Comparative Biochemistry and Physiology Part C 135(2003) 285–294

1532-0456/03/$ - see front matter� 2003 Elsevier Inc. All rights reserved.doi:10.1016/S1532-0456(03)00118-2

Hepatic metallothioneins in two neotenic salamanders,Proteusanguinus andNecturus maculosus (Amphibia, Caudata)

K. Dobrovoljc *, I. Falnoga , B. Bulog , M. Tusek-Znidaric , J. Scancara, b a b bˇˇˇ ˇ ˇ ˇ

Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, Ljubljana 1000, Sloveniaa ˇDepartment of Environmental Sciences, Jozef Stefan Institute, Jamova 39, Ljubljana 1000, Sloveniab ˇ

Received 4 December 2002; received in revised form 26 May 2003; accepted 27 May 2003

Abstract

The presence of metallothionein(MT) and the subcellular distribution of copper, zinc and cadmium were investigatedin livers of two neotenic salamanders,Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild,hepatic MTs were present as a single isoform of(Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to beapproximately 12 000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosoland of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs(80%), while zinc andcopper were also present in the regions of higher-molecular weight proteins. In laboratory bredN. maculosus, MTs wereisolated from the liver cytosol and extract of the pellet as(Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According tothe low amount of copper extracting from liver pellets ofN. maculosus, the presence of water insoluble aggregatedforms of Cu-thioneins should be checked in further investigations.� 2003 Elsevier Inc. All rights reserved.

Keywords: Amphibia; Cadmium; Copper; Liver; Metallothionein;Necturus maculosus; Proteus anguinus; Zinc

1. Introduction

Metallothioneins (MTs) are low molecularweight, cysteine-rich, metal-binding proteins, firstreported as Cd-binding proteins from equine renalcortex (Margoshes and Vallee, 1957). Because ofthe lack of a known enzymatic function, they areclassified according to their general structural fea-tures: low-molecular-mass(-9 kDa), high cyste-ine content(23–33 mol%), absence of aromaticamino acid residues, specific optical properties andhigh content of heavy metals(typically 7–12atomsymole) bound exclusively by clusters of

*Corresponding author. Tel.:q386-1-423-33-88; fax:q386-1-257-33-90.

E-mail address: katarina.dobrovoljc@uni-lj.si (K. Dobrovoljc).

thiolate bonds(Hamer, 1986; Kagi, 1993). The¨exact biological functions of MTs are not clearlyresolved; they are considered to be involved intransport, storage and homeostasis of essentialtrace elements, including Zn and Cu(Cherian andChan, 1993), detoxification of heavy metals, suchas Cd or Hg (Klaassen et al., 1999), and onaccount of their high cysteine content also inradical scavenging inside the cell(Sato and Brem-ner, 1993). In addition to MT induction by metalions, the syntheses of MT isoforms may also beinduced by oxidative stress, certain hormones,cytokines and various chemical or physical treat-ments(Kagi, 1993). Most vertebrate species con-¨tain two or more distinct MT isoforms designatedMT-I through MT-IV. MT-I and MT-II isoformshave been found in almost all tissues and cells

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where they are present mainly in the cytosol, butin some circumstances they can also be detectedin the cell nucleus(Cherian and Apostolova,2000), lysosomes(Klein et al., 1999) and mito-chondria(Ye et al., 2001). In contrast, the MT-IIIisoform is expressed mainly in the brain(Uchidaet al., 1991), while the MT-IV isoform is mostabundant in certain stratified epithelia(Quaife etal., 1994).

Amphibians are interesting species for studyingthe biological roles and phylogenetic changes ofMTs because they hold a phylogenetic positionbetween fishes and reptiles. In addition, the naturalhabitat of amphibians is often polluted with toxicsubstances as a consequence of industrializationand thus MTs, as cellular metal binding proteins,are of particular interest. Amphibian MTs havebeen studied in 10 species, mainly anurans(Suzukiet al., 1983; Suzuki and Akitomi, 1983; Suzukiand Tanaka, 1983; Yamamura and Suzuki, 1983;Suzuki and Kawamura, 1984; Suzuki et al., 1986),while the presence of MTs in tailed amphibians(order Caudata) has been reported in three species,Ambystoma maculatum (Suzuki and Ebihara,1984), Ambystoma mexicanum (Saint-Jacques etal., 1998) and Triturus pyrrhogaster (Suzuki etal., 1984).

Proteus anguinus (Amphibia, Caudata, Protei-dae) is the sole species of European Cave sala-mander. It maintains neotenic characteristicsthroughout its life and also reveals some troglo-morphic characteristics, typical of cave animalevolution(Bulog, 1994). According to its long lifespan(approx. 70 years) and occasional exposureto stress conditions in its underground habitat(starvation, low temperatures, hypoxia, potentiallytoxic pollutants like metals, pesticides, etc.), thefunctioning of the cellular mechanisms involvedin homeostasis and detoxification of metal ionsand radical scavenging in stress responses arefundamental for its existence. Thus, MTs couldplay an important role inP. anguinus physiologicalprocesses and tolerance mechanisms.

The aim of the present study was to investigatethe presence of MTs in the liver ofP. anguinusand their specific affinity toward copper, zinc andcadmium. Particular attention was given to thenumber of MT isoforms. In addition, the presenceof MTs and their affinity for binding copper, zincand cadmium was also investigated in the liver ofthe mudpuppy,Necturus maculosus (Amphibia,

Caudata), the closest living relative ofP. anguinus,found in rivers and large streams of the UnitedStates and Canada.P. anguinus specimens werecaught in nature, whileN. maculosus were rearedin the laboratory.

2. Materials and methods

2.1. Organisms

Two adult P. anguinus male specimens werecaptured with a net in Otovski breg near Crnomeljˇˇ(Slovenia). In the laboratory of the department ofbiology (Biotechnical Faculty, University of Lju-bljana), they were transferred to tanks with theoriginal field water, and kept in the dark at 108C.Approximately one day after capture, animals wereprepared for analyses. Due to the rigorous protec-tion of P. anguinus by national conservation laws,the number of captured animals available forresearch was limited. Two adult egg-bearingfemale mudpuppies,N. maculosus, were obtainedfrom Carolina biological supply company(USA).In the laboratory of the department of biology,they were maintained in glass aquaria with fieldwater at 108C and fed once per week with troutfry (Oncorhynchus mykiss) obtained from a localfish farm.

2.2. Tissue preparations

Animals (P. anguinus, ns2; N. maculosus, ns2) were anaesthetized with a 0.3% solution of MS-222 (ethyl 3-aminobenzoate methane sulfonic acidsalt, Sigma), weighed and killed by decapitation.Liver tissue was dissected, washed with distilledwater to remove blood residues, weighed, chilledin liquid nitrogen and stored aty70 8C. Sex, bodylength, body and liver mass, hepatosomatic index(HSIsliver massybody mass=100) of the speci-mens used in the study are presented in Table 1.

2.3. Biochemical procedures

2.3.1. Tissue homogenizationPooled liver samples(P. anguinus, ns2, ws

2.77 g; N. maculosus, ns2, ws10.11 g) werethawed and homogenized with a glass homogeniz-er and Teflon Polymix PX-OS 2000 pestle in ice-cold (1–4 8C) nitrogen-saturated 10 mM Tris–HCl buffer (pH 7.6) containing 25% saccharose,

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Table 1Mean morphological characteristics of adultP. anguinus andN. maculosus specimens

Species Sex Body length Body mass Liver mass HSI(mm) (g) (g)

Proteus anguinus � 280 33.6 1.293 3.85(ns2) � 273 33.1 1.759 5.31

Necturus maculosus �* 286 136.0 6.714 4.93(ns2) �* 280 112.2 7.980 7.11

HSI, hepatosomatic index(liver massybody mass=100).Egg-bearing female.*

0.1 mM phenylmethylsulfonyl fluoride(PMSF)and 10 mM dithiotreithol(DTT). The homogena-tes (33%) were centrifuged at 100 000g for 60min at 48C and the resulting supernatants(referredto as cytosol), pellets and fats used further.

2.3.2. Pellet extractionPellets were resuspended in 5–6 ml of homog-

enization buffer, chilled three times in liquid nitro-gen and centrifuged at 100 000g for 60 min at 48C. Supernatants(referred to as extract) were usedfurther.

2.3.3. Heat treatment of the cytosol and extractOne half of theN. maculosus cytosol was heat

treated at 708C for 10 min, allowed to cool onice and then centrifuged at 20 000g for 20 min at4 8C to remove precipitated proteins. Pellet extractsof both species were heat treated at 1008C for 2min and then treated in the same way as heat-treated cytosol ofN. maculosus.

2.3.4. Sephadex G-75 gel filtration chroma-tography

Aliquots (2.5 or 4.0 ml) of the cytosols andextracts obtained were applied to Sephadex G-75(Pharmacia) columns (1.7=60 cm or 2.4=65cm), standardized with marker proteins of knownmolecular weight(Serva, Pharmacia). Elution wascarried out with N saturated 10 or 20 mM Tris–2

HCl buffer (pH 8.0 or 7.6, 48C) containing 1mM DTT at a flow rate of 9.6 or 13.8 mlyh.Fractions were collected in;4 ml or 6–8 mlvolumes and assayed for Cu, Zn and Cd by flameAAS and by absorption at 280, 220 and 254 nm.

2.3.5. High performance liquid chromatography(HPLC)

Aliquots (200 ml) of P. anguinus liver cytosolfractions, corresponding to the MT peak from

Sephadex G-75 chromatography and MT II stan-dard (Rabbit liver MT II, Sigma) were used forthe HPLC determination of MT isoforms. HPLCwas performed on a Tosohaas column(TSKgelG3000SW, 0.75=60 cm, connected with a Toso-haas guard column of 0.75=7.5 cm). The columnwas eluted either with:(a) 50 mM Tris–HCl buffer(pH 8.0) or (b) 50 mM Tris–HCl buffer(pH 8.0)with 0.1% NaN at a flow rate of 1 mlymin.3

Absorbances at 215(a) or 254 (b) nm weredetermined by a UV detector. Under the chosenconditions the column and the buffer allowed theuse of size exclusion and ionic exchange propertiesfor the better separation of isoforms(Suzuki et al.,1980).

2.4. Determination of metal concentration

Radiochemical neutron activation analyses(RNAA; detection limit 10 ngyg) was used forthe determination of Cu, Zn and Cd concentrationsin the liver tissue and liver fractions(cytosol,pellet, fat and extract). Samples were irradiated inpolythene ampoules for 18–20 h in the carouselfacility of the TRIGA Mark II reactor, JSI, at athermal neutron flux of 1.1=10 ncm s .12 y2 y1

After irradiation simultaneous Cu, Zn and Cddetermination via wet digestion and carbamateextraction was used(Dermelj et al., 1979). Asuitable certified reference material(BCR CRM-186, Pig kidney) was also analyzed.

In the elution fractions after gel-filtration chro-matography on Sephadex G-75 the concentrationsof Cu, Zn and Cd were measured by flame atomicabsorption spectrometry(FAAS) in an air-acety-lene flame with deuterium correction of non-specific absorption on a Varian AA-5 atomicabsorption spectrometer. The detection limits for

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Table 2Concentration of metals(Cu, Zn, Cd) in pooled liver samples ofP. anguinus andN. maculosus and their percentage distribution withinthe liver cellular fractions

Species Metal

Tissue Percentage in thea Percentage in thea Percentage in thea

(mg g w wt)y1 cytosol pellet fat

Proteus anguinus Cu 3.18 27 47 26(ns2) Zn 7.09 56 25 19

Cd 0.31 77 13 10

Necturus maculosus Cu 19.60 54 42 4(ns2) Zn 8.45 76 20 4

Cd -dl – – –

dl, Detection limit of RNAA (10 ngyg).Percentage of metals regarding the sum of their content in cytosol, pellet and fat.a

metals were: Cuy20 ngyml, Zn y5 ngyml, Cdy10 ngyml.

3. Results

Table 1 shows the main morphological charac-teristics of the adultP. anguinus andN. maculosusspecimens used in our study. The concentrationsof Cu, Zn and Cd and their percentage distributionwithin the subcellular fractions ofP. anguinus andN. maculosus pooled liver samples are shown inTable 2. InP. anguinus relatively high amounts ofmetals were found in the fat, between 10 and 26%,where the highest amount of Cu was almost thesame as in the cytosol. In the pooled liver of themudpuppy,N. maculosus, the percentages of Cuand Zn associated with the cytosol were higherwhile the percentages of both metals in fat weremuch lower, approximately 4%(Table 2). Asexpected, the content of Cd was below the detec-tion limit (RNAA; 10 ngyg), likely due to thenon-contaminated conditions in the laboratoryenvironment. In the extracts obtained from liverpellets of both species, the amounts of water-soluble metals were very low or below the limitof detection(Cd). More than 90% of Cu and Znremained in the particulate phase(data not shown).

Fig. 1 presents Zn, Cu and Cd chromatographicprofiles of P. anguinus liver cytosol. The peaks ofZn, Cu and Cd found in the elution region ofcytochrome C (cyt C; M 12 400) indicate ther

characteristics of metallothioneins. Regarding thewhole chromatogram, 80% of Cd was associatedwith MTs, while Cu and particularly Zn were alsopresent in regions with higher molecular weight

proteins. The percentage metal distribution amongthe different cytosol proteins is given in Table 3.

The identity of MT in P. anguinus was alsoconfirmed by comparing its retention time(Rt)with a rabbit MT-II standard and by checking thepresence of MT isoforms. The MT elution fractionsobtained by Sephadex G-75 chromatography(frac-tion number 19–21; Ve 75.73–83.66, Fig. 1) andthe Rabbit liver MT-II standard(Sigma) was sub-jected to HPLC. From Fig. 2 it is evident that theisolated(Zn, Cu, Cd)-thioneins were eluted at thesame position as the MT II standard(Rt 20.2 or18.0), and found in only one isoform in conditionswhich should resolve at least two main isoforms(elution with 10 mM Tris–HCl buffer with 0.1%NaN ; Suzuki et al., 1980).3

In the mudpuppy, Sephadex G-75 chromatogra-phy of liver cytosol(Fig. 3a) revealed the presenceof (Cu, Zn)-thioneins. From Table 3 it is seen thatthe percentage of cytosolic Cu bound to MTs wassimilar as inP. anguinus, while the percentage ofZn was lower. This metal peak also showed hightemperature tolerance, illustrated in Fig. 3b. MTswere also detected in the extract of liver pellet as(Zn, Cu)-thioneins (Fig. 4). In these subcellularMTs, the order of contents of the two elementswas reversed and at the same time similar to thatin cytosol MTs ofP. anguinus.

Summarized metal composition of all isolatedMTs and apparent molecular weights from sizeexclusion chromatography are given in Table 4.The higherM for N. maculosus (Cu, Zn)-thioneinsr

could be the consequence of different structuralproperties of the MT molecule, due to its prevail-ing Cu content.

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Table 3Percentage(%) of cytosolic metals(Cu, Zn, Cd) bound to MTsand HMW proteins in pooled liver samples ofP. anguinus andN. maculosus

Species Metal Percentage of cyto-solic metals boundto:

MTs HMWP

Proteus anguinus Cu 36 31Zn 23 64Cd 80 9

Necturus maculosus Cu 36 41Zn 8 74Cd – –

MTs, metallothioneins; HMWP, high molecular weight pro-teins(M )80 000).r

Fig. 1. Gel-filtration chromatography ofP. anguinus liver cytosol (2.5 ml) on a Sephadex G-75 column(1.7=60 cm) eluted with 20mM Tris–HCl (pH 8.0, 48C, 1 mM DTT) at a flow rate of 9.6 mlyh. In the elution fractions(4 ml) absorbances(280, 220 nm) andmetals(Zn, Cu, Cd) were determined. The arrows represent ovalbumin(ov; M 43 000) and cytochromeC (cyt C; M 12 400); Ve,r r

elution volume.

4. Discussion

In this study the concentrations of liver Cu, Znand Cd, their subcellular distribution and the pres-ence of metallothionein(MT) in two neotenicsalamanders,P. anguinus and N. maculosus, wereinvestigated for the first time. An elevated levelof Cu in the liver of female specimens ofN.maculosus can be ascribed to egg production,because both animals were egg-bearing. It is wellknown that during spawning Cu accumulates inlarge quantities in the livers of female frogs(Pas-anen and Koskela, 1974) and also that amphibianeggs contain quite large amounts of Cu(Brownand Jr., 1964). In the liver of male specimens ofP. anguinus, the average hepatic Cu concentrationwas comparable with our previous data(Dobro-voljc, 2002). Furthermore, Cd was not detected inthe liver of the laboratory bredN. maculosus. Incontrast, its concentration in the liver ofP. anguin-us was higher than in the livers of other amphibi-ans of field origin (Suzuki and Akitomi, 1983;Suzuki and Kawamura, 1984; Suzuki et al., 1986;Vogiatzis and Loumbourdis, 1998).

Following uptake, Cu, Zn and Cd are distributedto different parts of the body. Our previous analy-ses of metal concentrations in skin, muscle, liverand kidneys of wild P. anguinus specimensrevealed that the liver contained the highestamounts of metals analyzed and, therefore, maybe considered a target organ(Bulog et al., 2002;

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Fig. 2. Chromatography of theP. anguinus liver cytosol MT elution fractions from Fig. 1(Fr. No. 19–21; Ve 75.73–83.66 ml; Fig. 1)and an animal MT II standard(Rabbit liver MT II, Sigma) on an HPLC column, eluted with:(a) 50 mM Tris–HCl, UV detection at215 nm, and(b) 10 mM Tris–HCl containing 0.1% NaN , UV detection at 254 nm.3

Dobrovoljc, 2002). In liver cells, a fundamentalsimilarity exists in the metabolism of Cu, Zn andCd at the subcellular level(Hogstrand and Haux,1991). When the intracellular amount of one ofthese elements increases, liver cells respond withthe de novo syntheses of MTs, which then chelatea significant portion of the excess metal enteringthe tissue.

In livers of adult amphibians MTs are usuallypresent as Cu, Zn-, Cu- or Zn-binding proteins,while exposure to Cd, Hg, Zn or Cu induces MTswith different ratios of bound metals(Mehra etal., 1980; Suzuki and Akitomi, 1983; Suzuki etal., 1983; Suzuki and Kawamura, 1984; Suzuki etal., 1984, 1986; Woodall and Maclean, 1992;Vogiatzis and Loumbourdis, 1998). In our studythe presence of MTs was identified in the livercytosol of P. anguinus and N. maculosus on thebasis of their molecular weight, the absence of UVabsorbance at 280 nm, and binding metals(Cu,Zn, Cd) in the eluted fractions after Sephadex G-75 chromatography. Thus,(Zn, Cu, Cd)-thioneinsof P. anguinus and (Cu, Zn)-thioneins of N.maculosus were identified(Table 4). They wereadditionally characterized by their heat stability(N. maculosus, Fig. 3b) and by HPLC rechroma-

tography of the isolated protein and by comparisonits HPLC retention time with that of an MT IIstandard(P. anguinus, Fig. 2). Since high levelsof metals remained in the pellets after homogeni-zation, we also attempted to isolate MT from thenucleus, lysosomes and mitochondria and weresuccessful in identifying(Zn, Cu)-thioneins fromthe pellet extract ofN. maculosus (Table 2 andFig. 4). In the case ofP. anguinus, the peak ofCu, Zn and Cd in the elution region of lowmolecular weight proteins was negligible and theelution chromatogram is not shown.

The isolated cytosolicP. anguinus MTs containan appreciable amount of Zn, which is often themost abundant metal in constitutive MTs becauseit is physiologically important for providing essen-tial structural and catalytic functions to a widevariety of proteins (Klaassen et al., 1999).Although the amount of Cd in MT was the lowest,it represents approximately 80% of the total cyto-solic Cd, indicating the significant protective roleof these proteins against toxic Cd ions inside2q

the cell. In amphibians, MTs seem also to be ofmajor importance for the frogs defence againsttoxicity associated with Cd, as there is a strongpositive correlation between the Cd concentration

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Fig. 3. Gel-filtration chromatography ofN. maculosus liver cytosol (2.5 ml) before(a) and after(b) heat treatment(70 8C, 10 min)on a Sephadex G-75 column(1.7=60 cm) eluted with 20 mM Tris–HCl(pH 8.0, 48C, 1 mM DTT) at a flow rate of 9.6 mlyh. Inthe elution fractions(4 ml) absorbances(280, 220 nm) and metals(Zn, Cu) were determined. The arrows represent ovalbumin(ov;M 43 000) and cytochromeC (cyt C; M 12 400); Ve, elution volume.r r

in the liver and the hepatic MT content(Vogiatzisand Loumbourdis, 1997, 1998).

In N. maculosus, MTs containing only Cu andZn were identified, as the animals were bred inlaboratory conditions and, therefore, unlikely to

have been exposed to environmental Cd. In con-trast to P. anguinus, the isolatedN. maculosusMTs contained a higher amount of Cu and a loweramount of Zn, which was also in accordance withthe tissue levels of metals. In spite of this, the

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Table 4Characteristics of the isolated metallothioneins from the liverof P. anguinus andN. maculosus

Species Tissue MTs Mr

fraction

Proteus anguinus cytosol Zn, Cu, Cd-thionein 12 000

Necturus maculosus cytosol Cu, Zn-thionein 17 000extract Zn, Cu-thionein 12 000

MTs, metallothioneins;M , molecular weight.r

Fig. 4. Gel-filtration chromatography ofN. maculosus extract(4 ml) obtained from the liver pellet on a Sephadex G-75 column(2.4=65cm) eluted with 10 mM Tris–HCl(pH 7.6, 48C, 1 mM DTT) at a flow rate of 13.8 mlyh. In the elution fractions(6–8 ml) absorbances(280, 254 nm) and metals(Zn, Cu) were determined. The arrow represents cytochromeC (cyt C; M 12 400); Ve, elution volume.r

percentage of total cytosolic Cu bound to MTswas similar in both species, while the percentageof Zn was different(Table 3).

Owing to the high percentage of metals in theliver pellet of both animals, especially Cu(N.maculosus-42%; P. anguinus-47%; Table 2), weattempted to isolate MTs additionally from theextract of the liver pellet. In the case ofN.maculosus, MTs were isolated but with the relativeratio of the metal concentration in the reverseorder of that in the liver cytosol of the samesample. In the cytosol MTs the main metal wasCu and in the pellet MTs Zn. Actually the majorpart of Cu still remained in water insoluble formin the cellular organelles. As mentioned at thebeginning, the high Cu levels in amphibian liverscan be attributed to increased Cu metabolismduring spawning(Pasanen and Koskela, 1974).Furthermore, changes in Cu metabolism can beassociated with MTs. It should be pointed out thatalthough they are cytoplasm proteins, they can alsoaccumulate in lysosomes and in some cases theyhave been observed in the cell nucleus and mito-chondria. Interesting data were found particularlywith Cu MTs (Bremner, 1991, 1993a; Cherian andChan, 1993). When liver Cu concentrations

increase greatly, the metal often accumulates inaggregated(polymeric) forms of copper MTs,which occur in the particulate fractions(includinglysosomes) of copper loaded tissue. These aggre-gated forms can be dissolved under reducing con-ditions (Bremner, 1993b; Klein et al., 1999). Inthis respect, further investigations of the waterinsoluble Cu found in the livers ofN. maculosusshould be performed.

In most vertebrates, hepatic MTs occur in twoisoforms, MT-I and MT-II (Kagi, 1993), while in¨amphibians the protein was shown to consist of asingle isoform, except in the bullfrog,Rana cates-beiana, where experimental Cd exposure-inducedtwo MT isoforms in the livers of larvae and adults.

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The relative ratios of the two isoforms werereversed between the adult and larval MTs(Suzukiand Akitomi, 1983). Under the conditions of thepresent study, the liver ofP. anguinus contained asingle MT isoform (Fig. 2), which was elutedactually at the same retention time as the RabbitMT-II liver standard, indicating that the two pro-teins are similar in molecular size and isoelectricpoint. Until now, the primary structure of amphib-ian hepatic MT is known only for the anuranXenopus laevis and the urodeleA. mexicanum(Muller et al., 1993; Saint-Jacques and Seguin,´1993; Saint-Jacques et al., 1995, 1998) and regard-ing the position of certain amino acid residues,X.laevis MT probably corresponds to the MT-IIisoform found in some vertebrates(Saint-Jacquesand Seguin, 1993).´

Acknowledgments

We are grateful to Dr Anthony R. Byrne forcritical reading and improvement of the manu-script. This study was supported by a grant fromthe Ministry of Education, Science and Sport,Republic of Slovenia.

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