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 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 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: naraoka@aomori-tech.go.jp 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 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 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 : 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 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 L of 10 mM sodium phosphate buffer (pH 7.4) at °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 °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 °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 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 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 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 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 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 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 to 1.0 M over 16.6 in 50 mM Tris/HCl (pH 7.0) was applied (fractions of 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 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 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 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 mM L-DOPA, 0.1 M sodium phosphate buffer (pH 6.8) and the enzyme solution in a total volume of mL The reaction took place in a cuvette with a path length of 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 lmol of L-DOPA per 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 mL of reaction mixture containing an appropriate concentration of substrate, 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 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 mM EDTA containing 10 mM Tricine/KOH buffer (pH 8.5), heated at 72 °C for 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 · PCR buffer under the following conditions: after holding (94 °C, 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, 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, s) and annealing/ elongation (72 °C, min), five cycles of denaturing (94 °C, s), annealing (70 °C, 10 s) and elongation (72 °C, min), followed by 25 cycles of denaturing (94 °C, s), annealing (68 °C, 10 s) and elongation (72 °C, 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 unit of KOD Plus DNA polymerase in the PCR buffer supplied PCR was performed under the following conditions: holding (94 °C, min), 30 cycles of denaturing (94 °C, s) and annealing/elongation (68 °C, 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 (http://www.ddbj.nig.ac.jp) 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 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 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 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 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 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 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 °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 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 °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 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 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 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 (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 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 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, 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 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 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), 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 lM was added to the reaction mixture The Vmax values are expressed as micromoles of substrate oxidized per 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 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 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 Comparison of the amino acid sequences at two putative copper-binding sites, Cu(A) and Cu(B), in ST94 and other type 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 (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 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 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 (http://www.ddbj.nig.ac.jp) Gaps were treated as missing data Bootstrap values (1000 replicates) are indicated at the nodes For abbreviated protein names, see Fig 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 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 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 The phylogenetic relationships among type 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 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cells Biochim Biophys Acta 1261, 151–154  ... 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. .. 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. .. 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