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Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors
Biochimica et Biophysica Acta 1478 (2000) 19^29 www.elsevier.com/locate/bba
Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors M. Anna Rosei a
a;
*, Ra¡aella Coccia a , Cesira Foppoli b , Carla Blarzino a , Chiara Cini c , M. Eugenia Schinina¨ a
Dipartimento di Scienze Biochimiche `A. Rossi Fanelli', Universita© `La Sapienza', P.le Aldo Moro 5, 00185 Roma, Italy b Centro di Biologia Molecolare del C.N.R., P.le Aldo Moro 5, 00185 Roma, Italy c Consiglio Nazionale delle Ricerche, P.le Aldo Moro 5, 00185 Roma, Italy Received 30 June 1999; received in revised form 15 October 1999; accepted 22 December 1999
Abstract The reaction of opioid peptides with mushroom tyrosinase in the presence of an excess of a thiol compound gives rise to cysteinyldopaenkephalins (CDEnks). The major product is represented by the 5-S-CDEnk (80%) and the minor one by the isomer 2-S-CDEnk (20%). The adducts between leucine-enkephalin (Leu-enk) and cysteine have been isolated by high performance liquid chromatography (HPLC) and identified by amino acid analysis and electrospray ion mass spectrometry. 5-S-CDEnk is able to bind to opioid receptors in bovine brain membranes. Its binding affinity is higher for N than for W receptors and about 8-fold lesser than that exploited by Leu-enk. In the presence of the peroxidase/H2 O2 system, CDEnks can be converted into the corresponding pheo-opiomelanins. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Tyrosinase; Synthesis; Enkephalin; Pheomelanin; Opioid receptor
1. Introduction Opioid peptides represent a large class among animal peptides. They can be divided into three groups: enkephalins, endorphins and dynorphins, all derived from larger protein precursors [1]. These compounds
Abbreviations: CD, cysteinyldopa; 5-S-CD, 5-S-cysteinyldopa; 2-S-CD, 2-S-cysteinyldopa; CDEnk, cysteinyldopaenkephalin; 5-S-CDEnk, 5-S-cysteinyldopaenkephalin; 2-S-CDEnk, 2-Scysteinyldopaenkephalin; Leu-enk, leucine-enkephalin; DPDPE, [D-Pen2;5 ]-enkephalin; DAGO, [D-Ala2 ,Me-Phe4 ,Gly5 -ol]-enkephalin; ESI, electrospray ionization; CID, collision-induced dissociation * Corresponding author. Fax: +39 (6) 4440062; E-mail: [email protected]
are of great interest because of their opiate-like activity. Their role as neurotransmitters and hormones has been demonstrated by a large number of investigations. Normally, opioid peptides are eliminated by peptidases that are able to bring about their complete cleavage and inactivation [2,3]. They exhibit as common feature the presence of a tyrosine residue at the amino-terminus, which is essential for their biological activity [4,5]. In recent years, a series of studies demonstrating that melanogenesis in vitro can occur with other substrates and other enzymes than those yet known has been carried out in our laboratory [6^9]. In particular, we demonstrated that opioid peptides are substrates in vitro of mushroom and sepia tyrosinase giving rise to synthetic melanins retaining the peptide
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 8 7 - 3
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Fig. 1. Tyrosinase-catalyzed formation of the CD derivatives of Leu-enk.
moiety, that we named opiomelanins [10^14]. Enzyme a¤nity for the peptidic substrates, with few exceptions, has been found to be greater than that for free tyrosine [15]. The sequence of the reactions we postulated to explain enkephalins and related peptides oxidation repeats essentially the Mason^ Raper pathway [16]. The brownish or yellowish melanopeptides obtained were completely soluble in hydrophilic solvents at neutral and basic pH. The high solubility of opiomelanins might be ascribed to the presence of the peptidic chain and in particular to the terminal carboxylic group [14]. It is acknowledged that in the presence of thiol groups, the dopaquinone resulting from the tyrosinase-catalyzed oxidation of tyrosine undergoes nucleophilic addition with the concomitant production of adducts, mainly 5-S-cysteinyldopa (5-S-CD) and 2-Scysteinyldopa (2-S-CD); these latter compounds are successively converted through the intermediate synthesis of benzothiazine derivatives into pheomelanin pigments [17]. 5-S-CD is considered the major metabolite in melanocytes [18] and its detection is routinely used in malignant melanoma for diagnostic purposes [19,20]. Also cysteinyldopamine and dopa-
mine have a considerable physiological importance, because they represent the starting point for pheomelanins production in skin and brain [21,22]. Pheomelanins synthesis can be catalyzed also by other enzymes, such as peroxidase [23] or lipoxygenase [7]. Some studies showed a loss of activity in enkephalins and other opioid peptides when the N-terminal tyrosine residue was absent [24,25] and in addition, Larsimont et al. [26] demonstrated that the dihydroxylated derivative of leucine-enkephalin (Leuenk) had a lower opioid receptor a¤nity with respect to the unmodi¢ed peptide. In order to ascertain if other physiological modi¢cations in the Tyr1 phenolic ring may exert an in£uence on the receptor binding, we synthesized a new class of adducts: the cysteinyldopaenkephalins (CDEnks). In the presence of thiol excess, it would be expected that the quinone resulting from enkephalin oxidation by tyrosinase undergoes nucleophilic addition with the sulfhydryl group giving rise to the formation of the adducts, as illustrated in Fig. 1. In this work, we report details on tyrosinase-catalyzed oxidation of enkephalins in the presence of cysteine, isolation and characterization of the prod-
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ucts and the binding of the major isomer (5-SCDEnk) to opioid receptors in bovine brain membranes.
pH 5.0, and ml/min. The tored at 254 the retention
2. Materials and methods
2.4. Ion-exchange chromatography
2.1. Materials
For amino acid analysis, the samples were hydrolyzed in sealed tubes with 6 N HCl at 110³C for 20 h. The hydrolysate was dried, dissolved, appropriately diluted and analyzed by a 3A30 Carlo Erba Amino Acid Analyzer, using a 15U0.46 cm column, ¢lled with 3AR/IC/6/10Li resin. The column was equilibrated with 0.2 M lithium citrate, pH 2.88, and eluted with (A) 0.2 M lithium citrate, pH 2.88, for 5 min; (B) 0.6 M lithium citrate, pH 3.0, for 31 min; (C) 1 M lithium citrate, pH 4.0, for 18 min. The column temperature was 45³C for 10 min, then 73³C. The £ow rates of bu¡er and ninhydrin were 30 and 20 ml/h, respectively.
Leu-enk, methionine-enkephalin, Tyr-Gly, TyrGly-Gly, kyotorphin, cysteine hydrochloride, glutathione reduced form, lipoic acid reduced form, tyrosinase from mushroom (3000 U/mg), peroxidase from horseradish (1100 U/mg), [D-Pen2;5 ]-enkephalin (DPDPE) and [D-Ala2 ,Me-Phe4 ,Gly5 -ol]-enkephalin (DAGO) were purchased from Sigma Chemical (St. Louis, MO, USA). [3 H]DPDPE and [3 H]DAGO were obtained from DuPont NEN (Boston, MA, USA). All other reagents were analytical grade products from Fluka (Buchs, Switzerland). 5-S-CD and 2S-CD were synthesized following the procedure of Ito and Prota [27]. 2.2. Spectrophotometric analysis Kinetic measurements were carried out by determining the rate of CD peptide formation at 293 nm. The absorbance was followed continuously using a Kontron Uvikon 930 Spectrophotometer in 1 cm light path thermostated cuvettes at 25³C. The standard incubation mixture contained 0.5 mM peptide, 1 mM cysteine and 50^200 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. The reaction was started by enzyme addition. Parallel samples without enzyme or with heat-inactivated enzyme were used as controls. The steady-state rate was de¢ned as the slope of the linear zone of the product accumulation curve. 2.3. High performance liquid chromatography (HPLC) analysis HPLC analysis was performed with a Waters Millipore apparatus (Milford, MA, USA). Samples were applied on a reverse-phase column (Novapak C18, 4 Wm, 15U0.39 cm). The chromatography was carried out in isocratic conditions at 30³C in a thermostated apparatus, utilizing as eluent a solution containing 88% 0.05 M citrate/phosphate bu¡er,
12% acetonitrile, at a £ow rate of 1.5 absorbance of the e¥uents was moniand 293 nm. In the above conditions, time of Leu-enk was 14.5 min.
2.5. Mass spectrometry Electrospray ion (ESI) mass spectra were obtained on a Finnigan LCQ ion trap mass spectrometer, in positive ion mode. The sample was dissolved in 50:50 (v/v) methanol-water solution containing 1% acetic acid to a concentration of 10 pmol/Wl and infused into the electrospray needle at a £ow rate of 5 Wl/min. ESI source conditions were as follows: sheath gas (nitrogen) £ow rate, 90 (arbitrary units); electrospray needle voltage, 50 kV; capillary voltage and temperature, 12 V and 260³C, respectively; electron multiplier and conversion dynode, 800 V and 15 kV, respectively. Full scan mass spectra were obtained in the peak continuum mode over the mass range of 50^1000 every 3 s and then summing each of the spectra. Collision-induced dissociation (CID) was achieved on selected trapped ions. A CID energy of 50% (arbitrary unit) was selected. 2.6. Membrane preparation and binding assay Bovine brain was obtained from the local slaughterhouse. The cortex was rapidly removed and membranes were prepared as described by Wood [28]. Brain (10 g) was homogenized in 100 ml of ice cold 50 mM Tris^HCl bu¡er, pH 7.7, in a Potter glass
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Fig. 2. Spectral modi¢cation of Leu-enk oxidized by tyrosinase in the presence of cysteine. Incubation mixture contained 0.5 mM Leu-enk, 1 mM cysteine and 100 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. Blank cuvette contained all the reagents except tyrosinase. Inset: reaction rate as a function of enzyme concentration. Incubation mixture contained 0.5 mM Leu-enk, 1 mM cysteine and the indicated amount of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4.
homogenizer. The homogenate was centrifuged at 49 000Ug for 15 min at 4³C. The pellet was homogenized in the original volume of the same bu¡er and recentrifuged at 49 000Ug for an identical time. The pellet obtained was newly homogenized, incubated at 37³C for 30 min and centrifuged in the same above conditions. The ¢nal pellet was resuspended by homogenization in 200 ml of 50 mM Tris^HCl bu¡er, pH 7.7. Aliquots of this suspension were frozen at 380³C until use. For the binding assay, brain membranes (0.3 mg of protein) were incubated at room temperature for 1 h in 1 ml of 50 mM Tris^HCl bu¡er, pH 7.7, containing 4 nM [3 H]DPDPE (for the assay of N opioid receptors) or [3 H]DAGO (for W receptors) as tracer, in the absence or presence of 10 WM unlabeled ligand (to estimate the non-speci¢c binding) or the tested enkephalins at the indicated concentrations. The incubation was stopped by ¢ltration under vacuum through Whatman GF/C glass micro¢ber ¢lters. The ¢lters were rinsed twice with 5 ml of ice cold 50 mM Tris^HCl bu¡er, pH 7.7, and placed in 7 ml of Ultima Gold Scintillation liquid (Packard).
Fig. 3. (A) Oxidation rate of various peptides by tyrosinase in the presence of cysteine. (B) Oxidation rate of Leu-enk by tyrosinase in the presence of various sulfhydryl compounds. The rate was determined as adduct formation, calculated as v of absorbance/min at 293 nm. Incubation mixture contained 0.5 mM peptide, 1 mM sulfhydryl compound and 50 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. All the adducts showed absorption spectra identical to those shown in Fig. 2.
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The radioactivity was determined in a Beckman LS6800 Scintillation Counter. Data are expressed as percentage of the maximum speci¢c binding and are means þ S.E.M. for six experiments with assays in triplicate. The inhibitory constants (Ki ) of the non-radioactive peptides were calculated by means of the equation Ki = IC50 / (1+L/Kd ), where IC50 is the concentration of peptide required to inhibit 50% of the speci¢c binding of radioligand, L is the free radioligand concentration used in the assay and Kd is the equilibrium binding a¤nity constant. The Kd values for DPDPE or DAGO were estimated from least-squares regression analysis of Scatchard transformation of the saturation isotherms. 3. Results 3.1. Kinetic measurements Enkephalins oxidation by tyrosinase in the presence of thiol compounds has been investigated spectrophotometrically. The spectral modi¢cation of Leu-enk incubated with the enzyme in the presence of an excess of cysteine is shown in Fig. 2. The reaction comes to an end within 20 min; during this time, a spectrum with an absorption maximum at 293 nm and a shoulder at 255 nm, characteristic of a CD moiety [29], could be evidenced. As shown in the inset of Fig. 2, the reaction rate was proportional to the tyrosinase concentration. The reaction occurred also for other peptides, whose CD derivatives exhibited absorption spectra equal to that shown in Fig. 2. Assuming that the extinction coe¤cients were similar for all the adducts, no signi¢cant di¡erence among the oxidation rates of the various peptides, except Tyr-Gly that seemed to be oxidized more quickly, could be observed (Fig. 3A). Besides cysteine, other sulfhydryl compounds are able to form adducts with enkephalins; in fact glutathione, lipoic acid and cysteamine are able to react with the dopaenkephalin-quinone, yielding the corresponding adducts (Fig. 3B). 3.2. Chromatographic analyses The products formed by Leu-enk and cysteine re-
Fig. 4. HPLC pro¢le of Leu-enk incubated with cysteine and tyrosinase at zero time (A) and after 20 min incubation (B). Incubation mixture contained 1 mM Leu-enk, 2 mM cysteine and 200 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. The chromatography was carried out in isocratic conditions at 30³C in a thermostated apparatus, utilizing as eluent a mixture containing 88% of 0.05 M sodium citrate/phosphate bu¡er, pH 5, and 12% acetonitrile with a £ow rate of 1.5 ml/min.
action in the presence of tyrosinase have been analyzed by HPLC. The chromatographic pro¢les of a reaction mixture at 0 time and after 20 min incubation are exhibited in Fig. 4. In chromatogram A, the peak with a retention time of 14.5 min (a) represents Leu-enk, as determined by comparison with a standard sample. In chromatogram B, the same peak signi¢cantly falls, whereas two peaks with retention times of 5 min (b) and 6.55 min (c) are clearly evidenced. On the basis of the reaction between tyrosine and tyrosinase in the presence of cysteine [27], the synthesis of two adducts would be expected: 5-SCDEnk and 2-S-CDEnk. In order to con¢rm this attribution, the eluates corresponding to peaks b and c have been collected separately and analyzed spectrophotometrically. In Fig. 5, the absorption spectra of eluate b and c are shown: eluate b exhibits the 2-S-CD moiety spectrum whereas eluate c shows that of the 5-S-CD moiety. The identi¢cation has been obtained through the di¡erent 255 nm/293 nm absorption ratios. They are in fact 0.8 for the 2-S-
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Fig. 5. Absorption spectra of b and c HPLC eluates. The eluates corresponding to the peaks b and c of the HPLC chromatogram were separately collected and assayed spectrophotometrically.
CD moiety and 1.25 for the 5-S-CD moiety, as reported in the literature [23]. Aliquots of eluates b and c have also been hydrolyzed and analyzed for amino acid composition. The chromatographic pro¢le of hydrolyzed peak c is shown in Fig. 6. The analysis unequivocally evidences the absence of Tyr and the presence of 5-S-CD. In the hydrolysate of peak b,
Fig. 6. Amino acid chromatogram of hydrolyzed 5-S-CDEnk. The eluate corresponding to peak c of the HPLC chromatogram was collected and hydrolyzed in sealed tubes with 6 N HCl at 110³C for 20 h. The hydrolysate was dried, dissolved, appropriately diluted and analyzed in a 3A30 Carlo Erba Amino Acid Analyzer under the conditions described in Section 2.
Fig. 7. (A) Decay of Leu-enk oxidized by tyrosinase in the presence of cysteine and (B) adducts formation. Incubation mixture contained 1 mM Leu-enk, 2 mM cysteine and 200 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. After incubation for the indicated time, 0.1 ml was subjected to HPLC analysis, under the conditions described in the legend of Fig. 4. Leu-enk (b), 5-SCDEnk (F), 2-S-CDEnk (R).
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Fig. 8. (A) ESI mass spectra of 5-S-CDEnk. (B) CID mass spectrum of pseudomolecular ion at m/z 691. The sample was dissolved in 50:50 (v/v) methanol-water solution containing 1% acetic acid to a concentration of 10 pmol/Wl. A CID energy of 50% (arbitrary unit) was selected for MS/MS experiments. Analytical conditions are described under Section 2.
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the presence of 2-S-CD has been detected (not shown). The Leu-enk decay (A) and the adducts formation (B), analyzed by HPLC, are shown in Fig. 7 as a function of incubation time. In the experimental conditions employed, about 20% of the initial Leu-enk was still present after 30 min of incubation; this percentage did not decrease even after prolonged incubation time. The major isomer 5-S-CDEnk, puri¢ed by HPLC, was subjected to further analyses. 3.3. ESI mass spectrometry In order to verify the cysteinyl adduct structure, 5S-CDEnk has been analyzed by ESI mass spectrometry. Full scan spray ion spectrum, acquired in single MS positive ion mode by infusion injection analysis, showed the predominant ions at m/z = 691 and m/z = 713, assigned to [M+H] and [M+Na] , respectively (Fig. 8A). These values, compared to those of ESI of Leu-enk (not shown), account for a 135 Da covalent adduct, in full agreement with a cysteinyl (119 Da) and oxygen (16 Da) bound. CID mass spectrum of pseudomolecular ion at m/z 691 has been collected and ion fragment masses measured (Fig. 8B). From the spectrum, several important sequence ions (m/z 271, 356, 413, 515, 560, 673) can be assigned, directly derived from the scheme shown at the bottom of Fig. 8. Ion series nomenclature is according to Roepstor¡ and Fohlman [30]. All the m/z values obtained in the CID of 5-S-CDEnk were 135 Da higher than the fragment obtained from CID of Leu-enk (not shown), con¢rming that the only modi¢cation would occur on the Tyr1 aromatic ring.
Fig. 9. Displacement of [3 H]DPDPE binding in bovine brain membranes by 5-S-CDEnk (F) and Leu-enk (b). Bovine brain membranes (0.3 mg of protein) were incubated at room temperature for 1 h in 1 ml of 50 mM Tris^HCl bu¡er, pH 7.7, containing 4 nM [3 H]DPDPE in the presence or absence of 10 WM unlabeled ligand and with enkephalins at the concentrations speci¢ed. Data are expressed as percentage of the maximum speci¢c binding.
3.4. Receptor binding a¤nity The binding of 5-S-CDEnk to N and W opioid receptors in membrane preparations from bovine cerebral cortex has been studied using [3 H]DPDPE or [3 H]DAGO as tracers, in comparison with the parent peptide Leu-enk. Displacement curves of the two enkephalins using [3 H]DPDPE are illustrated in Fig. 9. These curves, as the displacement curves of 5-SCDEnk and Leu-enk against [3 H]DAGO (not shown), are monophasic; thus envisaging the binding to a single site. Both 5-S-CDEnk and Leu-enk compete for the binding of [3 H]DPDPE or [3 H]DAGO with di¡erent potencies. The concentration of 5-SCDEnk required to inhibit 50% (IC50 ) of the speci¢c binding of 4 nM [3 H]DPDPE is about 0.14 WM. In contrast, a much greater concentration (IC50 about
Table 1 Summary of IC50 and Ki values obtained in bovine brain membrane receptor binding studies, using [3 H]DPDPE and [3 H]DAGO as tracers Substrate
IC50 (nM þ S.D.) 3
Leu-enk 5-S-CDEnk
Ki (nM þ S.D.) 3
[ H]DPDPE
[ H]DAGO
[3 H]DPDPE
[3 H]DAGO
18 þ 4 140 þ 39
261 þ 78 2114 þ 540
6.71 þ 1.39 52.23 þ 10.9
130 þ 32 1057 þ 210
The inhibitory constants (Ki ) were calculated from IC50 values, by means of the equation Ki = IC50 /(1+L/Kd ), where L is the free radioligand concentration used in the assay and Kd is the equilibrium binding a¤nity constant. The Kd values for DPDPE or DAGO were estimated from least-squares regression analysis of Scatchard transformation of the saturation isotherms.
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Fig. 10. Spectral modi¢cation of 5-S-CDEnk oxidized by the peroxidase/H2 O2 system. The incubation mixture contained 0.1 mM 5-S-CDEnk, 0.1 mM H2 O2 and 3.3 Wg of horseradish peroxidase in 1 ml of 100 mM phosphate bu¡er, pH 8.0. Blank cuvette contained all the reagents except peroxidase.
2.1 WM) is required to compete with the binding of [3 H]DAGO. By comparing the IC50 and Ki values for 5-S-CDEnk and Leu-enk (summarized in Table 1), it can be deduced that the presence of the cysteine residue and the additional hydroxyl group in the aromatic ring of Tyr1 of enkephalin causes an about 8fold decrease in binding a¤nity. 3.5. 5-S-CDEnk oxidation When 5-S-CDEnk was incubated with peroxidase in the presence of hydrogen peroxide, the spectral changes illustrated in Fig. 10 were evidenced. The absorption spectrum with a UV maximum around 304 nm, characteristic of the dihydro-1,4-benzothiazine chromophore [17], was very similar to that evidenced in the 5-S-CD oxidation catalyzed by peroxidase [23] or by lipoxygenase [7]. After 24 h, in the incubation mixture, the appearance of a brown coloring has been observed, indicating the formation of pheo-opiomelanins. Similarly to CD [23], 5-S-CDEnk is easily oxidized by peroxidase, whereas it represents a poor substrate for tyrosinase (not shown). 4. Discussion Normally, tyrosinase is inhibited by sulfhydryl
27
compounds which are able to bind copper at the enzyme active site [18]. However, in the presence of a proper amount of thiols (around 2 mM), the enzyme is not inhibited [31] and the thiol rapidly reacts with the o-quinone formed, giving adducts by the nucleophilic addition of the SH group on the aromatic ring. These compounds are the starting substrates of a speci¢c pathway leading to sulfur-containing melanins, i.e. pheomelanins [32]. We have demonstrated that the oxidation of enkephalins by tyrosinase in the presence of a thiol compound proceeds with the formation of adducts that show two UV absorption maxima at 255 and 293 nm, characteristic features of CD moiety absorption. The nucleophilic addition of SH compounds, such as cysteine or glutathione, to quinone is a very fast reaction. For this reason, only the enzymatic oxidation of enkephalin to quinone seems to be essential for the binding of cysteine to the peptide. In fact, the rate of the CD peptide formation was found to be actually the same as that of the quinone formation from enkephalin oxidation by tyrosinase in absence of thiols [15]. HPLC analysis shows the formation of two major enkephalin adducts. In the conditions used, the retention times of both products were lower than that of enkephalin, because of the presence of the cysteine residue which furnished to the adducts a minor hydrophobicity with respect to the parent peptide. HPLC utilization has allowed us to isolate the two enkephalin adducts; spectrophotometric analysis and amino acid determination in the hydrolysates of the HPLC eluates led us to identify the two adducts as 5S-CDEnk and 2-S-CDEnk. The 5-S-CDEnk structure was con¢rmed unequivocally by ESI mass spectrometry. 5-S-CDEnk was much more abundant (80%) than 2-S-CDEnk (20%), these percentages being very similar to the percent yields of the two major isomers formed in the tyrosinase-catalyzed synthesis of CD [27]. Various analogs of enkephalins with the insertion of methyl or hydroxyl groups have already been synthesized [26,33,34] but a sulfur natural derivative of Leu-enk, arising from the nucleophilic addition of cysteine to the tyrosine ring of the peptide, has never been described up to now. 5-S-CDEnk appears to bind to the opiate receptors of bovine brain mem-
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branes and, as well as enkephalins, it binds better to N than to W sites. The entry in the cell of cysteine or glutathione represents the ¢rst step of the overall phenomena linked to the oxidative stress [35]. For this reason, in vivo formation of these adducts in tissues, such as skin [18], melanoma cells [36] or brain [37,38], where tyrosinase and enkephalin immunoreactivities have been detected, cannot be ruled out. On the other hand, it is also to be noticed that a proximity between opioidergic and dopaminergic ¢bers in substantia nigra and locus coeruleus has been established [39]. Hence, the opiomelanins and pheoopiomelanins possible involvement in the process of Parkinson melanization cannot be entirely disregarded. The results of our experiments allow us to hypothesize the potential presence in the cell of two new groups of compounds: the CD opioid peptides and the melanins deriving from the oxidation of these adducts. As a matter of fact, though the existence in vivo of opiomelanins, CDEnks and pheoopiomelanins has to be demonstrated, these products represent per se the elements of an emerging area of genuine interest for general investigations in the ¢elds of ageing, dermatology and pigmentary systems. Acknowledgements This work was supported by grants from MURST and from National Research Council (C.N.R.). We wish also to thank Miss Di Sciullo and Mr. Peresempio for their technical assistance. References [1] B.M. Cox, Life Sci. 31 (1982) 1645^1658. [2] J.M. Hambrook, B.A. Morgan, M.J. Rance, C.F.C. Smith, Nature 262 (1976) 782^783. [3] E.G. Erdos, R.A. Skidgel, FASEB J. 3 (1989) 145^151. [4] L. Terenius, A. Wahlstrom, G. Lindeberg, S. Karlsson, U. Ragnarsson, Biochem. Biophys. Res. Commun. 71 (1976) 175^178. [5] J.T. Yang, T.A. Bewley, C.C. Chan, J. Med. Chem. 20 (1977) 323^327.
[6] M.A. Rosei, C. Blarzino, C. Foppoli, L. Mosca, R. Coccia, Biochem. Biophys. Res. Commun. 200 (1994) 344^350. [7] L. Mosca, C. Foppoli, R. Coccia, M.A. Rosei, Pigment. Cell Res. 9 (1996) 117^125. [8] L. Mosca, C. Blarzino, R. Coccia, C. Foppoli, M.A. Rosei, Free Radic. Biol. Med. 24 (1998) 161^167. [9] C. Blarzino, L. Mosca, C. Foppoli, R. Coccia, C. DeMarco, M.A. Rosei, Free Radic. Biol. Med. 26 (1999) 446^451. [10] M.A. Rosei, L. Mosca, C. DeMarco, Biochem. Biophys. Res. Commun. 184 (1992) 1190^1196. [11] M.A. Rosei, L. Mosca, R. Coccia, C. Blarzino, G. Musci, C. DeMarco, Biochim. Biophys. Acta 1199 (1994) 123^129. [12] M.A. Rosei, L. Mosca, C. DeMarco, Biochim. Biophys. Acta 1243 (1995) 71^77. [13] M.A. Rosei, L. Mosca and C. De Marco, in: Zeise, Chedeckel and Fitzpatrick (Eds.), Melanin: Its Role in Human Photoprotection, Valdenmar, Overland Park, KS, 1995, pp. 49^57. [14] M.A. Rosei, Pigment. Cell Res. 9 (1996) 273^280. [15] M.A. Rosei, L. Antonilli, R. Coccia, C. Foppoli, Biochem. Intern. 19 (1989) 1183^1193. [16] H.S. Mason, in: M. Gordon (Ed.), Pigment Cell Biology, Academic Press, New York, 1959, pp. 563^582. [17] G. Prota, in: Melanin and Melanogenesis, Academic Press, New York, 1992. [18] G. Prota, J. Invest. Dermatol. 100 (1993) 156^161. [19] H. Rorsman, G. Agrup, C. Hansson and E. Rosengren, in: R.H. MacKie (Ed.), Pigment Cell, Vol. 6, Karger, Basel, 1983, pp. 93^99. [20] T. Horikoshi, S. Ito, K. Wakamatsu, H. Onodera, H. Eguchi, Cancer 73 (1994) 629^636. [21] R. Carstam, C. Brinck, A. Hindemith-Augustsson, H. Rorsman, E. Rosengren, Biochim. Biophys. Acta 1097 (1991) 152^160. [22] L. Zecca, C. Mecacci, R. Seraglia, E. Parati, Biochim. Biophys. Acta 1138 (1992) 6^10. [23] S. Ito, K. Fujita, Biochim. Biophys. Acta 672 (1981) 151^ 157. [24] P.E. Hansen and B.A. Morgan, in: Udenfriend and Meierhofer (Eds.), Opioid peptides: Biology, Chemistry and Genetics, Vol. 6, Academic Press, Orlando, FL, 1984. [25] J. Shimohigashy, NIDA Res. Monograph. 69 (1986) 65^100. [26] V. Larsimont, L. Prokai, G. Hochaus, Biochim. Biophys. Acta 1222 (1994) 95^100. [27] S. Ito, G. Prota, Experientia 33 (1977) 1118^1119. [28] P.L. Wood, in: A.A. Boulton, G.B. Baker and P. Hrdina (Eds.), Neuromethods, Humana Press, Clifton, NJ, 1986, pp. 329^363. [29] H. Sanada, R. Suzue, Y. Nakashime, S. Kawada, Biochim. Biophys. Acta 261 (1972) 258^266. [30] P. Roepstor¡, J. Fohlman, Biochem. Mass Spectrom. 11 (1984) 601^606. [31] M. Seiji, T. Yoshida, M. Itakura, T. Irimajiri, J. Invest. Dermatol. 52 (1969) 280^298. [32] G. Prota, J. Invest. Dermatol. 75 (1988) 122^127.
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M. Anna Rosei et al. / Biochimica et Biophysica Acta 1478 (2000) 19^29 [33] D.W. Hansen, R.H. Mazur and M. Clare, in: Deber, Hruby and Kopple (Eds.), Peptides: Structure and Function, Pierce Chemical Company, Rockford, IL, 1985. [34] A.K. Judd, L.R. Toll, J.A. Lawson, E.T. Uyeno, W.E. Polgar and G.H. Loew, in: Deber, Hruby and Kopple (Eds.), Peptides: Structure and Function, Pierce Chemical Company, Rockford, IL, 1985. [35] H.M. Schipper, Neurobiol. Aging 17 (1996) 467^480.
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[36] A. Slominski, R. Paus, J. Wortsman, Experientia 48 (1991) 50^54. [37] H. Zhuo, S.J. Fung, V.K. Reddy, C.D. Barnes, J. Chem. Neuroanat. 5 (1992) 1^10. [38] S.R. Sesack, V.M. Pickel, J. Neurosci. 12 (1992) 1335^ 1350. [39] N. Zamir, M. Palkovits, E. Weber, E. Mezey, M.J. Brownstein, Nature 307 (1984) 643^645.
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Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors M. Anna Rosei a
a;
*, Ra¡aella Coccia a , Cesira Foppoli b , Carla Blarzino a , Chiara Cini c , M. Eugenia Schinina¨ a
Dipartimento di Scienze Biochimiche `A. Rossi Fanelli', Universita© `La Sapienza', P.le Aldo Moro 5, 00185 Roma, Italy b Centro di Biologia Molecolare del C.N.R., P.le Aldo Moro 5, 00185 Roma, Italy c Consiglio Nazionale delle Ricerche, P.le Aldo Moro 5, 00185 Roma, Italy Received 30 June 1999; received in revised form 15 October 1999; accepted 22 December 1999
Abstract The reaction of opioid peptides with mushroom tyrosinase in the presence of an excess of a thiol compound gives rise to cysteinyldopaenkephalins (CDEnks). The major product is represented by the 5-S-CDEnk (80%) and the minor one by the isomer 2-S-CDEnk (20%). The adducts between leucine-enkephalin (Leu-enk) and cysteine have been isolated by high performance liquid chromatography (HPLC) and identified by amino acid analysis and electrospray ion mass spectrometry. 5-S-CDEnk is able to bind to opioid receptors in bovine brain membranes. Its binding affinity is higher for N than for W receptors and about 8-fold lesser than that exploited by Leu-enk. In the presence of the peroxidase/H2 O2 system, CDEnks can be converted into the corresponding pheo-opiomelanins. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Tyrosinase; Synthesis; Enkephalin; Pheomelanin; Opioid receptor
1. Introduction Opioid peptides represent a large class among animal peptides. They can be divided into three groups: enkephalins, endorphins and dynorphins, all derived from larger protein precursors [1]. These compounds
Abbreviations: CD, cysteinyldopa; 5-S-CD, 5-S-cysteinyldopa; 2-S-CD, 2-S-cysteinyldopa; CDEnk, cysteinyldopaenkephalin; 5-S-CDEnk, 5-S-cysteinyldopaenkephalin; 2-S-CDEnk, 2-Scysteinyldopaenkephalin; Leu-enk, leucine-enkephalin; DPDPE, [D-Pen2;5 ]-enkephalin; DAGO, [D-Ala2 ,Me-Phe4 ,Gly5 -ol]-enkephalin; ESI, electrospray ionization; CID, collision-induced dissociation * Corresponding author. Fax: +39 (6) 4440062; E-mail: [email protected]
are of great interest because of their opiate-like activity. Their role as neurotransmitters and hormones has been demonstrated by a large number of investigations. Normally, opioid peptides are eliminated by peptidases that are able to bring about their complete cleavage and inactivation [2,3]. They exhibit as common feature the presence of a tyrosine residue at the amino-terminus, which is essential for their biological activity [4,5]. In recent years, a series of studies demonstrating that melanogenesis in vitro can occur with other substrates and other enzymes than those yet known has been carried out in our laboratory [6^9]. In particular, we demonstrated that opioid peptides are substrates in vitro of mushroom and sepia tyrosinase giving rise to synthetic melanins retaining the peptide
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 8 7 - 3
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Fig. 1. Tyrosinase-catalyzed formation of the CD derivatives of Leu-enk.
moiety, that we named opiomelanins [10^14]. Enzyme a¤nity for the peptidic substrates, with few exceptions, has been found to be greater than that for free tyrosine [15]. The sequence of the reactions we postulated to explain enkephalins and related peptides oxidation repeats essentially the Mason^ Raper pathway [16]. The brownish or yellowish melanopeptides obtained were completely soluble in hydrophilic solvents at neutral and basic pH. The high solubility of opiomelanins might be ascribed to the presence of the peptidic chain and in particular to the terminal carboxylic group [14]. It is acknowledged that in the presence of thiol groups, the dopaquinone resulting from the tyrosinase-catalyzed oxidation of tyrosine undergoes nucleophilic addition with the concomitant production of adducts, mainly 5-S-cysteinyldopa (5-S-CD) and 2-Scysteinyldopa (2-S-CD); these latter compounds are successively converted through the intermediate synthesis of benzothiazine derivatives into pheomelanin pigments [17]. 5-S-CD is considered the major metabolite in melanocytes [18] and its detection is routinely used in malignant melanoma for diagnostic purposes [19,20]. Also cysteinyldopamine and dopa-
mine have a considerable physiological importance, because they represent the starting point for pheomelanins production in skin and brain [21,22]. Pheomelanins synthesis can be catalyzed also by other enzymes, such as peroxidase [23] or lipoxygenase [7]. Some studies showed a loss of activity in enkephalins and other opioid peptides when the N-terminal tyrosine residue was absent [24,25] and in addition, Larsimont et al. [26] demonstrated that the dihydroxylated derivative of leucine-enkephalin (Leuenk) had a lower opioid receptor a¤nity with respect to the unmodi¢ed peptide. In order to ascertain if other physiological modi¢cations in the Tyr1 phenolic ring may exert an in£uence on the receptor binding, we synthesized a new class of adducts: the cysteinyldopaenkephalins (CDEnks). In the presence of thiol excess, it would be expected that the quinone resulting from enkephalin oxidation by tyrosinase undergoes nucleophilic addition with the sulfhydryl group giving rise to the formation of the adducts, as illustrated in Fig. 1. In this work, we report details on tyrosinase-catalyzed oxidation of enkephalins in the presence of cysteine, isolation and characterization of the prod-
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ucts and the binding of the major isomer (5-SCDEnk) to opioid receptors in bovine brain membranes.
pH 5.0, and ml/min. The tored at 254 the retention
2. Materials and methods
2.4. Ion-exchange chromatography
2.1. Materials
For amino acid analysis, the samples were hydrolyzed in sealed tubes with 6 N HCl at 110³C for 20 h. The hydrolysate was dried, dissolved, appropriately diluted and analyzed by a 3A30 Carlo Erba Amino Acid Analyzer, using a 15U0.46 cm column, ¢lled with 3AR/IC/6/10Li resin. The column was equilibrated with 0.2 M lithium citrate, pH 2.88, and eluted with (A) 0.2 M lithium citrate, pH 2.88, for 5 min; (B) 0.6 M lithium citrate, pH 3.0, for 31 min; (C) 1 M lithium citrate, pH 4.0, for 18 min. The column temperature was 45³C for 10 min, then 73³C. The £ow rates of bu¡er and ninhydrin were 30 and 20 ml/h, respectively.
Leu-enk, methionine-enkephalin, Tyr-Gly, TyrGly-Gly, kyotorphin, cysteine hydrochloride, glutathione reduced form, lipoic acid reduced form, tyrosinase from mushroom (3000 U/mg), peroxidase from horseradish (1100 U/mg), [D-Pen2;5 ]-enkephalin (DPDPE) and [D-Ala2 ,Me-Phe4 ,Gly5 -ol]-enkephalin (DAGO) were purchased from Sigma Chemical (St. Louis, MO, USA). [3 H]DPDPE and [3 H]DAGO were obtained from DuPont NEN (Boston, MA, USA). All other reagents were analytical grade products from Fluka (Buchs, Switzerland). 5-S-CD and 2S-CD were synthesized following the procedure of Ito and Prota [27]. 2.2. Spectrophotometric analysis Kinetic measurements were carried out by determining the rate of CD peptide formation at 293 nm. The absorbance was followed continuously using a Kontron Uvikon 930 Spectrophotometer in 1 cm light path thermostated cuvettes at 25³C. The standard incubation mixture contained 0.5 mM peptide, 1 mM cysteine and 50^200 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. The reaction was started by enzyme addition. Parallel samples without enzyme or with heat-inactivated enzyme were used as controls. The steady-state rate was de¢ned as the slope of the linear zone of the product accumulation curve. 2.3. High performance liquid chromatography (HPLC) analysis HPLC analysis was performed with a Waters Millipore apparatus (Milford, MA, USA). Samples were applied on a reverse-phase column (Novapak C18, 4 Wm, 15U0.39 cm). The chromatography was carried out in isocratic conditions at 30³C in a thermostated apparatus, utilizing as eluent a solution containing 88% 0.05 M citrate/phosphate bu¡er,
12% acetonitrile, at a £ow rate of 1.5 absorbance of the e¥uents was moniand 293 nm. In the above conditions, time of Leu-enk was 14.5 min.
2.5. Mass spectrometry Electrospray ion (ESI) mass spectra were obtained on a Finnigan LCQ ion trap mass spectrometer, in positive ion mode. The sample was dissolved in 50:50 (v/v) methanol-water solution containing 1% acetic acid to a concentration of 10 pmol/Wl and infused into the electrospray needle at a £ow rate of 5 Wl/min. ESI source conditions were as follows: sheath gas (nitrogen) £ow rate, 90 (arbitrary units); electrospray needle voltage, 50 kV; capillary voltage and temperature, 12 V and 260³C, respectively; electron multiplier and conversion dynode, 800 V and 15 kV, respectively. Full scan mass spectra were obtained in the peak continuum mode over the mass range of 50^1000 every 3 s and then summing each of the spectra. Collision-induced dissociation (CID) was achieved on selected trapped ions. A CID energy of 50% (arbitrary unit) was selected. 2.6. Membrane preparation and binding assay Bovine brain was obtained from the local slaughterhouse. The cortex was rapidly removed and membranes were prepared as described by Wood [28]. Brain (10 g) was homogenized in 100 ml of ice cold 50 mM Tris^HCl bu¡er, pH 7.7, in a Potter glass
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Fig. 2. Spectral modi¢cation of Leu-enk oxidized by tyrosinase in the presence of cysteine. Incubation mixture contained 0.5 mM Leu-enk, 1 mM cysteine and 100 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. Blank cuvette contained all the reagents except tyrosinase. Inset: reaction rate as a function of enzyme concentration. Incubation mixture contained 0.5 mM Leu-enk, 1 mM cysteine and the indicated amount of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4.
homogenizer. The homogenate was centrifuged at 49 000Ug for 15 min at 4³C. The pellet was homogenized in the original volume of the same bu¡er and recentrifuged at 49 000Ug for an identical time. The pellet obtained was newly homogenized, incubated at 37³C for 30 min and centrifuged in the same above conditions. The ¢nal pellet was resuspended by homogenization in 200 ml of 50 mM Tris^HCl bu¡er, pH 7.7. Aliquots of this suspension were frozen at 380³C until use. For the binding assay, brain membranes (0.3 mg of protein) were incubated at room temperature for 1 h in 1 ml of 50 mM Tris^HCl bu¡er, pH 7.7, containing 4 nM [3 H]DPDPE (for the assay of N opioid receptors) or [3 H]DAGO (for W receptors) as tracer, in the absence or presence of 10 WM unlabeled ligand (to estimate the non-speci¢c binding) or the tested enkephalins at the indicated concentrations. The incubation was stopped by ¢ltration under vacuum through Whatman GF/C glass micro¢ber ¢lters. The ¢lters were rinsed twice with 5 ml of ice cold 50 mM Tris^HCl bu¡er, pH 7.7, and placed in 7 ml of Ultima Gold Scintillation liquid (Packard).
Fig. 3. (A) Oxidation rate of various peptides by tyrosinase in the presence of cysteine. (B) Oxidation rate of Leu-enk by tyrosinase in the presence of various sulfhydryl compounds. The rate was determined as adduct formation, calculated as v of absorbance/min at 293 nm. Incubation mixture contained 0.5 mM peptide, 1 mM sulfhydryl compound and 50 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. All the adducts showed absorption spectra identical to those shown in Fig. 2.
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The radioactivity was determined in a Beckman LS6800 Scintillation Counter. Data are expressed as percentage of the maximum speci¢c binding and are means þ S.E.M. for six experiments with assays in triplicate. The inhibitory constants (Ki ) of the non-radioactive peptides were calculated by means of the equation Ki = IC50 / (1+L/Kd ), where IC50 is the concentration of peptide required to inhibit 50% of the speci¢c binding of radioligand, L is the free radioligand concentration used in the assay and Kd is the equilibrium binding a¤nity constant. The Kd values for DPDPE or DAGO were estimated from least-squares regression analysis of Scatchard transformation of the saturation isotherms. 3. Results 3.1. Kinetic measurements Enkephalins oxidation by tyrosinase in the presence of thiol compounds has been investigated spectrophotometrically. The spectral modi¢cation of Leu-enk incubated with the enzyme in the presence of an excess of cysteine is shown in Fig. 2. The reaction comes to an end within 20 min; during this time, a spectrum with an absorption maximum at 293 nm and a shoulder at 255 nm, characteristic of a CD moiety [29], could be evidenced. As shown in the inset of Fig. 2, the reaction rate was proportional to the tyrosinase concentration. The reaction occurred also for other peptides, whose CD derivatives exhibited absorption spectra equal to that shown in Fig. 2. Assuming that the extinction coe¤cients were similar for all the adducts, no signi¢cant di¡erence among the oxidation rates of the various peptides, except Tyr-Gly that seemed to be oxidized more quickly, could be observed (Fig. 3A). Besides cysteine, other sulfhydryl compounds are able to form adducts with enkephalins; in fact glutathione, lipoic acid and cysteamine are able to react with the dopaenkephalin-quinone, yielding the corresponding adducts (Fig. 3B). 3.2. Chromatographic analyses The products formed by Leu-enk and cysteine re-
Fig. 4. HPLC pro¢le of Leu-enk incubated with cysteine and tyrosinase at zero time (A) and after 20 min incubation (B). Incubation mixture contained 1 mM Leu-enk, 2 mM cysteine and 200 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. The chromatography was carried out in isocratic conditions at 30³C in a thermostated apparatus, utilizing as eluent a mixture containing 88% of 0.05 M sodium citrate/phosphate bu¡er, pH 5, and 12% acetonitrile with a £ow rate of 1.5 ml/min.
action in the presence of tyrosinase have been analyzed by HPLC. The chromatographic pro¢les of a reaction mixture at 0 time and after 20 min incubation are exhibited in Fig. 4. In chromatogram A, the peak with a retention time of 14.5 min (a) represents Leu-enk, as determined by comparison with a standard sample. In chromatogram B, the same peak signi¢cantly falls, whereas two peaks with retention times of 5 min (b) and 6.55 min (c) are clearly evidenced. On the basis of the reaction between tyrosine and tyrosinase in the presence of cysteine [27], the synthesis of two adducts would be expected: 5-SCDEnk and 2-S-CDEnk. In order to con¢rm this attribution, the eluates corresponding to peaks b and c have been collected separately and analyzed spectrophotometrically. In Fig. 5, the absorption spectra of eluate b and c are shown: eluate b exhibits the 2-S-CD moiety spectrum whereas eluate c shows that of the 5-S-CD moiety. The identi¢cation has been obtained through the di¡erent 255 nm/293 nm absorption ratios. They are in fact 0.8 for the 2-S-
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Fig. 5. Absorption spectra of b and c HPLC eluates. The eluates corresponding to the peaks b and c of the HPLC chromatogram were separately collected and assayed spectrophotometrically.
CD moiety and 1.25 for the 5-S-CD moiety, as reported in the literature [23]. Aliquots of eluates b and c have also been hydrolyzed and analyzed for amino acid composition. The chromatographic pro¢le of hydrolyzed peak c is shown in Fig. 6. The analysis unequivocally evidences the absence of Tyr and the presence of 5-S-CD. In the hydrolysate of peak b,
Fig. 6. Amino acid chromatogram of hydrolyzed 5-S-CDEnk. The eluate corresponding to peak c of the HPLC chromatogram was collected and hydrolyzed in sealed tubes with 6 N HCl at 110³C for 20 h. The hydrolysate was dried, dissolved, appropriately diluted and analyzed in a 3A30 Carlo Erba Amino Acid Analyzer under the conditions described in Section 2.
Fig. 7. (A) Decay of Leu-enk oxidized by tyrosinase in the presence of cysteine and (B) adducts formation. Incubation mixture contained 1 mM Leu-enk, 2 mM cysteine and 200 Wg of tyrosinase in 1 ml of 0.05 M phosphate bu¡er, pH 7.4. After incubation for the indicated time, 0.1 ml was subjected to HPLC analysis, under the conditions described in the legend of Fig. 4. Leu-enk (b), 5-SCDEnk (F), 2-S-CDEnk (R).
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Fig. 8. (A) ESI mass spectra of 5-S-CDEnk. (B) CID mass spectrum of pseudomolecular ion at m/z 691. The sample was dissolved in 50:50 (v/v) methanol-water solution containing 1% acetic acid to a concentration of 10 pmol/Wl. A CID energy of 50% (arbitrary unit) was selected for MS/MS experiments. Analytical conditions are described under Section 2.
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the presence of 2-S-CD has been detected (not shown). The Leu-enk decay (A) and the adducts formation (B), analyzed by HPLC, are shown in Fig. 7 as a function of incubation time. In the experimental conditions employed, about 20% of the initial Leu-enk was still present after 30 min of incubation; this percentage did not decrease even after prolonged incubation time. The major isomer 5-S-CDEnk, puri¢ed by HPLC, was subjected to further analyses. 3.3. ESI mass spectrometry In order to verify the cysteinyl adduct structure, 5S-CDEnk has been analyzed by ESI mass spectrometry. Full scan spray ion spectrum, acquired in single MS positive ion mode by infusion injection analysis, showed the predominant ions at m/z = 691 and m/z = 713, assigned to [M+H] and [M+Na] , respectively (Fig. 8A). These values, compared to those of ESI of Leu-enk (not shown), account for a 135 Da covalent adduct, in full agreement with a cysteinyl (119 Da) and oxygen (16 Da) bound. CID mass spectrum of pseudomolecular ion at m/z 691 has been collected and ion fragment masses measured (Fig. 8B). From the spectrum, several important sequence ions (m/z 271, 356, 413, 515, 560, 673) can be assigned, directly derived from the scheme shown at the bottom of Fig. 8. Ion series nomenclature is according to Roepstor¡ and Fohlman [30]. All the m/z values obtained in the CID of 5-S-CDEnk were 135 Da higher than the fragment obtained from CID of Leu-enk (not shown), con¢rming that the only modi¢cation would occur on the Tyr1 aromatic ring.
Fig. 9. Displacement of [3 H]DPDPE binding in bovine brain membranes by 5-S-CDEnk (F) and Leu-enk (b). Bovine brain membranes (0.3 mg of protein) were incubated at room temperature for 1 h in 1 ml of 50 mM Tris^HCl bu¡er, pH 7.7, containing 4 nM [3 H]DPDPE in the presence or absence of 10 WM unlabeled ligand and with enkephalins at the concentrations speci¢ed. Data are expressed as percentage of the maximum speci¢c binding.
3.4. Receptor binding a¤nity The binding of 5-S-CDEnk to N and W opioid receptors in membrane preparations from bovine cerebral cortex has been studied using [3 H]DPDPE or [3 H]DAGO as tracers, in comparison with the parent peptide Leu-enk. Displacement curves of the two enkephalins using [3 H]DPDPE are illustrated in Fig. 9. These curves, as the displacement curves of 5-SCDEnk and Leu-enk against [3 H]DAGO (not shown), are monophasic; thus envisaging the binding to a single site. Both 5-S-CDEnk and Leu-enk compete for the binding of [3 H]DPDPE or [3 H]DAGO with di¡erent potencies. The concentration of 5-SCDEnk required to inhibit 50% (IC50 ) of the speci¢c binding of 4 nM [3 H]DPDPE is about 0.14 WM. In contrast, a much greater concentration (IC50 about
Table 1 Summary of IC50 and Ki values obtained in bovine brain membrane receptor binding studies, using [3 H]DPDPE and [3 H]DAGO as tracers Substrate
IC50 (nM þ S.D.) 3
Leu-enk 5-S-CDEnk
Ki (nM þ S.D.) 3
[ H]DPDPE
[ H]DAGO
[3 H]DPDPE
[3 H]DAGO
18 þ 4 140 þ 39
261 þ 78 2114 þ 540
6.71 þ 1.39 52.23 þ 10.9
130 þ 32 1057 þ 210
The inhibitory constants (Ki ) were calculated from IC50 values, by means of the equation Ki = IC50 /(1+L/Kd ), where L is the free radioligand concentration used in the assay and Kd is the equilibrium binding a¤nity constant. The Kd values for DPDPE or DAGO were estimated from least-squares regression analysis of Scatchard transformation of the saturation isotherms.
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Fig. 10. Spectral modi¢cation of 5-S-CDEnk oxidized by the peroxidase/H2 O2 system. The incubation mixture contained 0.1 mM 5-S-CDEnk, 0.1 mM H2 O2 and 3.3 Wg of horseradish peroxidase in 1 ml of 100 mM phosphate bu¡er, pH 8.0. Blank cuvette contained all the reagents except peroxidase.
2.1 WM) is required to compete with the binding of [3 H]DAGO. By comparing the IC50 and Ki values for 5-S-CDEnk and Leu-enk (summarized in Table 1), it can be deduced that the presence of the cysteine residue and the additional hydroxyl group in the aromatic ring of Tyr1 of enkephalin causes an about 8fold decrease in binding a¤nity. 3.5. 5-S-CDEnk oxidation When 5-S-CDEnk was incubated with peroxidase in the presence of hydrogen peroxide, the spectral changes illustrated in Fig. 10 were evidenced. The absorption spectrum with a UV maximum around 304 nm, characteristic of the dihydro-1,4-benzothiazine chromophore [17], was very similar to that evidenced in the 5-S-CD oxidation catalyzed by peroxidase [23] or by lipoxygenase [7]. After 24 h, in the incubation mixture, the appearance of a brown coloring has been observed, indicating the formation of pheo-opiomelanins. Similarly to CD [23], 5-S-CDEnk is easily oxidized by peroxidase, whereas it represents a poor substrate for tyrosinase (not shown). 4. Discussion Normally, tyrosinase is inhibited by sulfhydryl
27
compounds which are able to bind copper at the enzyme active site [18]. However, in the presence of a proper amount of thiols (around 2 mM), the enzyme is not inhibited [31] and the thiol rapidly reacts with the o-quinone formed, giving adducts by the nucleophilic addition of the SH group on the aromatic ring. These compounds are the starting substrates of a speci¢c pathway leading to sulfur-containing melanins, i.e. pheomelanins [32]. We have demonstrated that the oxidation of enkephalins by tyrosinase in the presence of a thiol compound proceeds with the formation of adducts that show two UV absorption maxima at 255 and 293 nm, characteristic features of CD moiety absorption. The nucleophilic addition of SH compounds, such as cysteine or glutathione, to quinone is a very fast reaction. For this reason, only the enzymatic oxidation of enkephalin to quinone seems to be essential for the binding of cysteine to the peptide. In fact, the rate of the CD peptide formation was found to be actually the same as that of the quinone formation from enkephalin oxidation by tyrosinase in absence of thiols [15]. HPLC analysis shows the formation of two major enkephalin adducts. In the conditions used, the retention times of both products were lower than that of enkephalin, because of the presence of the cysteine residue which furnished to the adducts a minor hydrophobicity with respect to the parent peptide. HPLC utilization has allowed us to isolate the two enkephalin adducts; spectrophotometric analysis and amino acid determination in the hydrolysates of the HPLC eluates led us to identify the two adducts as 5S-CDEnk and 2-S-CDEnk. The 5-S-CDEnk structure was con¢rmed unequivocally by ESI mass spectrometry. 5-S-CDEnk was much more abundant (80%) than 2-S-CDEnk (20%), these percentages being very similar to the percent yields of the two major isomers formed in the tyrosinase-catalyzed synthesis of CD [27]. Various analogs of enkephalins with the insertion of methyl or hydroxyl groups have already been synthesized [26,33,34] but a sulfur natural derivative of Leu-enk, arising from the nucleophilic addition of cysteine to the tyrosine ring of the peptide, has never been described up to now. 5-S-CDEnk appears to bind to the opiate receptors of bovine brain mem-
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M. Anna Rosei et al. / Biochimica et Biophysica Acta 1478 (2000) 19^29
branes and, as well as enkephalins, it binds better to N than to W sites. The entry in the cell of cysteine or glutathione represents the ¢rst step of the overall phenomena linked to the oxidative stress [35]. For this reason, in vivo formation of these adducts in tissues, such as skin [18], melanoma cells [36] or brain [37,38], where tyrosinase and enkephalin immunoreactivities have been detected, cannot be ruled out. On the other hand, it is also to be noticed that a proximity between opioidergic and dopaminergic ¢bers in substantia nigra and locus coeruleus has been established [39]. Hence, the opiomelanins and pheoopiomelanins possible involvement in the process of Parkinson melanization cannot be entirely disregarded. The results of our experiments allow us to hypothesize the potential presence in the cell of two new groups of compounds: the CD opioid peptides and the melanins deriving from the oxidation of these adducts. As a matter of fact, though the existence in vivo of opiomelanins, CDEnks and pheoopiomelanins has to be demonstrated, these products represent per se the elements of an emerging area of genuine interest for general investigations in the ¢elds of ageing, dermatology and pigmentary systems. Acknowledgements This work was supported by grants from MURST and from National Research Council (C.N.R.). We wish also to thank Miss Di Sciullo and Mr. Peresempio for their technical assistance. References [1] B.M. Cox, Life Sci. 31 (1982) 1645^1658. [2] J.M. Hambrook, B.A. Morgan, M.J. Rance, C.F.C. Smith, Nature 262 (1976) 782^783. [3] E.G. Erdos, R.A. Skidgel, FASEB J. 3 (1989) 145^151. [4] L. Terenius, A. Wahlstrom, G. Lindeberg, S. Karlsson, U. Ragnarsson, Biochem. Biophys. Res. Commun. 71 (1976) 175^178. [5] J.T. Yang, T.A. Bewley, C.C. Chan, J. Med. Chem. 20 (1977) 323^327.
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