咪唑二肽Imidazole dipeptide

Purification and characterization of a novel imidazole dipeptide synthasefrom the muscle of the Japanese eel Anguilla japonicaShiori Tsubone,Naoko Yoshikawa,Shigeru Okada,Hiroki Abe ⁎Department of Aquatic Bioscience,Graduate School of Agricultural and Life Sciences,The University of Tokyo,Bunkyo,Tokyo 113-8657,JapanReceived 14August 2006;received in revised form 5December 2006;accepted 6December 2006Available online 12December 2006AbstractWe have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides,such as carnosine (β-alanyl-L -histidine),anserine (β-alanyl-π-methyl-L -histidine),and balenine (ophidine;β-alanyl-τ-methyl-L -histidine),directly from their precursors.The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies.Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine,the anserine synthesizing activity was by far the highest.From gel permeation chromatography,the molecular mass of the enzyme was calculated to be 275kDa.SDS-PAGE of the purified enzyme represented a band around 43kDa,suggesting that the native enzyme is a hexamer or heptamer.The optimal pH and temperature were around 9.5and 60°C,respectively.K m values for β-alanine and π-methyl-L -histidine were 44and 89mM,respectively.The enzyme was greatly activated by Zn 2+and inhibited by EDTA.The N-terminal amino acid sequence of 25residues of the purified enzyme showed 52%amino acid identity to 38–62residues of zebrafish haptoglobin precursor.The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.©2007Elsevier Inc.All rights reserved.Keywords:Imidazole dipeptides;Carnosine;Anserine;Balenine;Imidazole dipeptide synthase;Japanese eel;Muscle;Anguilla japonica1.IntroductionImidazole dipeptides in vertebrates,i.e.,carnosine (β-alanyl-L -histidine),anserine (β-alanyl-π-methyl-L -histidine),and bale-nine (ophidine;β-alanyl-τ-methyl-L -histidine),have long been known to exist in excitable tissues such as skeletal muscle and brain,and are proposed to have versatile physiological func-tions.Of these dipeptides,carnosine received special attention as a research target for functional analyses because of the availability of the reagent.Carnosine has been proven to work as a proton buffering constituent in fast-twitch white muscle (Abe et al.,1985;Abe and Okuma,1991;Abe,2000),as an antioxidant and scavenger for reactive oxygen species (Kohen et al.,1988;Boldyrev et al.,1989,2004;Boldyrev and Abe,1999),as a neurotransmitter or modulator (Margolis,1974;Bonfanti et al.,1999),as a neuroprotector (Hipkiss et al.,1997;Boldyrev et al.,2004),and as an anti-aging agent (Hipkiss,2000;Hipkiss et al.,2001).A large amount of carnosine is found in human muscle (10mM)and in sprinting mammals such as horses (34mM)and oxen (20mM)(Abe,1995).Although the distribution of carnosine in fish muscle is rather restricted,a high concentration of carnosine is resident in eel muscle (20mM).Anserine,on the other hand,is abundant in chicken pectoral muscle (43mM)and the white muscle of fast swimming fish such as tunas (27mM),skipjack tuna (51mM),and blue marlin (105mM).Copious concentrations of balenine is found only in whales (78mM)and snakes (13mM),although balenine has been found in trace amounts of all vertebrate muscles thus far examined (Abe,1995).Apparently,the distribution of these imidazole dipep-tides in vertebrate muscle is highly species specific and does not have any phylogenetic relationship.Although many researchers have focused on the physiolog-ical functions,information on the biosynthetic pathway and the constituent enzymes that catalyze formation of these dipeptides has remained deficient.Carnosine is known to be synthesized from β-alanine and L -histidine by carnosine synthetase (EC 6.3.2.11)in the presence of ATP,which has been characterized from chick pectoral muscle (Kalyankar and Meister,1959;Comparative Biochemistry and Physiology,Part B 146(2007)560–567/locate/cbpb⁎Corresponding author.Tel.:+81358415296;fax:+81358418166.E-mail address:aabe@mail.ecc.u-tokyo.ac.jp (H.Abe).1096-4959/$-see front matter ©2007Elsevier Inc.All rights reserved.doi:10.1016/j.cbpb.2006.12.002Winnick and Winnick,1959,1960;Stenesh and Winnick,1960; Bauer and Schulz,1994),mouse olfactory system(Harding and Margolis,1976),and rat and mouse brains(Skaper et al.,1973; Bauer et al.,1979,1982).No information,however,has been published on the primary structure of carnosine synthetase.Anserine,on the other hand,has been proposed to be syn-thesized by two different pathways.The first is the direct meth-ylation of carnosine catalyzed by carnosine-N-methyltransferase (EC2.1.1.22)in the presence of S-adenosyl-L-methionine,which was confirmed in chick pectoral muscle(McManus,1962;Bauer and Schulz,1994).The second pathway is the A TP-dependent condensation of the precursors,β-alanine andπ-methyl-L-histidine,by carnosine synthetase or some closely related enzyme (Winnick and Winnick,1959,1960;Stenesh and Winnick,1960).In contrast to the synthesis of imidazole dipeptides,their hydrolysis has been elucidated in more detail.Carnosine is hydrolyzed by carnosinases(Rosenberg,1960;Harding and Margolis,1976;Lenney,1976)which are classified into two groups,tissue carnosinase(cytosolic non-specific dipeptidase; EC3.4.13.18)and serum carnosinase(EC3.4.13.20),both of which belong to a large family of metalloproteases. Recently,cDNAs of human brain carnosinase and cytosolic non-specific dipeptidase were cloned and characterized(Teufel et al.,2003).Moreover,an anserine hydrolyzing enzyme, anserinase(EC3.4.13.5),was purified from the brain of Nile tilapia Oreochromis niloticus and its nucleotide and amino acid sequences were identified(Yamada et al.,2005).For these enigmatic imidazole dipeptides,their physiological functions and synthetic pathways,as well as the reasons for their species specific distribution remain unsolved.In this report,we describe the purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica,which catalyzes both the synthesis and hydrolysis of these three dipeptides.2.Materials and methods2.1.MaterialsLive Japanese eel(Anguilla japonica)were obtained from the Tokyo Metropolitan Central Wholesale Market(Tokyo, Japan).β-Alanine was purchased from Wako Pure Chemical Industries(Osaka,Japan).L-Histidine,π-methyl-L-histidine(1-methyl-L-histidine),carnosine,andγ-aminobutyric acid were from Sigma-Aldrich(St.Louis,MO,USA).τ-Methyl-L-histidine(3-methyl-L-histidine)was obtained from Bachem (Bubendorf,Switzerland).Anserine and balenine were prepared from the skeletal muscle of big-eye tuna Thunnus obesus and minke whale Balaenoptera acutorostrata,respectively(Abe and Okuma,1991).TSK-gel DEAE-Toyopearl650M and Ether-Toyoperl650M were obtained from Tosoh(Tokyo, Japan).His-Trap HP,MonoQ HR5/5,and Superdex20010/ 300were obtained from Amersham Biosciences(Uppsala, Sweden).Macro-prep Ceramic Hydroxyapatite Type1(CHT-1) column was from Bio-Rad Laboratories(Hercules,CA,USA). All other reagents were of analytical grade and purchased either from Sigma-Aldrich or Wako unless otherwise stated.2.2.Assay of imidazole dipeptide synthetic activitiesDipeptide synthetic activities were assayed by measuring the formation of products by high performance liquid chromatography(HPLC)according to the method of Togashi et al.(1998)with some modifications.The standard incubation mixture for the assay of anserine synthesis consisted of160mM π-methyl-L-histidine,250mMβ-alanine,and2mM ZnCl2in 50mM Tris–HCl,pH7.5.For carnosine or balenine synthesis,L-histidine orτ-methyl-L-histidine was used,respectively,instead ofπ-methyl-L-histidine.γ-Aminobutyric acid and L-histidine were used as substrates for homocarnosine synthesis.The enzyme preparation was added to the reagent mixture prepared on ice and incubated for1h at37°C unless otherwise described.The reaction was stopped by placing the tubes in a boiling water bath for3min followed by centrifugation at2300×g for3min.The supernatant was filtered with Ultrafree-MC(Millipore;Bedford,MA,USA) and used for HPLC analysis.For a time course experiment, aliquots of reaction mixture were taken out at appropriate time intervals(0to24h)and treated as above.The HPLC determination of dipeptides was carried out on a Jasco HPLC system(Jasco;Tokyo,Japan)with a Superiorex ODS column(φ4.6×250mm,Shiseido;Tokyo,Japan).Elution was performed isocratically with50mM KH2PO4,pH 3.4, containing6mM1-heptanesulfonic acid and2%acetonitrile at a flow rate of1mL/min at50°C and monitored at210nm.Protein concentration was measured with a protein assay dye solution(Bio-Rad Laboratories)using bovine serum albumin as a standard.During enzyme purification,protein elution was monitored by the absorbance at280nm using a micro plate reader,SpectraMax M2(Molecular Devices;Sunnyvale,CA, USA).2.3.Purification of imidazole dipeptide synthaseDuring the purification,the enzyme activity was checked for anserine synthesis.All purification procedures were performed at4°C.The white muscle of Japanese eel,weighing about 400g,was homogenized with5vol of50mM Tris–HCl,pH7.5, and the homogenate was centrifuged at12,000×g for15min.Ammonium sulfate was added to the supernatant up to50% saturation.After being stirred for2h,the suspension was centrifuged as above and the supernatant was brought to75% saturation with ammonium sulfate.The resultant precipitate (50–75%fraction)was dialyzed overnight against10mM Tris–HCl,pH8.0.After centrifugation as above,the supernatant was applied onto a DEAE-Toyopearl column(3×50cm)previously equilibrated with the same buffer,and the column was washed with500mL of the same buffer.The enzyme was eluted with a linear gradient of0−250mM NaCl at a flow rate of2mL/min. The active fractions were collected and ammonium sulfate was added to the solution at a final concentration of2M.This pooled solution was loaded onto an Ether-Toyopearl column(3×15cm)equilibrated with the same buffer containing 2M ammonium sulfate,and the column was washed with the same buffer.The enzyme was eluted with a linear gradient of2–0M ammonium sulfate in the same buffer at a flow rate of2mL/min.561S.Tsubone et al./Comparative Biochemistry and Physiology,Part B146(2007)560–567The pooled active fractions were desalted and concentrated by ultrafiltration using a Vivaspin concentrator (Vivascience;Stone-house,UK).The enzyme solution was subjected to a His-Trap 5mL column (1.6×2.5cm)previously equilibrated with 20mM phosphate buffer,pH 7.4,containing 500mM NaCl.From this step,purification was carried out on an automated liquid chromatography system,ÄKTA purifier 10/100(Amersham Bioscience).The protein was eluted with 10mM imidazole in the above phosphate buffer.The active fractions were applied onto a CHT-1column (7×52mm)equilibrated with 5mM phosphate buffer,pH 7.0,and then eluted with a linear gradient of 5–175mM phosphate buffer.The active fractions obtained were applied to a MonoQ HR5/5column (5×50mm)previously equilibrated with 10mM phosphate buffer,pH 8.0,and the protein was then eluted with a linear gradient of 0–300mM NaCl.The pooled active fractions were concentrated by ultrafiltration using Ultrafree-MC to approximately 100μL.Finally,this fraction was loaded onto a Superdex 20010/300column (1×30cm)and the enzyme was eluted with 50mM phosphate buffer,pH 7.2,containing 500mM NaCl at a flow rate of 0.4mL/min.The active fractions were collected and stored frozen as a final preparation.2.4.ElectrophoresisSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)was carried out according to the method of Laemmli (1970),using 4.75%acrylamide in the stacking gel and 10%in the separating gel.Protein bands were visualized by staining with Coomassie Brilliant Blue R-250.2.5.N-terminal amino acid analysisThe purified enzyme was subjected to SDS-PAGE under reducing conditions,and the protein band on the gel was elec-trophoretically blotted onto a polyvinylidene difluoride (PVDF)membrane with a transfer buffer (20mM Tris and 190mM glycine with 20%methanol).The protein band was applied to a protein sequencer,Procise (Applied Biosystems;Foster,CA,USA).2.6.Determination of enzymatic properties of the purified imidazole dipeptide synthaseThe effect of pH on the enzyme activity for anserine synthesis was investigated over the pH range of 5–12at 37°C.The optimal temperature of the purified enzyme was determined by incubating the enzyme –substrate mixture in 50mM Tris –HCl,pH 7.5,for 1h on the heat block at various temperatures from 20to 80°C.The apparent K m and V max values for anserine synthesis against β-alanine and π-methyl-L -histidine were determined by Lineweaver –Burk plots using the substrate concentrations from 10to 250mM.Effects of metal ions on anserine synthetic activity were determined by incubating the reaction mixture containing β-alanine,π-methyl-L -histidine,and the purified enzyme solu-tion with 2to 10mM of metal ions or 1mM of EDTA.2.7.Enzymatic hydrolysis of imidazole dipeptides by the purified enzymeAs substrates,0.8mM carnosine,anserine,or balenine were individually incubated at 37°C with the purified enzyme preparation in 50mM Tris –HCl,pH 7.5,and the amount of remaining dipeptide was measured at each sampling time (0to 60min)by the above described HPLC method.Inhibitory effects on anserine hydrolyzing activity were determined by the incubation of 4mM anserine with the purified enzyme preparation and 2mM inhibitors including phenyl-methylsulfonyl fluoride (PMSF),p -chloromercuribenzoic acidTable 1Purification of imidazole dipeptide synthase from eel muscle PurificationProtein (mg)Total activity (μmol/min)Yield (%)Specific activity (nmol/min mg protein)Purification(fold)50–75%(NH 4)2SO 4558832.3100 5.831DEAE-Toyopearl 49316.751.533.8 5.80Ether-Toyopearl 145 2.537.8517.53.02His-Trap 8.5 1.92 5.9522739.2Hydroxyapatite 3.85 1.35 4.1835060.6MonoQ 0.94 1.43 4.451535265Superdex 2000.0780.521.5965671126Purification was performed based on the anserine syntheticactivity.Fig.1.SDS-PAGE patterns of each purification step of imidazole dipeptide synthase.M,molecular weight marker;lane 1,50–75%(NH 4)2SO 4fraction;lane 2,DEAE-Toyopearl;lane 3,Ether-Toyopearl;lane 4,His-Trap;lane 5,Hydroxyapatite;lane 6,MonoQ;lane 7,Superdex 200.562S.Tsubone et al./Comparative Biochemistry and Physiology,Part B 146(2007)560–567(PCMB),1,10-o -phenanthroline monohydrate (o -phenanthro-line),bestatin,and 10mM EDTA.3.Results3.1.Purification of imidazole dipeptide synthaseThe activity of imidazole dipeptide synthase could hardly be detected in the crude enzyme solution of eel white muscle butclearly detected in 50–75%ammonium sulfate fraction.Carno-sine was synthesized by the enzyme in this fraction from its constituents,β-alanine and L -histidine,with a specific activity of 0.52nmol/min d mg protein.Anserine and balenine were also synthesized from β-alanine and either π-methyl-L -histidine or τ-methyl-L -histidine,respectively.Specific activities for anse-rine and balenine syntheses were 5.8and 0.78nmol/min d mg protein,respectively.ATP was not required in these reactions.The purification of imidazole dipeptide synthase is summa-rized in Table 1.The enzyme was finally purified about 1130-fold with 1.6%yield from ammonium sulfate fraction by six steps of column chromatography.The specific activity for the anserine synthesis of the final preparation was 6.5μmol/min d mg protein.The specific activities for carnosine and balenine syntheses were 0.25and 0.53μmol/min .mg protein,respectively.The native molecular mass of the enzyme was estimated to be approximately 275kDa by the final Superdex gel permeation chromatography.The highest activity peak coincided with the largest protein peak.The major protein band of the purified enzyme preparation on SDS-PAGE gel showed an estimated molecular mass of approximately 43kDa (Fig.1).Although a minor smear band was also found on the SDS-PAGE gel,the final preparation was subjected to subsequent analyses without further purification.3.2.N-terminal amino acid sequenceThe amino acid sequence of the N-terminal region was determined on the main protein band of purified imidazole dipeptide synthase.The N-terminal amino acid sequence of 25residues was estimated to be LVGGSLTRNVPWQVLLQFSD-SVLYG.A homology search using the BLAST algorithm gave 52%amino acid identity to residues 38–62of the haptoglobin precursor from the zebrafish Danio rerio .The N-terminal amino acid sequence of 16residues of the smear band in Fig.1gave high homologies to some proteins which were not related to the metabolism of thedipeptides.Fig.2.Time course of the dipeptide syntheses by the purified imidazole dipeptide synthase.Amounts (mmol)synthesized per mg of protein for anserine (•),carnosine (▴),and balenine (○)were shown.Mean±SE for triplicatedeterminations.Fig.3.Effects of pH and temperature on anserine synthetic activity of imidazole dipeptide synthase.(A)Optimal pH.The activity was assayed in 50mM acetate (▪),50mM MES (•),50mM Tris –HCl (▴),and 500mM CAPS (□)buffers.(B)Optimal temperature.The reaction time is 1h at each temperature.Mean±SE for triplicate determinations.563S.Tsubone et al./Comparative Biochemistry and Physiology,Part B 146(2007)560–5673.3.Characteristics of purified imidazole dipeptide synthaseTime course analyses of the purified enzyme for carnosine, anserine,and balenine syntheses are depicted in Fig.2.The enzyme clearly shows time-dependent syntheses of these three imidazole dipeptides.After24h of incubation,carnosine, anserine,and balenine synthesized by the enzyme amounted to 0.2,2.4,and0.35mmol/mg protein,respectively.Anserine synthetic activity was by far the highest at each incubation time.The effects of pH on anserine synthetic activity of the purified enzyme are shown in Fig.3A.The optimal pH for anserine synthesis is around9.5.The activity decreases sharply below pH6and above pH9.5.The enzyme shows the highest activity of anserine synthesis at60°C(Fig.3B).A significant decrease of the activity was observed above 70°C.The apparent K m and V max values of the purified enzyme for anserine synthesis were determined by Lineweaver–Burk plots (Fig.4A and B).The apparent K m values againstβ-alanine and π-methyl-L-histidine were calculated to be44and89mM,respec-tively.The V max values forβ-alanine andπ-methyl-L-histidine were7.4and11μmol/min d mg protein,respectively.The effects of metal ions and EDTA are presented in Table2. Anserine synthesis is greatly activated by2mM Zn2+and Co2+, more than5-and4-fold,respectively.However,10mM of both metal ions largely reduced the activating effects on the enzyme. Mg2+and Mn2+ions gave a slight activating effect on the enzyme at each concentration.The activity was strongly inhibited to58%of the control by1mMEDTA.Fig.4.Lineweaver–Burk plots of imidazole dipeptide synthase againstβ-alanine(A)andπ-methyl-L-histidine(B).Mean±SE for triplicate determinations.Table2Effects of metal ions and EDTA on anserine synthetic activities of imidazole dipeptide synthaseCompound Concentration(mM)Relative activity(%) Control100±3.76Zn2+2525±23.410119±2.19Mg2+2112±2.7710123±5.72Mn2+2140±11.610140±31.4Co2+2423±7.4510196±13.7EDTA158.3±0.78Mean±SE for triplicatedeterminations.Fig.5.Time course of hydrolyses of dipeptides by the purified imidazole dipeptide synthase.As substrates,0.8mM each of anserine(•),carnosine(▴), and balenine(○)was used.Mean±SE for triplicate determinations.564S.Tsubone et al./Comparative Biochemistry and Physiology,Part B146(2007)560–5673.4.Enzymatic hydrolysis of imidazole dipeptides by the purified enzyme preparationCarnosine,anserine,and balenine were hydrolyzed to their constituents,β-alanine and L-histidine or its derivatives by the purified imidazole dipeptide synthase(Fig.5).The increase of the hydrolytic reaction products,L-histidine and its methyl derivatives,were confirmed on the same HPLC chromatograms (data not shown).Anserine was the most susceptible to hydrolysis and hydrolyzed completely within10-min incuba-tion,with a specific activity of1.95μmol/min d mg protein. Carnosine and balenine were also hydrolyzed although at a slower rate than anserine.Specific activities for carnosine and balenine hydrolysis were0.86and0.43μmol/min d mg protein, respectively.The inhibition of anserine hydrolytic activity by several protease inhibitors are shown in Table3.A metal chelator,o-phenanthroline,completely inhibited the anserine hydrolyzing activity at2mM,whereas the inhibitory effect of EDTA was not clear.The serine protease inhibitor,PMSF,gave a low inhibitory effect on the activity.Anserine hydrolytic activity was inhibited about70%by bestatin,a specific inhibitor of various amino-and di-peptidases including serum carnosinase.The cysteine protease inhibitor,PCMB,inhibited the anserine hydrolytic activity about50%.4.DiscussionThe enzyme purified from eel muscle in the present study catalyzed the synthesis of carnosine,anserine,and balenine in the absence of ATP.Watanabe and Konosu(1979)have demonstrated the incorporation of14C-histidine into carnosine in eel muscle24h after intramuscular injection of labeled histidine,but no enzymatic evidence for the dipeptide syntheses in fish has been obtained hitherto.Since the purified enzyme did not require ATP for dipeptide synthesis,the enzyme found in this study might be distinct from carnosine synthetase and may be a new type synthase.Unexpectedly,anserine synthetic activity was the highest of all activities for these dipeptides syntheses,although eel contains a large amount of carnosine and only trace amounts of anserine and balenine.Thus,the enzyme is thought to prefer π-methyl-L-histidine to L-histidine orτ-methyl-L-histidine.In addition to these three dipeptides,a weak activity for homo-carnosine(γ-aminobutyryl-L-histidine)synthesis was also detected (data not shown).The substrate affinity forγ-aminobutyric acid was supposed to be extremely lower than that forβ-alanine.From these data,it is possible that the enzyme catalyzes the syntheses of any of the three dipeptides depending on the availability of substrate,histidine or its methyl derivatives.Anserine synthetic activity was also found in50–75% ammonium sulfate fraction of the white muscle of big-eye tuna Thunnus obesus which contains a high-level of anserine(data not shown).We also detected balenine synthetic activity in the same fraction of the skeletal muscle of minke whale Balaenoptera acutorostrata which contains high concentrations of balenine, although the activity was very weak(data also not shown). Evidently,the imidazole dipeptide synthase distributes in many vertebrates having large amounts of the dipeptides in muscle.The molecular mass of the native enzyme was estimated to be approximately275kDa and that of subunit was to be43kDa (Fig.1),suggesting that the native enzyme is a hexamer or heptamer.Wood and Johnson(1981)reported that the purified carnosine synthetase from chick pectoral muscle was a homodimer composed of119kDa monomer.There is a clear difference between the chick carnosine synthetase and eel dipeptide synthase based upon their molecular size.As seen in Table2,anserine synthetic activity was greatly elevated by2mM Zn2+and inhibited by a metal chelator, EDTA.Therefore,the enzyme is believed to be divalent cation-dependent or a metalloenzyme.Carnosine synthetase in chick muscle is reported to require2mM Mg2+or Mn2+for optimal activity whereas higher concentrations are inhibitory(Stenesh and Winnick,1960).For rat brain enzyme,the maximal activity is attained with4to6mM Mg2+with equimolar amounts of ATP(Skaper et al.,1973).The purified enzyme showed a maximal pH at about9.5 (Fig.3A)which was different from that for carnosine synthetase, at pH7.5(Winnick and Winnick,1959).Surprisingly,the enzyme displays an optimal activity at a temperature of60°C which is higher than expected for fish enzymes.For anserine synthesis,K m values againstβ-alanine andπ-methyl-L-histidine was43.8and 88.5mM,respectively,suggesting the very low substrate affinity of the enzyme.π-Methyl-L-histidine exists only in trace amounts in eel muscle whereas L-histidine is much higher,around1mM (Abe,1983).Thus,it remains unclear whether these dipeptides are synthesized by this enzyme in eel muscle under low substrate concentrations and high K m values.However,the rate of incorporation of labeled L-histidine into carnosine and anserine has been reported to be very low in rainbow trout(Abe and Hochachka,1987)and skipjack tuna(Abe et al.,1986).The N-terminal amino acid sequence of the purified imidazole dipeptide synthase showed52%amino acid identity to the partial sequence of haptoglobin precursor of zebrafish. Haptoglobin is a plasma glycoprotein known to have an ability to bind hemoglobin released on hemolysis and protect the tissues from oxidative damage(Wicher and Fries,2004).Human haptoglobin is composed of two short and two long polypep-tides,α-andβ-chains,respectively,which are synthesized as a single precursor polypeptide and processed post-translationally (Raugei et al.,1983;Yang et al.,1983).Theβ-chain is related toTable3Effects of inhibitors on anserine hydrolytic activity by the purified enzyme preparationCompound Concentration(mM)Relative activity(%) Control100±0.1EDTA1089.4±14.7o-Phenanthroline20±1.58PMSF281.2±0.69PCMB247.9±1.58 Bestatin229.7±3.24Mean±SE for triplicate determinations.o-Phenanthroline,1,10-o-phenanthro-line;PMSF,phenylmethylsulfonyl fluoride;PCMB,p-chloromercuribenzoic acid.565S.Tsubone et al./Comparative Biochemistry and Physiology,Part B146(2007)560–567trypsine-like serine protease but does not possess the essential catalytic amino acid residues(Kurosky et al.,1980).It is suggested that haptoglobin is evolutionarily related to serine proteases(Barnett et al.,1970,1972;Kurosky et al.,1980).Thus,we predicted that imidazole dipeptide synthase purified in the present study was a kind of protease and could hydrolyze dipeptides.To solve the question,the hydrolytic activity of purified enzyme against these three dipeptides was examined. These dipeptides were all hydrolyzed by the purified preparation (Fig.5).Anserine was hydrolyzed markedly,and carnosine and balenine were also hydrolyzed with a slower rate than anserine, as was seen in the order of synthetic activity.Thus,we propose that one enzyme catalyzes both the synthesis and hydrolysis of imidazole dipeptides.However,the N-terminal amino acid sequence showed no similarity to human carnosinase,cytosolic non-specific dipeptidase(Teufel et al.,2003),or Nile tilapia anserinase(Yamada et al.,2005).The mechanisms of dipeptide synthesis without ATP remain unclear.However,one possibility is that proteases are known to catalyze the reverse condensation reaction from amino acids leading to peptides which are called plastein(Watanabe and Arai,1992).More detailed research is required to unambiguously ascertain the mechanisms of dipeptide synthesis and hydrolysis.As seen in Table3,the hydrolytic activity was inhibited completely by a metal chelator,o-phenanthroline,and about 70%by bestatin,a specific inhibitor of various amino-and di-peptidases,and50%by PCMB for cysteine protease.From these data,the enzyme was thought to be at least a metalloprotease-like enzyme.It is not clear how the enzyme regulates the synthesis and hydrolysis of imidazole dipeptides in eel muscle. One possibility is that dipeptide synthase is located in some subcellular organelle other than cytosol where dipeptides exist in large amount.Due to the scarce amount of the purified enzyme obtained,further experiments could not be performed on the properties of hydrolyzing activity.In conclusion,we purified for the first time a novel enzyme from eel white muscle,which synthesizes and hydrolyzes imidazole dipeptides.Further studies are needed to clarify the enzyme kinetics for both synthesis and hydrolysis of dipeptides, as well as the regulatory and structural characteristics of the enzyme.Additional research is also required to explain the distribution of the enzyme in various vertebrates containing these dipeptides in large amount.These lines of research may pave the way for understanding the reasons for the species specific distribution of these imidazole dipeptides in vertebrates. 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