Tarantula Hemocyanin Shows Phenoloxidase Activity
1998; Elsevier BV; Volume: 273; Issue: 40 Linguagem: Inglês
10.1074/jbc.273.40.25889
ISSN1083-351X
Autores Tópico(s)Hemoglobin structure and function
ResumoAn enzyme generally catalyzes one well defined reaction with high specificity and efficiency. We report here in contrast that the copper protein hemocyanin of the tarantulaEurypelma californicum exhibits two different functions. These occur at the same active site. While hemocyanin usually is an oxygen carrier, its function can be transformed totally to monophenoloxidase and o-diphenoloxidase activity after limited proteolysis with trypsin or chymotrypsin.N-acetyldopamine (NADA) is more effectively oxidized thanl-dopa or dopamine. This irreversible functional switch of tarantula hemocyanin function is limited to the two subunitsb and c of its seven subunit types. A conserved phenylalanine in the hemocyanin molecule acts as a placeholder for other substrates that are phenylalanine derivatives. The proteolytic cleavage removes an N-terminal fragment, including the critical phenylalanine residue, which opens an entrance for substrates. Therefore no new arrangement of the active site, with its two copper atoms and the μ − η2:η2 bound O2 molecule, is necessary to develop the catalytic function. An enzyme generally catalyzes one well defined reaction with high specificity and efficiency. We report here in contrast that the copper protein hemocyanin of the tarantulaEurypelma californicum exhibits two different functions. These occur at the same active site. While hemocyanin usually is an oxygen carrier, its function can be transformed totally to monophenoloxidase and o-diphenoloxidase activity after limited proteolysis with trypsin or chymotrypsin.N-acetyldopamine (NADA) is more effectively oxidized thanl-dopa or dopamine. This irreversible functional switch of tarantula hemocyanin function is limited to the two subunitsb and c of its seven subunit types. A conserved phenylalanine in the hemocyanin molecule acts as a placeholder for other substrates that are phenylalanine derivatives. The proteolytic cleavage removes an N-terminal fragment, including the critical phenylalanine residue, which opens an entrance for substrates. Therefore no new arrangement of the active site, with its two copper atoms and the μ − η2:η2 bound O2 molecule, is necessary to develop the catalytic function. N-acetyldopamine. Although hemocyanins and phenoloxidases, both extracellular proteins in the hemolymph of arthropods, bind oxygen in a μ − η2:η2 coordination, their biological functions are very different (1Kitajima N. Morooka Y. Chem. Rev. 1994; 94: 737-757Crossref Scopus (876) Google Scholar, 2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar). Hemocyanins serve as oxygen carriers for many chelicerates and crustaceans (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 4Salvato B. Beltramini M. Life Chem. Rep. 1990; 8: 1-47Google Scholar, 5Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). This function is well understood on the basis of the known structure of several hemocyanins (6Gaykema W.P.J. Hol W.G. Vereijken J.M. Soeter N.M. Bak H.J. Beintema J.J. Nature. 1984; 309: 23-29Crossref Scopus (308) Google Scholar, 7Volbeda A. Hol W.G.J. J. Mol. Biol. 1989; 209: 249-279Crossref PubMed Scopus (382) Google Scholar, 8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). Phenoloxidases are found in an inactive form in the hemolymph of Crustacea and insects (10Hernandez-Lopez J. Gollas-Galvan T. Vargas-Albores F. Comp. Biochem. Physiol. 1996; 113C: 61-66Google Scholar, 11Kopacek P. Weise C. Gotz P. Insect Biochem. Mol. Biol. 1995; 25: 1081-1091Crossref PubMed Scopus (96) Google Scholar, 12Nellaiappan K. Sugumaran M. Comp. Biochem. Physiol. 1996; 113B: 163-168Crossref Scopus (62) Google Scholar, 13Sanchez-Ferrer A. Rodriguez-Lopez J.N. Garcia-Canovas F. Garcia-Carmona F. Biochim. Biophys. Acta. 1995; 1247: 1-11Crossref PubMed Scopus (1134) Google Scholar, 14Fujimoto K. Okino N. Kawabata S. Iwanaga S. Ohnishi E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7769-7773Crossref PubMed Scopus (139) Google Scholar, 15Aspan A. Huang T.S. Cerenius L. Soderhall K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (232) Google Scholar, 16Brivio M.F. Mazzei C. Scari G. Comp. Biochem. Physiol. 1996; 113B: 281-287Crossref Scopus (17) Google Scholar, 17van Gelder C.W.G. Flurkey W.H. Wicherts H.J. Phytochemistry. 1997; 45: 1309-1323Crossref PubMed Scopus (412) Google Scholar, 18Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1127) Google Scholar). After limited proteolysis, the phenoloxidase so produced shows both a monophenoloxidase and ano-diphenoloxidase activity. Phenoloxidase is widespread in animals and plants as well as in fungi; it starts the synthesis of melanin, is involved in defense reactions (19Prota G. Melanins and Melanogenesis. Academic Press, San Diego1992Google Scholar, 20Sugumaran M. Kanost M. Beckage N.E. Thompson S.N. Federici A.B. Parasites and Pathogens of Insects. Academic Press, San Diego1993: 317-342Crossref Scopus (81) Google Scholar), and is also crucial for arthropod sclerotization, using N-acetyldopamine (NADA)1 as substrate (21Anderson S.O. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 123-140Google Scholar,22Sugumaran M. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 141-168Google Scholar). Although much is known about the biological functions of phenoloxidase, its molecular mechanism and regulation are not well understood because of the lack of any known structure (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar, 13Sanchez-Ferrer A. Rodriguez-Lopez J.N. Garcia-Canovas F. Garcia-Carmona F. Biochim. Biophys. Acta. 1995; 1247: 1-11Crossref PubMed Scopus (1134) Google Scholar). o-Diphenoloxidase and monophenoloxidase activity were measured spectrophotometrically by recording the formation of dopachrome, with an absorption maximum at 475 nm. Iterative spectra in the region of 300–600 nm were recorded to exclude other by-products. The assay medium contained 4 mml-dopa in the case of o-diphenol oxidation and 2 mm tyrosine in the case of tyrosinase activity, which includes the monophenoloxidase and o-diphenoloxidase activities, in 0.1 m potassium phosphate buffer at pH 6.8 at 20 °C. Native polyacrylamide gel electrophoresis (5%) and crossed immuno gel electrophoresis were performed according to Lamyet al. (32Lamy J. Lamy J. Weill J. Markl J. Schneider H.-J. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 889-895Crossref PubMed Scopus (42) Google Scholar) and stained by incubation in 4 mml-dopa, pH 6.8, and 0.1 m potassium phosphate at room temperature. When the protein was to be activated by the staining solution itself, then 1 mg/ml trypsin was also added. After staining, the gels were dried or stored in methanol/acetic acid/water. Limited proteolysis was performed with bovine trypsin and bovine chymotrypsin (Sigma) in 0.1 mTris/HCl buffer, pH 7.8 (in the case of the native 24-mer) and pH 9.6 (in the case of subunits) at room temperature. The ratio between hemocyanin and protease varied between 1:1 and 10:1 (w/w). Proteolysis was terminated by addition of soybean trypsin inhibitor (Sigma). The x-ray structures of subunit II of Limulus polyphemus hemocyanin(Protein Data Bank code 1oxy) and the hexameric hemocyanin of Panulirus interruptus (Protein Data Bank code 1hcy) were obtained from the Protein Data Bank, Brookhaven National Laboratory. Sequence comparison was performed using DNAstar, based on the clustal method. The various peptide sequences were obtained from the Protein Data Bank as indicated previously (28Beintema J.J. Stam W.T. Hazes B. Smidt M.P. Mol. Biol. Evol. 1994; 11: 493-503PubMed Google Scholar, 29Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar, 30Basyoni M. On the Sequence of Subunit b from Tarantula HemocyaninPh.D. thesis. University of Munich, 1990Google Scholar). The hemocyanin of Eurypelma californicum hemolymph was obtained by dorsal puncture of the heart. All samples were immediately diluted 1:2 (v/v) with 0.2 m Tris/HCl, pH 8.0, 10 mmCaCl2, 10 mm MgCl2 to stabilize the protein. The samples were then centrifuged at low speed for 10 min at room temperature using a table centrifuge to remove blood cells. The hemocyanin was purified by gel filtration (TSK-HW 55 F; 0.1m Tris/HCl, pH 8.0, 5 mm CaCl2, 5 mm MgCl2 at 20 °C). The large leading peak contained purified 24-meric hemocyanin, as verified by UV spectroscopy and two-dimensional immuno gel electrophoresis. 24-meric hemocyanin was dissociated by dialysis against 0.02 m glycine/NaOH buffer, pH 9.6, at 4 °C for 4–5 days. The protein concentration was below 1 mg/ml to avoid reassociation. The native 24-meric hemocyanin of the tarantula E. californicum (23Markl J. Kempter B. Linzen B. Bijlholt M.M.C. van Bruggen E.F.J. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1631-1641Crossref PubMed Scopus (69) Google Scholar, 24Decker H. Sterner R. J. Mol. Biol. 1990; 211: 281-293Crossref PubMed Scopus (64) Google Scholar) seems incapable of oxidizingo-diphenols such as l-dopa and monophenols such as l-tyrosine, even after addition of urea or perchlorate at any concentration. An activity similar to phenoloxidase has recently been observed for the hemocyanins from the crustaceans Homarus americanus and Carcinus maenas and from the molluscOctopus vulgaris using catechol (25Zlateva T. di Muro P. Salvato B. Beltramini M. FEBS Lett. 1996; 384: 251-254Crossref PubMed Scopus (84) Google Scholar, 26Salvato B., Santamaria, M., Beltramini, M., Alzuet, G., and Casella, L. (1998) Biochemistry, in pressGoogle Scholar). We have now observed monophenoloxidase activity as well aso-diphenoloxidase activity for a hemocyanin from a chelicerate, the tarantula E. californicum, after limited proteolysis with serine proteases such as trypsin and chymotrypsin, by following the enzyme-specific formation of dopachrome at 475 nm (Fig. 1, A and B). Monophenoloxidase activity of activated tarantula hemocyanin starts with a long lag phase that is also characteristic for tyrosinases but not well understood (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar, 13Sanchez-Ferrer A. Rodriguez-Lopez J.N. Garcia-Canovas F. Garcia-Carmona F. Biochim. Biophys. Acta. 1995; 1247: 1-11Crossref PubMed Scopus (1134) Google Scholar). The o-diphenoloxidase activity has no lag phase although the active sites are identical for both activities, assessed by means of absorption spectroscopy (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar). Compared with the activity of tyrosinase from mushrooms, the activity of hemocyanin is lower by almost two orders of magnitude (Fig. 1 C). To prove that an oxygen consuming process occurs during phenoloxidase activity, oxygen consumption was followed using protease-treated 24-meric hemocyanin with various o-diphenol derivatives such as l-dopa, dopamine, and NADA (Fig. 1 D). These data represent an absolute measure for comparing the phenoloxidase activity of different substrates. Although dopamine is oxidized at the same rate as l-dopa, NADA is oxidized significantly faster. The phenoloxidase activity of tarantula hemocyanin is not dependent on the quaternary structure of the 24-mer. When investigated on polyacrylamide gel electrophoresis under conditions where destabilized 24-mers tend to dissociate, pH 8.8, 24-mers are detected as well as various intermediates and monomers (27Savel-Niemann A. Markl J. Linzen B. J. Mol. Biol. 1988; 204: 385-395Crossref PubMed Scopus (31) Google Scholar). The 24-, the 12-, and the 7-mer dissociation intermediates show phenoloxidase activity after incubation with a phenoloxidase-specific activity staining assay (Fig. 2 panel A). Among the subunits, only the broad band containing the subunit d and heterodimerbc shows phenoloxidase activity. Using crossed immuno gel electrophoresis (Fig. 2, panel B) only the heterodimer bc shows phenoloxidase activity. This observation was also confirmed by separating the heterodimerbc from the monomeric subunits a, d, e, f, and g by size-exclusion gel chromatography (TSK HW 55), where again only the heterodimerbc showed phenoloxidase activity. We believe that both theb and c subunits possess enzymic activity. Incubation in 3 and 4 m urea results in a partial dissociation of b and c from the heterodimer (Fig. 2, panel B). At 3 m urea, subunitsb and c are both visible and both show phenoloxidase activity. At higher concentrations of urea, however, all subunits become unstable, in particular subunit c becomes undetectable. This exclusive phenoloxidase activity of the heterodimerbc of tarantula hemocyanin is observed after limited proteoloysis with trypsin as well as with chymotrypsin. Based on sequence alignments (8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 28Beintema J.J. Stam W.T. Hazes B. Smidt M.P. Mol. Biol. Evol. 1994; 11: 493-503PubMed Google Scholar, 29Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar), including the sequence of subunitb (30Basyoni M. On the Sequence of Subunit b from Tarantula HemocyaninPh.D. thesis. University of Munich, 1990Google Scholar) (subunit c is not sequenced yet), and the x-ray structures of L. polyphemus hemocyanin subunit II (chelicerate) and of P. interruptus hemocyanin (crustacean) (6Gaykema W.P.J. Hol W.G. Vereijken J.M. Soeter N.M. Bak H.J. Beintema J.J. Nature. 1984; 309: 23-29Crossref Scopus (308) Google Scholar, 7Volbeda A. Hol W.G.J. J. Mol. Biol. 1989; 209: 249-279Crossref PubMed Scopus (382) Google Scholar, 8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar), several cleavage positions for chymotrypsin and trypsin have been detected that are located on the surface of a chelicerate hemocyanin subunit. According to the sequence numbering of Limulus subunit II, trypsin may cleave at Arg-81 (subunitb: Arg-68), Lys-130 (subunit b: Lys-0117) and Arg-133 (subunit b: Arg-120). Chymotrypsin may cleave at Phe-83 (subunit b: Tyr-70). The most promising candidate seems to be Arg-120 on subunit b, which corresponds to Arg-176 of the closely related prophenoloxidases of the crustaceanPacifastacus leniusculus (15Aspan A. Huang T.S. Cerenius L. Soderhall K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (232) Google Scholar). This enzyme, withM r = 81,000, is activated by trypsin at Arg-176, yielding an active fragment of about 60 kDa. The other tarantula hemocyanin subunits do not have an Arg but a Lys at this position, but surprisingly they are not activated. The prophenoloxidase subunits from the insect Galleria mellonella are of similar sizes toPacifastacus prophenoloxidase, with masses of 80 and 83 kDa. After proteolysis with chymotrypsin, two active fragments with 67 and 50 kDa were found. Phenoloxidase-active Eurypelma hemocyanin fragments also have comparable masses. SDS-gel electrophoretic analysis reveals that the heterodimer bc splits into peptides with two major components of M r ≈ 50,000 and M r ≈ 55,000 after cleavage with chymotrypsin and one major component with M r ≈ 55,000 after cleavage with trypsin (Fig. 2, panel C). However, any procedure to obtain a purified pseudo-native 55-kDa fragment with full enzymatic properties has not been successful so far. In the case of tarantula hemocyanin from E. californicum, the cleavage of an N-terminal peptide including Phe-49 from the two subunits b and c seems to open the entrance to the active site, with its copper atoms, for phenolic substrates. This is independent of the association status of the subunits (Fig. 3). This highly conserved phenylalanine in hemocyanins is at a distance of 3.5 Å from the active site and serves as an allosteric trigger during the oxygenation process (8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). We think that Phe-49 may also act as a placeholder for phenolic substrates during the time when hemocyanin is acting only as an oxygen carrier, to conserve the hidden function as a phenoloxidase in the native but uncleaved state. Assuming the same orientation of possible substrates for the phenoloxidase activity as Phe-49, the phenyl ring lies almost perpendicular to the Cu-Cu-O2 plane, with close contact of the ortho position to one of the two oxygen atoms (Fig. 4). In this geometry, the hydroxyl group may bind to the copper atoms to initialize the cleavage of the oxygen molecule (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar). The optimal arrangement of the substrate may be sterically hindered by Thr-351 (according to the numbering of L. polyphemus hemocyanin). This would explain the low o-phenoloxidase activity of hemocyanin with respect to other phenoloxidase and tyrosinase.Figure 4The orientation of a hypothetical substrate HO-Phe-49 replacing Phe-49 at the oxygen binding site. The hemocyanin subunit II (L. polyphemus) is used (9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). The two copper atoms (blue) are complexed by six histidines. The dioxygen (red) is bound in a μ − η2:η2 coordination. A hypothetical hydroxyl group is added to Phe-49 to design a hypothetical tyrosine. Note that Phe-49 as well as the HO-Phe-49 are closer to the dioxygen and Cu(A) than to Cu(B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thus, the explanation for the activation of phenoloxidase activity of tarantula hemocyanin by limited proteolysis seems to be based not on a rearrangement of the active site but by providing a free access for various substrates to the active site, following removal of the nonreactive substrate analogue Phe-49. The greater accessibility of the active site of mushroom tyrosinase compared with hemocyanin can also be deduced from kinetic removal of the copper atoms by cyanide in both proteins (31Beltramini M. Salvato B. Santamaria M. Lerch K. Biochim. Biophys. Acta. 1990; 1040: 365-372Crossref PubMed Scopus (23) Google Scholar). This hypothesis about a free entrance is supported by a recent study (25Zlateva T. di Muro P. Salvato B. Beltramini M. FEBS Lett. 1996; 384: 251-254Crossref PubMed Scopus (84) Google Scholar) which shows that access for catechol to the active site is potentiated by the presence of salts of the Hofmeister series such as high concentrations of perchlorate. The salts increase the low phenoloxidase activity of crustacean hemocyanins (H. americanus and C. maenas) by a factor of two (25Zlateva T. di Muro P. Salvato B. Beltramini M. FEBS Lett. 1996; 384: 251-254Crossref PubMed Scopus (84) Google Scholar). However, we have not been able to induce any significant phenoloxidase activity in the crustacean hemocyanins from P. interruptusor H. americanus by proteolysis, using tyrosine orl-dopa as substrates. This difference in the hemocyanins from Crustacea and Chelicerata may be explained on the basis of their x-ray structures (8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). Although the active sites are highly conserved, Thr-351 belonging to the active site of Limulushemocyanin is replaced by the larger Phe-371 in Panulirushemocyanin (Fig. 3), which sterically hinders any substrate larger than oxygen from coming close to the active site, even after removal of the N-terminal fragment. Thr-351 is conserved in Chelicerata hemocyanins. In the case of Eurypelma hemocyanin, however, a reason for the exclusive phenoloxidase activity of subunits b and c remains unknown. We may make three significant deductions from our results. First, our findings may explain the high conservation of quaternary structure of the 24-meric tarantula hemocyanin throughout evolution in this ancient animal. Subunits b and c bridge the two structurally identical 2 × 6-meric half-molecules (23Markl J. Kempter B. Linzen B. Bijlholt M.M.C. van Bruggen E.F.J. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1631-1641Crossref PubMed Scopus (69) Google Scholar). In the case of crustacean hemocyanins, these linker subunit types are not present: only 6-mers and 2 × 6-mers are found in the hemolymph of modern arthropods (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 4Salvato B. Beltramini M. Life Chem. Rep. 1990; 8: 1-47Google Scholar, 5Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). In addition, species of two large groups among arthropods, Crustacea and Insecta, have evolved prophenoloxidases, most probably by gene duplication of hemocyanins, which is indicated by the close structural relationship between phenoloxidase and hemocyanin (14Fujimoto K. Okino N. Kawabata S. Iwanaga S. Ohnishi E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7769-7773Crossref PubMed Scopus (139) Google Scholar, 15Aspan A. Huang T.S. Cerenius L. Soderhall K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (232) Google Scholar), and consecutive optimization of a physiologically beneficial phenoloxidase activity. Second, evolution seems to have stabilized a double function in the hemocyanin from the ancient tarantula Eurypelma californicum. This is a further example in contrast to the dogma: one gene—one protein—one function with respect to the active site. We suggest that the Eurypelma hemocyanin can develop two different functions consecutively using the same active site. During normal life, the hemocyanins serve as oxygen carriers. At a crucial moment of the life of arthropods, i.e. the growing phase, hemocyanin function may switch to phenoloxidase activity by limitedin vivo proteolysis. Such phenoloxidase activity is known to be necessary to harden the exoskeleton rapidly after molting (i.e. within minutes to hours) for protection against predators. The "new function" of hemocyanin may contribute toward catalyzing the first steps in the sclerotization process, using NADA as the main starter substrate, as is the case for many insect phenoloxidases (21Anderson S.O. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 123-140Google Scholar, 22Sugumaran M. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 141-168Google Scholar). This hypothesis may be supported by the more efficient oxidation rate of NADA compared with l-dopa or dopamine (Fig. 1). The enzymatic activity is low, but it may be compensated for by the extremely high concentration of hemocyanin in the hemolymph of the tarantula, ranging between 5 and 120 g/liter. It should be mentioned that we were not able to detect other phenoloxidase activity in the hemolymph of tarantula under our experimental conditions. We tested fresh hemolymph with intact cells and crude but cell-free hemolymph by gel-electrophoresis but not spectroscopically because of the high turbidity of the samples. However, we did not look for prophenoloxidase activity within any cells. Thus, we cannot exclude the existence of any prophenoloxidase either in the blood cells or in other tissue cells. Experiments to demonstrate the physiological relevance of our experiments in vivo are in progress. Third, prophenoloxidases have been identified in the hemocytes of insects and a crustacean but have not been looked for in chelicerates yet (18Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1127) Google Scholar). Nevertheless, as cheliceratean hemocyanin are also synthesized in the cytoplasm of hemocytes in contrast to crustacean hemocyanins, which are synthesized in cells of the hepatopancreas (5Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar), we could make the following very tentative hypothesis. In ancestral arthropods, hemocyanin was synthesized in hemocytes and had both functions, the oxygen transport and the oxidation of aromatic compounds after proteolytic cleavage. This ancestral state is still found in the chelicerates. But after divergence of chelicerates from other arthropods like crustaceans and insects, a gene duplication has occurred leading to crustacean hemocyanins and hexamerins (28Beintema J.J. Stam W.T. Hazes B. Smidt M.P. Mol. Biol. Evol. 1994; 11: 493-503PubMed Google Scholar, 29Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar). These hemocyanins no longer possess the capability to become phenoloxidases by proteolytic cleavage and become secondary proteins synthesized elsewhere in the body. The other line has lost the capability to form hexamers and to transport oxygen and has become the prophenoloxidases identified presently in insects and crustaceans. Finally, these observations suggest a system whereby one may examine the structural basis of phenoloxidase activity because no crystal structure of any phenoloxidase is available. We thank Drs. F. Tuczek, J. Beintema, and N. Terwilliger for intensive discussions and Dr. K. Miller for reading the manuscript and help with the English language.
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