) and that of ST94t (MSPQEYIQ), respectively. Sense (TYF1 and TYF2) and antisense primers
(TYR1 and TYR2) were designed from these sequences to
lower the degeneracy at the 3¢ end regions: TYF1, 5¢-ATGG
TNGAYGTNWSNCARTCNGA-3¢; TYF2, 5¢-ATGGT
NGAYGTNWSNCARAGYGA-3¢; TYR1, 5¢-TGDATR
TAYTCYTGNGGNGACA-3¢; TYR2, 5¢-TGDATRTA
YTCYTGNGGRCTCA-3¢. PCR was carried out using a
TITANIUM Taq DNA polymerase (Clontech) in 25 lL of
reaction mixture composed of the cDNA (0.5 lL), 25 pmol
of sense primer (TYF1 or TYF2), 25 pmol of antisense
primer (TYR1 or TYR2), 0.2 mM dNTPs, 0.5 lL of the Taq
DNA polymerase and 1 · PCR buffer under the following
conditions: after holding (94 °C, 1 min), 30 cycles of
denaturing (94 °C, 10 s), annealing (56 °C, 30 s) and
elongation (72 °C, 30 s), followed by holding (72 °C,
3 min). The amplified product (about 200 bp) that occurred
in the reaction mixture containing the primers TYF1 and
TYR1 was subcloned into pT7 Blue T-vector (Novagen,
Madison, WI, USA) following purification by 2% (m/v)
agarose gel electrophoresis using a MiniElute purification kit
(Qiagen, Tokyo, Japan). The clones were subjected to DNA
sequencing on both strands by the dideoxy chain termination method. The sequencing reaction was performed using
a Thermo Sequenase Primer Cycle Sequencing kit (Amersham Biosciences) with Texas red-labeled M13 forward

primer () 21) or M13 reverse primer () 29). The samples
were analyzed with a DNA sequencer SQ-5500 (Hitachi).
For 5¢-RACE of ST94 cDNA, the specific primer,
TYR3 (5¢-CGTCTGCCGATTTCCAATTCTTCTG-3¢),
was designed from the sequence of part of the RT-PCR
product. 5¢-RACE was carried out using a SMART RACE
cDNA Amplification kit according to the manufacturer’s
instructions with 0.25 lL of the first strand cDNA for


Ó FEBS 2003

Tyrosinase from the mollusk, Illex argentinus (Eur. J. Biochem. 270) 4029

5¢-RACE as a template and 10 pmol of TYR3 in 50 lL of
the reaction mixture. Touchdown PCR was performed as
follows: five cycles of denaturing (94 °C, 5 s) and annealing/
elongation (72 °C, 2 min), five cycles of denaturing (94 °C,
5 s), annealing (70 °C, 10 s) and elongation (72 °C, 2 min),
followed by 25 cycles of denaturing (94 °C, 5 s), annealing
(68 °C, 10 s) and elongation (72 °C, 2 min). The amplified
products were subcloned into pT7 Blue following purification by agarose gel electrophoresis and subjected to DNA
sequencing as described above.
3¢-RACE was performed to amplify the whole ST94
cDNA. A primer, TYFW (5¢-GATATGAGGATGAA
ACCACACTTGG-3¢), corresponding to nucleotide 4–28
in the cDNA sequences (Fig. 6), was designed from the
sequence of the largest 5¢-RACE product. PCR was carried
out using a KOD Plus DNA polymerase (Toyobo, Osaka,
Japan) to ensure high fidelity in 50 lL of reaction mixture

containing 2.5 lL of the first strand cDNA for 3¢-RACE,
15 pmol of TYFW, 7.5 lL of 10 · universal primer A mix
(a component of a SMART RACE cDNA Amplification
kit), 0.2 mM dNTPs, 0.8 mM MgSO4 and 1 unit of KOD
Plus DNA polymerase in the PCR buffer supplied. PCR
was performed under the following conditions: holding
(94 °C, 1 min), 30 cycles of denaturing (94 °C, 5 s) and
annealing/elongation (68 °C, 3 min). The PCR products
about 2.2 kbp in length were subcloned into pT7 Blue with a
Perfectly Blunt Cloning kit (Novagen) following purification by agarose gel electrophoresis. Clones of the amplified
fragments were subjected to DNA sequencing on both
strands by primer walking using a BigDye Terminator cycle
sequencing kit with a DNA sequencer, ABI PRISM 3100
(Applied Biosystems). The nucleotide sequence data are
available in the DDBJ/EMBL/GenBank databases under
the accession numbers AB107880 and AB107881 for the
squid tyrosinase ST94 cDNA-1 and cDNA-2, respectively.
Sequence analysis
Sequences were analyzed using a software DNASIS (Hitachi
Software, Tokyo, Japan). A homology search was carried
out with NCBI-BLAST 2.0 program available at DDBJ web
server (). Phylogenetic tree was
deduced by a neighbor-joining analysis based on the
alignment of amino acid sequences constructed using the
CLUSTALW program available at DDBJ web server.

Results and discussion
Purification of tyrosinase ST94
ST94 was isolated from the ink of I. argentinus by
ammonium sulfate fractionation, and Phenyl-Sepharose

and anion-exchange chromatography. The ammonium
sulfate fractionation recovered 95% of tyrosinase activity
in the ink as a precipitate (fraction AS60): the dialyzed
solution of AS60 (300 mL) obtained from 100 g of defatted
ink powder contained 2.9 g of protein, 0.29 g of hexose,
0.25 g of uronic acid, 0.12 g of methylpentose and 5400
units of tyrosinase activity. The compositional analysis
revealed that the IPG [23–25] of the ink was also concentrated in fraction AS60. The elution profile of fraction AS60
on Phenyl-Sepharose CL-4B chromatography is shown in

Fig. 1. Chromatography of squid tyrosinase on a Phenyl-Sepharose
CL-4B column. (A) Fraction AS60 (100 mL) was applied to a PhenylSepharose CL-4B column (2.6 · 28 cm), and eluted with a linear
gradient of 0.5–0 M ammonium sulfate in 10 mM sodium phosphate
(pH 7.4) (flow rate 1 mLỈmin)1, fractions of 10 mL). d, tyrosinase
activity, absorbance of L-DOPA oxidation reaction mixture at
492 nm; s, uronic acid, absorbance of assay reaction mixture at
530 nm; ——, absorbance at 280 nm. Fractions containing ST94,
indicated with horizontal bar, were pooled. (B) Native-PAGE of the
tyrosinase-active fractions. Samples were run on 10–15% gradient gels
and stained with CBB (left), with L-DOPA (center) and with L-tyrosine
(right). Lane M, marker proteins, thyroglobulin (669 kDa), ferritin
(440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and
BSA (66.4 kDa); Lane 1, fraction no. 170; lane 2, fraction no. 180; lane
3, fraction no. 190; lane 4, fraction no. 198; lane 5, fraction no. 204.
The arrow indicates the band of ST94 (lanes 3 and 4). (C) IEF-native
2D-PAGE of the pooled fraction containing ST94. The first dimension, IEF (pH 3-9); the second dimension, native-PAGE (10–15%
gradient gel). The gels were stained with CBB (left) and with L-DOPA
(right). Lane M, mass marker proteins. Positions of pI marker proteins
were indicated at the top; amyloglucosidase (pI 3.50), soybean trypsin
inhibitor (pI 4.55), b-lactoglobulin A (pI 5.20), bovine carbonic

anhydrase B (pI 5.85), horse myoglobin (pI 7.35) and lentil lectin
(pI 8.65). The arrow indicates the spot of ST94.


Ó FEBS 2003

4030 T. Naraoka et al. (Eur. J. Biochem. 270)

Fig. 2. PAGE analyses of ST94 and ST94t. Purified ST94 and the trypsin treatment reaction mixture of ST94 were subjected to PAGE analyses.
Lane M, molecular mass markers; lane 1, ST94; lane 2, the trypsin treatment reaction mixture of ST94. The closed arrow and the open arrow
indicate the band of ST94 and that of ST94t, respectively. (A) Native-PAGE. Samples were run on 10–15% gradient gel with the same marker
proteins as in Fig. 1B, and stained with CBB (left) and with L-DOPA (right). (B) SDS/PAGE under reducing conditions. Samples were run on
12.5% gel and stained with CBB. Marker proteins used were, phosphorylase b (97.2 kDa), BSA (66.4 kDa), ovalbumin (45.0 kDa), carbonic
anhydrase (29.0 kDa), soybean trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa). (C) SDS/PAGE under nonreducing conditions. Samples
were run on 4–15% gradient gel and stained with CBB. Marker proteins used were, myosin (212 kDa), a2-macroglobulin (170 kDa), b-galactosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa).

Fig. 1A. Most of the IPG was eluted in the breakthrough
fraction and separated from tyrosinase; these two components could not be separated by anion-exchange chromatography and gel permeation chromatography [26]. In
native-PAGE of the tyrosinase-active fractions (Fig. 1B),
the protein bands were not observed in the position
corresponding to the strong enzyme activities, which yielded
wide, and smeared bands. As ST94 showed weak activity
compared with other tyrosinase-active components, ST94
was detected by PAGE analyses during purification, as
shown in Fig. 1B. On isoelectric focusing (IEF)-native 2DPAGE, ST94 was separated from other tyrosinase-active
components and was observed as a clear spot corresponding
to the position of a protein with pI 4.1 and a molecular mass
of 94 kDa. The tyrosinase activity of ST94 was also
observed by activity staining (Fig. 1C). ST94 was further
purified by anion-exchange HPLC and obtained as a

homogeneous preparation (seen in Fig. 2) with a yield of
0.73 mg protein from 100 g of defatted ink powder and a
specific tyrosinase activity of 23.5 U per mg protein.
Activation of ST94 by treatment with trypsin
As described above, most of the tyrosinase activity in the ink
of I. argentinus originated from tyrosinase-active molecules
showing a broad smeared band on native-PAGE followed
by activity staining. Despite their high tyrosinase activities,
these molecules showed indistinct bands when stained with
CBB, which suggests that their specific activity was high
compared with that of ST94. From these observations,
ST94 was presumed to be a partially activated molecule that
was capable of becoming a more active molecule. As
arthropod prophenoloxidases [10,11,14–16] and a bivalve
tyrosinase [34] have been shown to be activated by

proteolysis, we examined the effect of trypsin treatment on ST94. During the treatment, the tyrosinase activity
was increased to about four times the maximum after
incubation for 2 h under the conditions described in
Materials and methods. As shown in Fig. 2A, ST94
generated a proteolyte, termed ST94t, showing slightly
higher mobility than ST94 on native-PAGE. ST94t in the
proteolysate of ST94 was purified as the enzyme preparation with a yield of 0.23 mg protein from 0.30 mg of ST94
and a specific activity of 103 U per mg protein by gel
permeation HPLC. The results indicated that ST94t was an
activated tyrosinase molecule bearing the stable catalytic
domain of ST94.
Molecular mass of the squid tyrosinase
ST94 electrophoresed to a position corresponding to that of
a protein with a molecular mass of about 70 kDa on SDS/

PAGE under reducing conditions, whereas it migrated
as a  140 kDa protein under nonreducing conditions
(Fig. 2B,C). In MALDI-TOF mass spectrometry, a parent
ion signal of ST94 was observed at m/z 140.2 kDa. These
results indicated that ST94 is a 140.2-kDa protein composed
of two 70.1-kDa subunits that are linked, probably by a
disulfide bond. These estimates of the molecular mass of
ST94 were supported by the result of gel permeation HPLC
(Fig. 3). There was a minor band between 140 and 232 kDa
in the native-PAGE of Fig. 2A (lane 1), implying that ST94
enables to form an oligomeric protein. On SDS/PAGE,
ST94t electrophoresed to a position corresponding to that
of a protein of about 65 kDa under reducing conditions,
while migrated as an  130 kDa protein under nonreducing
conditions (Fig. 2B,C). In MALDI-TOF mass spectrometry, the trypsin-treated reaction mixture of ST94 showed


Ó FEBS 2003

Tyrosinase from the mollusk, Illex argentinus (Eur. J. Biochem. 270) 4031

Fig. 3. Molecular mass estimation of ST94 by gel permeation HPLC.
ST94 (2 lg) and molecular mass standard proteins (each 2.5 lg) were
chromatographed on a column of G3000SWXL (7.8 · 300 mm)
equilibrated with 0.2 M NaCl in 0.1 M sodium phosphate (pH 7.0)
(flow rate 0.5 mLỈmin)1). The eluate was monitored at 205 nm.
Molecular mass standard proteins used were: 1, catalase (232 kDa); 2,
lactate dehydrogenase (140 kDa); 3, BSA (66.4 kDa); 4, ovalbumin
(45.0 kDa); 5, chymotrypsinogen (25.0 kDa). The arrow indicates the
elution volume of ST94.

a parent ion signal of ST94t at m/z 127.6 kDa. These results
indicated that ST94 was digested by trypsin to generate
ST94t as a 127.6-kDa protein composed of two 63.8-kDa
subunits that remained linked after treatment with trypsin.
The molecular masses of the subunits of ST94 and ST94t
were similar to those of the proenzymes ( 70–80 kDa) and
the activated enzymes by proteolysis ( 60–70 kDa) of
a bivalve tyrosinase [34] and arthropod phenoloxidases
[10–13,16].

ST94 was found to contain copper atoms at a content of
0.18 ± 0.01% (by mass): the copper concentration of ST94
solution of 28 lg proteinỈmL)1 was determined to be
50.2 ± 1.5 ngỈmL)1. The result indicates that ST94 has
two copper atoms per a subunit of 70.1 kDa. For control
experiment, the copper content of KLH was also analyzed
by the same procedure and determined to be 0.24% (by
mass) in good agreement with the value calculated from the
literature [7] (16 copper atoms per subunit): 17.5 ± 0.3 ng
copperỈmL)1 was detected in KLH solution of 7.3 lg
proteinỈmL)1. The ST94 solution was also subjected to the
analysis of manganese, but no manganese was detected.
The N-terminal amino acid sequences of ST94 and ST94t
were shown to be NH2-MVDVSQSDGLQSXLDRFADD
(X represents an amino acid undetermined) and NH2ISTLATMSPQEYIQ, respectively, which indicated that the
N-terminal region of ST94 was truncated with trypsin to
generate the N-terminal of ST94t. Each analysis for ST94
and for ST94t showed a single N-terminal sequence,
allowing us to speculate that ST94 is a homodimeric protein.

Enzymatic properties of the squid tyrosinase
Effects of pH and temperature on stability and activity. As ST94 and ST94t showed the identical results on
pH- and temperature-effects, we describe the data of ST94t in
this section. ST94t retained more than 90% of its activity
after incubation at 4 °C for 24 h within a pH range of 6.5–11
(Fig. 4A). The optimum pH for o-diphenolase activity of
ST94t was determined to be pH 8.0 (Fig. 4B) with correction
by subtraction of the increasing baseline caused by spontaneous oxidation of L-DOPA; we routinely assayed the
tyrosinase activity at pH 6.8 to avoid spontaneous oxidation
of o-diphenols and their oxidized products (o-quinones)
observed particularly under alkaline conditions.
ST94t was shown to be stable up to 30 °C and complete
inactivation was observed at 70 °C when ST94t was

Fig. 4. Effects of pH on stability (A) and o-diphenolase activity (B) of ST94t. (A) ST94t solution (6.8 lgỈmL)1of 20 mM buffer with various pH) was
incubated for 24 h at 4 °C, followed by measurement of residual activity by the dopachrome method. (B) The o-diphenolase activity of ST94t was
measured under various pH conditions at 25 °C; the assay mixture (3 mL) contained 5 mM L-DOPA and ST94t (0.36 lg) in 0.1 M buffer (pH 3.6–
9.2). The reaction was monitored at 475 nm. The data were corrected by subtraction of the increase caused by auto-oxidation of L-DOPA. The
following buffers were used: d, sodium acetate buffer (pH 3.6–5.6); s, sodium phosphate buffer (pH 5.7–8.0); j, Tris/HCl buffer (pH 8.0–9.0);
h, sodium carbonate buffer (pH 9.2–10.8); m, sodium phosphate buffer (pH 11.2–11.9).


Ó FEBS 2003

4032 T. Naraoka et al. (Eur. J. Biochem. 270)

Fig. 5. Thermostability (A) and temperature-dependency of o-diphenolase activity (B) of ST94t. (A) ST94t solution (6.8 lgỈmL)1 of 20 mM sodium
phosphate buffer, pH 7.4) was incubated at 20–75 °C for 20 min and residual activity was measured by the dopachrome method. Activity after
incubation on ice is taken as 100%. (B) The o-diphenolase activity of ST94t (d) was measured at 4–50 °C using the dopachrome method (0.30 lg
per assay). Mushroom tyrosinase (s) was also examined for comparison (3.8 lg per assay). The reaction rates, v (DA475Ỉmin)1 per mg protein) were

plotted according to the Arrhenius equation (T, absolute temperature).

incubated at pH 7.4 for 20 min (Fig. 5A). Similar stability
toward temperature has been reported for the subunit of
hemocyanin from a gastropod, Rapana thomasiana grosse
[45]. The conformational stability of hemocyanin was
influenced generally by the aggregation state; the association
of structural subunits to hemocyanin increased the stability
[45]. Covalently linked dimeric form, the characteristic
structure of ST94 possibly contributes to the stability.
The effects of temperature on the o-diphenolase activity
of ST94t were investigated in the range 4–50 °C using
L-DOPA as a substrate. Tyrosinase from mushroom was
also examined for comparison with ST94t. As shown in
Fig. 5B, the o-diphenolase activities of both enzymes
correlated linearly with temperature according to the
Arrhenius equation, within the range from 4 to 40 °C.
When the reactions were carried out above 45 °C, however,
the reaction rates could not be determined accurately due to
inactivation of the enzymes. It was noteworthy that the
rate of decline for o-diphenolase activity of ST94t was
considerably smaller than that of mushroom tyrosinase.
Although no other cephalopod tyrosinase for comparison
has been characterized so far, the high activity of I. argentinus tyrosinase at low temperature seems to be an
adaptation to the cold living environment of the squid
[46]. For example, it has been reported that hemocyanin of
the Antarctic octopod, Megaleledone senoi, showed the
highest level of oxygen affinity among the cephalopods, and
the thermal dependence of the affinity was remarkably
smaller than those of temperate cephalopod hemocyanins

[47].
Effects of inhibitors. As shown in Table 1, the L-DOPA
oxidizing activities of ST94 and ST94t were inhibited by
phenylthiourea, tropolone, kojic acid and arbutin, potent
inhibitors of tyrosinase [10,35,48,49]. EDTA, which is
known to inhibit some tyrosinases [49], did not affect the
activities of ST94 and ST94t.

Table 1. Effects of tyrosinase inhibitors on the o-diphenolase activities of
ST94 and ST94t. The o-diphenolase activities of ST94 and ST94t were
measured by the dopachrome method in the presence of inhibitor at
25 °C. Conditions were, 5 mM L-DOPA, 0.1 M sodium phosphate
buffer (pH 6.8), several concentrations of inhibitor and the enzyme
(ST94, 0.35 lgỈmL)1; ST94t, 0.10 lgỈmL)1). The IC50 value represents
the concentration of inhibitor needed to inhibit the o-diphenolase
activity by 50%, which was obtained from each graph of the reciprocal
of reaction rate vs. inhibitor concentration.
IC50 (lM)
Inhibitor

ST94

ST94t

Phenylthiourea
Tropolone
Kojic acid
Arbutin
EDTA

0.17
2.1
48
1170
>105

0.18
2.2
48
1020
>105

Substrate specificity. The substrate specificity of ST94t
was investigated using five diphenols and three monophenols as substrates and compared with that of ST94. The rate
parameters for oxidation reactions of these substrates by
ST94 and ST94t are summarized in Table 2. Both enzymes
were shown to be able to catalyze the oxidation of all
monophenols and o-diphenols tested. The oxidation of
monophenol by these enzymes showed a characteristic lag
period, as reported for other tyrosinases, which was
shortened by addition of each mono-oxygenation product
(diphenol) as a cofactor [2,28]. From the comparison of
reaction efficiency (k0/Km), dopamine appeared to be
oxidized most effectively by ST94 as well as by ST94t,
whereas DHPPA, which is known to be good substrate for
several tyrosinases [42,43], was shown to be a poor substrate
for these enzymes. The Km value for L-tyrosine was higher


Ó FEBS 2003

Tyrosinase from the mollusk, Illex argentinus (Eur. J. Biochem. 270) 4033

Table 2. Rate parameters for the oxidation of several o-diphenols and monophenols catalyzed by ST94 and ST94t. The steady-state rate of the
oxidation of substrate was measured by the MBTH method at 25 °C. Assay conditions were 50 mM sodium phosphate buffer (pH 6.8), 5 mM
MBTH, 2% N,N-dimethylformamide, differing substrate concentrations and the enzyme (0.1–0.7 lgỈmL)1). In the analyses for monophenols, the
corresponding o-diphenol at a final concentration of 1 lM was added to the reaction mixture. The Vmax values are expressed as micromoles of
substrate oxidized per min per mg of protein. The k0 values were calculated using molecular masses of 140.2 kDa and 127.6 kDa for ST94 and
ST94t, respectively.
ST94

Substrate
L-DOPA
D-DOPA

Dopamine
DHPPA
Pyrocatechol
L-Tyrosine
D-Tyrosine
Tyramine

ST94t

Km
(mM)

Vmax
(lmolỈmin)1Ỉmg)1)

k0
(s)1)

k0/Km
(mM)1Ỉs)1)

Km
(mM)

Vmax
(lmolỈmin)1Ỉmg)1)

k0
(s)1)

k0/Km
(mM)1Ỉs)1)

Ratio of k0/Km
(ST94t/ST94)

6.8
3.2
1.3
0.49
9.3
0.57
0.32
0.32

55.4
29.4
51.2
0.478
28.8
0.194
0.216
1.59

129
68.7
120
1.12
67.3
0.453
0.505
3.72

19
21
92
2.3
7.2
0.79
1.6
12

6.5
2.7
0.39

0.24
7.3
0.35
0.24
0.12

268
136
185
3.56
197
8.74
7.98
27.6

570
289
393
7.57
418
18.6
17.0
58.7

88
110
1000
32
57
53

71
490

4.6
5.2
11
14
7.9
67
44
41

than that for the D-isomer. The same tendency was observed
for isomers of DOPA. This catalytic stereospecificity of
I. argentinus tyrosinase was similar to that reported for
S. officinalis tyrosinase [28]. From the comparison of ST94
and ST94t, it appeared that the trypsin treatment of ST94
caused a fall in the Km value and a rise in the k0 value,
resulting in approximately five to 70 times higher reaction
efficiency of ST94t for oxidation of each substrate. These
results suggest that a limited proteolysis for activation is
involved in the natural regulation system of tyrosinase in
cephalopods, as in the case of arthropods [14–17].
Molecular cloning of ST94 cDNA
The cDNA cloning of tyrosinase ST94 was carried out
by degenerate RT-PCR and RACE using the first strand
cDNA of poly(A)+ RNA extracted from an ink sac of
I. argentinus as a template. First, in order to detect the target
cDNA, RT-PCR was carried out using degenerate primers
designed from the N-terminal amino acid sequences of ST94

and ST94t. For lowering degeneracy at the 3¢ side of the
primers, each two primers for sense and for antisense, which
differed only in the triplet corresponding to the Ser codon at
the 3¢ side, were prepared and used for RT-PCR. The
amplified product about 200 bp in length was observed only
in the reaction carried out using the pair of degenerate
primers TYF1 and TYR1. The DNA sequence analysis of
the RT-PCR products revealed that the two distinct DNA
fragments of 194 bp corresponding to nucleotide 176–369 in
the cDNAs (Fig. 6) were amplified. On the 5¢-RACE
performed using the specific primer TYR3 (nucleotide 290–
314 in the cDNAs), designed from the internal sequence of
the RT-PCR products, DNA fragments of about 310 bp,
which contained the sequences of nucleotide 62–314 in the
cDNAs with an additional universal primer sequence, were
mainly amplified. Fragments about 370 bp in length, which
contained the sequences of nucleotide 1–314 in the cDNAs
(Fig. 6) were obtained as the largest product in this study.
Finally, using a primer TYFW corresponding to nucleotide
4–28 in the cDNAs, the full-length cDNAs of about 2.2 kbp
were amplified by 3¢-RACE. The complete nucleotide

sequences were determined for 12 plasmid clones of the
full-length cDNAs.
Comparison of the cDNA sequences revealed that two
distinct messages for the squid tyrosinase, represented as
cDNA-1 and cDNA-2, were expressed in the ink sac of
I. argentinus used for extraction of poly(A)+ RNA in this
study, as summarized in Fig. 6; seven and five clones carried
cDNA-1 and cDNA-2, respectively. These two cDNA

sequences were also confirmed by other PCR experiments
performed using the first strand cDNA independently
prepared from the same poly(A)+ RNA preparation as a
template. Both of the cDNAs covered the complete open
reading frames of 1878 bp (nucleotide 122–1999) encoding
putative 625-amino acid proteins with 121 bp of the 5¢
untranslated regions and the 3¢ untranslated regions
containing polyadenylation signals (AATAAA) at three
positions and the poly(A)-tails. The criteria for a consensus
translation initiation site were observed around the putative
initiator ATG codon (CCGAAATGG) of the largest open
reading frames [50]. The molecular mass numbers calculated
for the encoded proteins in the open reading frames of
cDNA-1 and cDNA-2 were 70 975 and 71 046, respectively.
Deduced amino acid sequences
The amino acid sequence deduced from the nucleotide
sequence of cDNA-1 was shown to contain sequences that
agreed with the N-terminal amino acid sequences of ST94
and ST94t determined by Edman degradation, whereas the
nucleotide sequence of cDNA-2 was different from that of
cDNA-1 at 15 positions, resulting in amino acid substitutions at four positions (Fig. 6). In particular, the substitution from Gly27 to Glu27 caused by the single base
substitution, which did not agree with the result obtained
from the N-terminal amino acid sequence analysis of ST94,
was observed in cDNA-2. In the N-terminal sequence
analysis, a portion of the ST94 preparation isolated from
about 200 ink sacs was subjected to Edman degradation,
but no similar amount of phenylthiohydantoin (PTH)-Glu
to PTH-Gly was detected at the corresponding cycle in this
study despite the similar existence ratios of the two cDNAs.

4034 T. Naraoka et al. (Eur. J. Biochem. 270)

Ó FEBS 2003

Fig. 6. Nucleotide and deduced protein sequences of ST94 cDNA-1. The nucleotides (upper) are numbered from the first base; the amino acids
(lower) are numbered from the initiating methionine. Base substitutions at 15 positions and amino acid substitutions at four positions observed in
cDNA-2 are shown in italics. The N-terminal amino acid sequences of ST94 and ST94t obtained by Edman degradation are underlined. The
putative copper ligands are circled. A potential N-linked glycosylation site is shown by an asterisk. Cysteine residues are indicated by d. The
putative polyadenylation signals are indicated by double underlining.

Furthermore, the nucleotide sequences of cDNA-1 and
cDNA-2 were almost identical: the calculated homology
was 99.3% except for the poly(A)-tails. Therefore, the
cDNA-2 was thought to be of an allelic variant message for
tyrosinase ST94, expressed in the individual of I. argentinus
used for poly(A)+ RNA extraction in this study. Both

amino acid sequences deduced from the two cDNAs were
revealed to possess two putative copper-binding sites
(critical regions for tyrosinase activity), as described below,
and no amino acid substitution was observed in these two
sites (Figs 6 and 7). Therefore, the variant of ST94 was
thought to be able to function as tyrosinase as well as ST94.


Ó FEBS 2003

Tyrosinase from the mollusk, Illex argentinus (Eur. J. Biochem. 270) 4035

Fig. 7. Comparison of the amino acid sequences at two putative copper-binding sites, Cu(A) and Cu(B), in ST94 and other type 3 copper proteins.
Numbers indicate positions of the amino acid residues in each sequence. Gaps (–) have been introduced to optimize the alignment. The putative
copper ligands of histidine residues conserved in all proteins are labeled with d, those conserved in molluscan proteins and tyrosinases with an s,
and those conserved in arthropod proteins with an h. The identical residues are shaded. IaY, I. argentinus tyrosinase ST94; OdHc and OdHe,
Octopus dofleini hemocyanin functional unit c and e, respectively (SWISS-PROT, accession No. O61363); SoHh, S. officinalis hemocyanin unit h
(SWISS-PROT, P56826); HpHg, Helix pomatia bc-hemocyanin unit g (SWISS-PROT, P56823); McH2c, M. crenulata hemocyanin unit 2-c
(SWISS-PROT, P81732); MmY, Mus musculus tyrosinase (SWISS-PROT, P11344); GgY, Gallus gallus tyrosinase (DDBJ, D88349); CeY,
C. elegans hypothetical protein K08E3.1 (DDBJ, Z81568); AoY, Aspergillus oryzae tyrosinase (DDBJ, D37929); SgY, Streptomyces glaucescens
tyrosinase (SWISS-PROT, P06845); BmP, B. mori prophenoloxidase subunit 1 (DDBJ, D49370); PlP, Pacifastacus leniusculus prophenoloxidase
(DDBJ, X83494); PiH, Panulirus interruptus hemocyanin subunit a (SWISS-PROT, P04254); LpH, Limulus polyphemus hemocyanin II (SWISSPROT, P04253).

These findings suggested the occurrence of other ST94
variants in the gene pool of I. argentinus. Sequence polymorphism of the tyrosinase gene has also been observed in
Neurospora crassa [51].
The N-terminal sequence of ST94 was found to start at
the 19th amino acid residue in the open reading frame
encoded in cDNA-1, which was preceded by 18 amino acid
residues, indicating that the subunit of ST94 was expressed
as a premature 625-amino acid protein, followed by excision
of the preceding 18 amino acid residues considered to be a
signal sequence to form the mature subunit polypeptide.
Thus, the subunit of ST94 was thought to consist of 607
amino acid residues from Met19 to Lys625 with a molecular
mass of 68 993 Da, on the basis of the amino acid sequence
deduced from cDNA-1. The N-terminal sequence of ST94t
was shown to start at the 70th amino acid residue, indicating
that the N-terminal 51 amino acid residues of ST94 were
digested with trypsin to generate the N-terminal of ST94t,
resulting in a reduction in molecular mass of 5807 Da per
subunit. However, the molecular mass of ST94 was reduced

by 6.3 kDa per subunit by trypsin treatment, in the mass
analyses. Therefore, ST94 seemed to be digested by trypsin
not only at the N-terminal but also (a few amino acids) at
the C-terminal. Although there is no evidence, one candidate for trypsin cleavage site is at Arg620-Asn621 in the
C-terminal region to release a peptide of five residues

(Asn621 to Lys625). The molecular mass of the N- and
C-terminal-truncated subunit, a 551-amino acid polypeptide
composed of Ile70 to Arg620, was calculated to be
62 644 Da, which gives a value for the reduction in
molecular mass (6349 Da per subunit) concordant with
that observed in the mass analyses.
Both molecular mass numbers deduced for the subunits
of ST94 (68 993 Da) and ST94t (62 644 Da) from cDNA-1
were approximately 1.1 kDa less than those obtained from
the mass spectrometry (70.1 kDa for ST94 and 63.8 kDa
for ST94t). The cause of the difference of about 1 kDa
except for mass of two copper atoms (127 Da) remains
unclear; however, the difference was presumed to be due to
post-translational modifications, for example, glycosylation
at the unique potential glycosylation site for N-linked
carbohydrate found at Asn333.
It was demonstrated that mature ST94 subunit could
undergo the digestion of N-terminal 51 amino acid residues
by trypsin. In prophenoloxidases in two insects, Bombyx
mori and Manduca sexta, the N-terminal region consisting
of 51 amino acid residues were also cleaved by the
prophenoloxidase-activating enzyme (PPAE) to generate
active phenoloxidases [11,13]. This similarity suggests
that the conformational change of ST94 caused by the

elimination of the N-terminal region is required principally
for its activation. Furthermore, the cleavage site of ST94


4036 T. Naraoka et al. (Eur. J. Biochem. 270)

(Lys69-Ile70, Fig. 6) was consistent with those for the
activation of pro-PPAE (zymogen of PPAE) to PPAE in
arthropods (predominantly between Lys-Ile and Arg-Ile)
[17,52]. The N-terminal cleavage site of ST94 seems to be in
the specific conformational environment (for example, in
surface-exposed loop) which urges the site to be attacked
easily by trypsin-type serine proteases. This cleavage site of
ST94 may function as the activation site also in the natural
activation system, if the trypsin-type serine proteases are
involved in the system as in the case of the arthropod
proteins.
It remains unclear whether C-terminal cleavage is
involved in the activation of ST94. For tyrosinases from
plant and fungi, the proteolytic cleavage of C-terminal part
is necessary for activation, of which mechanism was
different from arthropod prophenoloxidases requiring
N-terminal deletion [6,53]. In addition, it was reported
recently that an antibacterial peptide was generated from
the C-terminal region of crayfish hemocyanin [54], implying
the unknown physiological functions of the peptides cleaved
from other tyrosinases and hemocyanins.
Fifteen cysteine residues were found in the amino acid
sequence of mature ST94 subunit; two residues were in
the N-terminal region and others were clustered in the

C-terminal region (Fig. 6). As ST94 remained a covalently
linked dimer after elimination of the N-terminal region
containing two cysteine residues by proteolysis with trypsin,
at least one of 13 cysteine residues in the C-terminal region
was thought to be responsible for the linkage between
two subunits. Some of the other cysteine residues may
be involved in intramolecular disulfide bridges, creating a
packed structure in the C-terminal region similar to the
C-terminal domain of hemocyanin [3,6]. As described
above, the I. argentinus tyrosinase was found to exhibit
temperature-dependency of o-diphenolase activity like a
psychrophilic enzyme. As there is no cysteine residue
around the copper-binding sites of ST94, we suggest that
the active center of ST94 is more flexible, which allows this
enzyme to retain higher activity at low temperatures [46].

Ó FEBS 2003

Structural similarity of ST94 to other tyrosinases
and hemocyanins

Fig. 8. Phylogenetic tree of copper-binding regions. The tree was
deduced by a neighbor-joining analysis based on the alignment of the
amino acid sequences of copper-binding regions ranging from the
N-terminal of Cu(A) site to the C-terminal of Cu(B) site shown in
Fig. 7, which was carried out by the CLUSTALW program available at
DDBJ web server (). Gaps were treated as
missing data. Bootstrap values (1000 replicates) are indicated at the
nodes. For abbreviated protein names, see Fig. 7.

BLAST homology search of the amino acid sequence of
ST94 deduced from cDNA-1 revealed that two putative
copper-binding sites for Cu(A) and Cu(B), characteristically
conserved regions in type 3 copper proteins, were present in
ST94 [6]. Comparison of these sites of ST94 with those of
tyrosinases, hemocyanins and phenoloxidases from other
organisms are shown in Fig. 7. These two copper-binding
sites of ST94 were similar to those of molluscan hemocyanins as well as tyrosinases from microorganisms, a
nematode and vertebrates, and less similar to those of
phenoloxidases and hemocyanins from arthropods. Six
histidine residues in these sites of ST94 assumed to be
copper-binding ligands could be arranged in similar positions with those of molluscan hemocyanins and tyrosinases
[7,9]; highly conserved amino acid sequences and residues
around these copper ligands in molluscan hemocyanins and
tyrosinases were also well conserved in ST94. On the other
hand, molluscan hemocyanins revealed that one of the
ligands in the Cu(A) site is involved in the cysteine-histidine

thioether bridge [55]. This unusual linkage in the copperbinding site has also been observed in ascomycete tyrosinases, allowing the hypothesis that molluscan hemocyanin
evolved from ancient tyrosinase [51,53,56]. However, no
cysteine residue was found around the copper-binding sites
of ST94 as mentioned above. Furthermore, for the whole
amino acid sequence, the similarity among ST94 and
molluscan hemocyanins and other tyrosinases was not high,
compared with that between phenoloxidases and hemocyanins in arthropods; ST94 showed the highest homology
(30%) with the hypothetical tyrosinase-related protein of
C. elegans. From these observations we suggest that there
are great evolutional distances between molluscan
tyrosinases and molluscan hemocyanins as well as other
tyrosinases, as shown in Fig. 8. The phylogenetic relationships among type 3 copper proteins will be deduced more

accurately by using the structural information of other
molluscan tyrosinases obtained hereafter.


Ó FEBS 2003

Tyrosinase from the mollusk, Illex argentinus (Eur. J. Biochem. 270) 4037

Evolutional scenarios of hemocyanins and tyrosinases
have been proposed [7–9]. It has been suggested, for
example, that molluscan hemocyanins and tyrosinases
genetically diverged from their common ancestor, separately from the evolutional line of hemocyanins and
phenoloxidases in arthropods, and that the divergence of
molluscan hemocyanins and tyrosinases occurred earlier
than that of arthropod hemocyanins and phenoloxidases, at
an extremely early evolutional stage of life similar to that
when aerobic metabolism was established and metazoans
emerged. As described above, the structural features of
molluscan tyrosinase ST94, the nearest relative of molluscan
hemocyanins, seem to support these evolutional scenarios.

Acknowledgements
The authors wish to thank Dr Isoshi Nukatsuka, Hirosaki University
and Ms Junko Murakami, Aomori Prefectural Environmental and
Health Center, for excellent technical support and valuable advice on
metal analysis.

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