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Differences in the polymorphic forms of metallothionein
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 214, No. 1, March, pp. 80-88, 1982
Differences
in the Polymorphic
DENNIS
Forms
R. WINGE2 AND KATHY-ANNE
University of Utah Medical Center, Salt
Luke
of Metallothionein’ MIKLOSSY City, Utah 84132
Received on July 20, 1981, and in revised form October 16, 1981 Metallothionein induced in rat liver by metal ions can be resolved into two forms, referred to as isoforms I and II. These polymorphic forms differ in several properties. Sequence analysis of the CNBr-digested thionein isoforms revealed seven differences in the first 25 amino acids between isoforms I and II of Cd-induced thionein. The sequence of the first 25 amino acids in isoform II of Cu-induced thionein was identical to that of isoform II of Cd-induced thionein, suggesting that the same polymorphic forms are induced with different metal ions. Isoform II of Cd,Zn-thionein exhibited a reproducibly greater Stokes radius (1’7 A) by gel filtration compared to isoform I (16.2 A), whereas isoform II of Cu-thionein eluted with a Stokes radius of 16.2 A. The isoforms also differ in Zn-binding affinity. Isoform I of several preparations of Cd,Zn-thionein and Zn-thionein invariably reconstituted greater esterase activity in apo-carbonic anhydrase than to apodid isoform II. In Cd,Zn-thionein samples, only the Zn ‘+ ions were transferred carbonic anhydrase. Similarily, the Zn ‘+ ions in isoform I were more reactive with EDTA compared to isoform II. The greater reactivity of isoform I with EDTA was evident kinetically from spectrophotometric assays as well as thermodynamically from equilibrium dialysis experiments. The significance of these differences is unclear, but it is conceivable that the dissimilarities in the conformational and Zn-binding affinity of rat liver metallothionein may correlate with a functional distinction.
unclear whether the Cu-thionein isoforms differed structurally from the Cd,Zn-thionein isoforms. The significance of the polymorphism remains unknown. Recently, Suzuki and Yamamura (18-20) observed that the ratio of the two isometallothioneins changed with time after injection of the inducing metal ion regardless of whether the inducing ion was Cd, Zn, or Cu. These results suggest that the turnover of the two forms may be dissimilar which raises the possibility that they may also differ functionally. In the course of investigating the extent and significance of the thionein polymorphism, we observed that the two isomeric forms of Zn-thionein vary in their ability to reconstitute apo-carbonic anhydrase. Zn ion transfer from Zn-thionein to apometalloenzymes has been recently reported by Udom and Brady (21) and Li et al. (22). Their results have indicated that metallothionein may function in intracel-
Metallothionein is a protein of unknown function that was first described in 1957 by Margoshes and Vallee (1). The protein is capable of being induced in cultured cells or tissues by administration of various metal ions, e.g., Zn2+, Cd2+, and Cu2+ (2-11). Two major variants of equine metallothionein have been characterized that differ in sequence in 7 out of the 61 total amino acids (12, 13). Each structurally distinct polymorphic form of Cd,Zn-thionein can bind 7 metal ions (usually 5 Cd ions and 2 Zn ions), whereas the Cu-thionein isoforms bind 10 Cu ions (14). The metal ions are ligated in cysteine-metal ion clusters (15-17). The isoforms induced by Cd, Zn, Hg, and Ag appear to be similar, whereas the forms induced by Cu differ from those induced by these other metal ions in their electrophoretic mobility and the number of metal ions bound. It was 1 Supported by Grant AM 20207-04. ’ To whom correspondence should be addressed. 0003-9861/82/030080-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
80
POLYMORPHIC
FORMS OF METALLOTHIONEIN
lular Zn transfer. In the present study we have used this transfer reaction with apocarbonic anhydrase as an assay system to study the diversity between metallothionein isoforms. We report that the two forms of metallothionein appear to differ in their intrinsic Zn-binding affinity. This variation in the isometallothioneins may correlate with the difference in turnover of the two forms observed by Suzuki and Yamamura. MATERIALS
AND METHODS
Cd,Zn-thionein, Zn-thionein, and Cu-thionein were purified from the livers of rats injected subcutanteously with CdClz (3 mg Cd/kg body wt), ZnSOl (8 mg Zn/kg), or CuS04 (2.5 mg Cu/kg), respectively. The animals were Sprague-Dawley males from Simmonsen. The preparation of the metallothioneins was performed as described previously (14) with NP-saturated buffers. DEAE-cellulose chromatography of Cd,Zn-thionein or Zn-thionein was carried out in 10 mM Tris-Cl, pH 8.6, using an elution gradient of 0 to 25 mM NaCI. Elution of Cu-thionein was accomplished with a gradient of 2.5 to 25 mM potassium phosphate, pH ‘7. Metallothionein fractions located by metal analysis were concentrated by lyophilization and desalted by Sephadex G-25 column chromatography. Purity of the protein samples was assessed by polyacrylamide gel electrophoresis and amino acid analysis. Protein concentrations were determined by quantitative amino acid analysis on a Beckman 120 C analyzer. Metal analysis was carried out on a Perkin-Elmer Model 303 spectrometer. Sulfhydryl titrations were done with 2,2’-dithiodipyridine (Pys$ purchased from Sigma in 0.2 M sodium acetate, pH 4, containing 1% sodium dodecyl sulfate and 1 mM EDTA at 25°C. The final Psy, concentration was 128 PM, whereas metallothionein concentrations ranged from 1.5 to 2.5 pM. The absorbance at 343 nm was recorded after 60 min of incubation, and calculations were performed using a molar extinction coefficient of 7.06 X lo3 (23). Analytical gel filtration was done on a Sephadex G-75 superfine column (2.5 X 110 cm) in 50 mM potassium phosphate, pH 7.8, at 25°C. Protein standards and metallothionein samples were chromatographed in varying combinations. Blue Dextran and NaCl were added to mark the excluded and included volumes, respectively. Thionein samples were prepared for amino acid 3 Abbreviations used: Pys,, 2,2’-dithiodipyridine; Th, thionein; CA, carbonic anhydrase. (the two polymorphic forms of metallothionein (M-Th) are referred to as isoforms I and II); HPLC, high-performance liquid chromatography.
81
sequencing by CNBr digestion followed by carboxymethylation. Five milligrams of CNBr was added to 150-200 nmol of thionein in 70% formic acid. The mixtures were incubated for about 60 h at 25°C in the dark. The samples were then diluted and dried by lyophilization. Carboxymethylation in 6 M guanidine hydrochloride and 0.2 M Tris-Cl, pH 8.6, containing 2 mM EDTA, was performed according to Hirs (24). The carboxymethylated thioneins were desalted by gel filtration in 5 mM HCI, dried, and loaded in the sequencer cup with 50% acetic acid. Automated Edman degradations were carried out with a Beckman 89OB sequencer using a 0.1 M Quadrol program (12078). Polybrene was included in each sequencer run, and five pretreatment cycles were run in the presence of the dipeptide lysyl-glycine prior to loading thionein (25). Fractions recovered from each cycle were automatically converted to phenylthiohydantoins with a Sequemat P-6 converter. Phenylthiohydantoins were analyzed by HPLC using a Waters SISP-710A instrument and certain assignments were confirmed by amino acid analysis after back hydrolysis in 5.7 N HCl containing 0.1% SnC&
(26). Carbonic anhydrase was purified from bovine blood according to the procedure of Lindskog (27). The assay employed was the esterase activity toward p-nitrophenylacetate (2 mM final concentration) in 50 mM Tris-Cl, pH 7.5, containing 0.5 mM EDTA (28). Zinc was removed by dialysis of carbonic anhydrase in 0.2 M potassium phosphate, pH 7, containing 50 mM dipicolinic acid for 2 days followed by a 24-h dialysis in only the buffer (29). The resulting sample was 96% apo as determined by Zn analysis and enzymatic activity measurements. Protein concentrations were calculated from the molar extinction coefficient at 280 nm of 5.6 X lo4 (27). The apoprotein could be readily reactivated with ZnSO( as reported (29-31). All protein samples were stored at -80°C. The reaction of metallothionein with EDTA (Sigma) was monitored spectrophotometrically at 215 nm where Zn-thionein exhibits an absorption maximum (32). The reaction mixture contained concentrations of metallothionein ranging from 7 to 10 PM and 0.1 to 1 mM EDTA in 0.1 M Tris-Cl, pH 7.5. After various periods of incubation, the mixtures were chromatographed on Sephadex G-25 (1 X 26 cm) to separate Zn-thionein from Zn-EDTA. The elution buffer was 0.01 M Tris-Cl, pH 7.5. Equilibrium dialysis of metallothionein with EDTA was carried out in 0.1 M Tris-Cl, pH 7.5, at 25°C with a Spectrapor 3 dialysis membrane separating the two compartments. Equal volumes of Cd,Zn-thionein (25 PM Zn) and EDTA (50 pM) were initially in the separated compartments. The chambers were filled with liquid and sealed to exclude air. The partition of EDTA assayed as [14C]EDTA across the membrane was completed within 38 h.
82
WINGE
AND MIKLOSSY
RESULTS
Characterization Metallothionein
of the Isoform
of
Ion-exchange chromatography was used to resolve the two major polymorphic forms of Zn-thionein and of Cd,Zn-thionein. The same forms exist regardless of whether Cd or Zn is used as the inducing metal ion (5). Metallothionein-I eluted from DEAE-cellulose at a NaCl concentration of 4.5 mM, whereas form II eluted at a salt concentration of 15.5 mM. The metal ion content of the two polymorphic forms was similar. Both Cd,Zn-thionein I and II averaged 5 g atoms Cd/mol protein and 2 g atoms Zn/mol. Zn-thionein I and II contained over 5 g atoms Zn/mol. The lower metal content of the Zn-thioneins was a result of slight contaminants present in the sample. Rechromatography of such a sample on DEAE-cellulose gave a Zn-thionein with 6.0 to 6.5 g atoms Zn bound/m01 protein. The Cu content of the two Zn-thionein forms was less than 0.3 g atoms/mol, whereas that of the two Cd,Zn-thionein forms was less than 0.1 g atom Cu/mol. The polymorphic forms of both Zn-thionein and Cd,Zn-thionein had molar ratios of titratable sulfhydryls to metal ions of 2.8 to 3.1. The number of reduced thiols per molecule was between 18 to 20. The isoforms were chromatographed on a gel filtration column calibrated with proteins of known Stokes radii. Cd,Zn-thionein- eluted with a Kd of 0.55 f .003 (&SD, n = 5) which corresponded to a Stokes radius of 16.2 A, whereas form II eluted with a Kd of 0.535 f 0.005 (n = 4) which corresponded to a Stokes radius of 1’7 A. Chromatography of mixtures of Cd,Zn-thionein I and II with one isoform labeled with [14C]glycine resulted in separation of the two forms according to their Stokes radii. Since the two isoforms have the same number of amino acids (12), the difference in Stokes radius suggests that the two forms are slightly dissimilar in tertiary conformation. Cu-thionein was also resolved into two forms by ion-exchange chromatography, but the elution positions differed from
those of the two Cd,Zn-thioneins. Under the same conditions used for separation of the two isoforms of Cu-thionein, Cu-Th II eluted at a potassium phosphate concentration of 6 mM, whereas Cd,Zn-Th II eluted at a concentration of 4 mM. On analytical gel filtration, Cu-Th II exhibited an apparent Stokes radius of 16.2 A compared to the Stokes radius of 17 A for Cd,Zn-Th II. As described previously (14), Cu-thionein bound 10 g atoms Cu/mol protein. Since the metal ion content and ionexchange elution position of Cu-Th II differed from those of Cd,Zn-Th II, the question arose whether the Cu-thionein polypeptides were dissimilar from the Cd,Znthionein polypeptides. To resolve this question, the two isoforms of Cd,Zn-Th and Cu-Th II were digested with CNBr and subjected to automated Edman degradation. Kojima et al. (12) showed that CNBr digestion in formic acid released the acetylated N-terminal methionine. The remainder of the 61 amino acid polypeptide is therefore free to react with phenylisothiocyanate. Degradations were continued for 25 cycles. The sequences of Cd,Zn-Th II and Cu-Th II were found to be the same, whereas there were differences between Cd,Zn-Th isoforms I and II (Table I). The data suggest that the differing Stokes radii and chromatographic behavior did not reflect differing primary structures but instead another property associated with the different metals bound. There appear to be only two major polymorphic forms of thionein rat liver regardless of the metal ion used to induce the protein. Therefore, in our studies on the significance of the polymorphism, we chose to use the isoforms of Zn-Th and Cd,Zn-Th as models of thionein polymorphism. Metallothionein-dependent Reconstitution of Apo-Carbonic Anhydrase
Since the two isoforms of Cd,Zn-Th bind the same number of metal ions but fold in differing tertiary conformations, it is conceivable that the geometry of the metal binding sites of the isoforms may differ. One approach to investigate Zn binding in metallothionein is to test the ability of the
POLYMORPHIC
83
FORMS OF METALLOTHIONEIN TABLE
I
AUTOMATED SEQUENCE ANALYSIS OF ISOFORMS OF METALLOTHIONEIN Cd,Zn-Th Cycle
Residue
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
ASP Pro Asn CYS Ser CYS Ser Thr GUY GUY Ser CYS Thr CYS Ser Thr Ser CYS GUY CYS LYS Asn CYS LYS CYS
Cd,Zn-Th
I Yield 52 63 54 44 14 36 11 8 30 88 10 34 29 26 5 14 6 39 45 43 41 18 27 30 28
Cu-Th II
II
Residue
Yield
Residue
Yield
ASP Pro Asn CYS Ser CYS Ala Thr ASP GUY Ser CYS Ser CYS Ala GUY Ser CYS LYS CYS LYS Gln CYS LYS CYS
108 115 63 53 28 68 90 33 41 63 7 36 10 41 54 30 5 34 33 27 51 23 24 42 24
ASP Pro Asn CYS Ser CYS Ala Thr ASP GUY Ser CYS Ser CYS Ala GUY Ser CYS LYS CYS LYS Gln CYS LYS CYS
25 26 16 11 7 12 23 8 25 24 5 11 4 11 14 13 3 9 9 6 5 3 3 6 4
Note. Each sequence was repeated twice with carboxymethylated or carboxamidomethylated cyanogen bromide-treated thionein. PTH-amino acids were identified and quantitated by HPLC analysis. For each thionein isoform, first cycle yields of the samples in the polybrene-treated cup were about 20% of the quantities loaded.
isoforms to reactivate apocarbonic anhy- tween preparations, but this was not a drase (CA). The isoforms of Cu-thionein result of partially oxidized thionein molwere not included since anaerobically pre- ecule, since all samples had the same numpared Cu-Th does not reactivate apome- ber of titratable sulfhydryls per metal ion. talloenzymes (33). The reaction system in- The kinetics of the reconstitution of apovolved incubation of 6.6 PM of apo-CA with CA by the two forms of Zn-thionein (sammetallothionein in 0.1 M Tris-Cl, pH 7.5. ple 0126) at three different thionein conThe reconstitution observed with 5 eq centrations are shown in Fig. 1. The conZnSOJmol apo-CA was defined as 100%. trast in the ability of the two isoforms to Results shown in Table II with metalloreconstitute apo-CA is apparent throughthionein as the Zn donor are expressed as out the time of the three experiments. In the percentages of reactivation seen with similar kinetic experiments, a second ZnSO, after a 60-min incubation. Isoform preparation of Zn-thionein I (sample 0204) I of metallothionein invariably reacti- and Cd,Zn-thionein I from several prepavated apo-CA to a greater extent than did rations, restored greater esterase activity isoform II. This was true with both Zn-Th than did their corresponding isoforms II and Cd,Zn-Th. Diversity was apparent be- at all time points. Zn-thionein samples
84
WINGE TABLE
AND MIKLOSSY
II
RECONSTITUTION OF APO-CARBONIC ANHYDRASE Reconstitution of apo-CA (% ) Zn-Th/CA ratio
Sample Cd, Zn-Th 0807 Cd, Zn-Th 0121 Zn-Th 1112 Zn-Th 1119 Zn-Th 1106 Zn-Th 0106 Zn-Th 0204 Zn-Th 0126
I
II
5 5 1 2 1 5 2
28 25 86 105 83 72 28
14 13 30 63 30 41 21
2 5
29 85
15 42
J 8
Note. The reconstitution assay was performed as described in the text. The percentage reactivation by various preparations of metallothionein after a 60min incubation with apo-CA is listed. The concentration of metallothionein varied from equimolar concentrations of Zn as Zn-thionein and apo-CA to a fivefold molar Zn excess.
Ix
60. 40-
0
IO
20
30 TIME
showed greater variation than did Cd,Znthionein. This may occur due to a greater instability of Zn-thionein compared to Cd,Zn-thionein or to a variation in the occupancy of the metal-binding sites. The significant observation is that within a given preparation in which the isoforms of Zn-Th or Cd,Zn-Th were exposed to identical conditions, the two isoforms show dissimilar reactivities toward apo-CA. In the incubations of Cd,Zn-Th with apo-CA, no Cd was transferred to the enzyme, since additions of ZnSOl to the mixture after the incubation period reactivated apo-CA completely. Any Cd binding by apo-CA would prevent full reactivation. Reactivity
of Metallothionein
with EDTA
Since polymorphic form I of metallothionein reconstituted apo-CA more rapidly than did form II, we asked whether its Zn ions were also more readily removed with EDTA. Li et al. (22) showed that under pseudo-first-order conditions for EDTA, the reaction could be followed spectrophotometrically at 215 nm. The second part of the triphasic reaction was
40
50
60
70
(MIN.)
FIG. 1. Reactivation of apo-carbonic anhydrase by Zn-thionein. APO-CA (6.6 GM) in 0.1 M Tris-Cl, pH 7.5, was incubated with different concentrations of Zn-thionein (sample 0126) isoforms I (0) and II (A). Concentrations used include 1 eq Zn/mol apoCA, twofold and fivefold molar excess.
monitored in the first 20 min because the initial phase of the reaction was already completed during mixing. In the second phase of the reaction, isoform I reacted more rapidly with EDTA than did isoform II. This was true with Zn-thioneins (Fig. 2) and Cd,Zn-thioneins (Fig. 3). Psuedofirst-order rate constants were calculated from the plots (Table III). Differences seen between metallothionein preparations in the reconstitution of e&erase activity are also apparent in the reactivity with EDTA. At intervals, samples of the mixtures of metallothionein and EDTA were subjected to gel filtration on Sephadex G-25 to separate the two components. Fractions were analyzed for Zn ion concentration. Metallothionein eluted in the excluded volume of the column, whereas Zn-EDTA eluted in the internal volume (Fig. 4). Results for several preparations show that the greater
85
POLYMORPHIC FORMS OF METALLOTHIONEIN 0.5
Cd, Zn-Th
(08151
Cd,Zn-Th
(Ol2l)
0.3I
04
FIG. 2. Kinetics of the reactivity
of Zn-thionein with EDTA. Zn-thionein isoforms I (0) and II (A) of two preparations, sample 0126 (---) and sample 0204 (- - -), were incubated at 25°C in 0.1 M Tris-Cl, pH 7.5, containing 0.2 mM EDTA. The Zn concentration was 46 pM. The reaction was monitored at 215
rate of loss of Zn by isoform I compared to isoform II correlated with a greater retention of Zn by isoform II on gel filtration (Table III). Results with Cd,Zn-Th are more reproducible than those with Zn-Th as was observed in the reconstitution studies with apo-CA. The difference between isoforms of Zn-thionein (sample 0204), which was slight in the spectrophotometric data, was greater in the gel filtration experiments. In experiments with Cd,Znthionein, there was little Cd exchange between thionein and EDTA. This was demonstrated by observing only a slight absorbance change at 254 nm which is the wavelength maximum of Cd-mercaptides (32) and, secondly, by recovering over 85% of the Cd in an incubation as Cd-thionein (Fig. 5). To determine whether the isoform variation in reactivity with apo-carbonic anhydrase and EDTA was due to thermodynamic differences in Zn-binding affinity, Cd,Zn-thionein I and II were placed in sealed equilibrium dialysis cells for a com-
1
I
0 2 4
,
6
8
1
I
IO
12
I
14
I
I
I
I6
18
20
8
1
22 24
MINUTES
FIG. 3. Kinetics of the reactivity of Cd,Zn-thionein with EDTA. Cd,Zn-Th isoforms I (0) and II (A) of two preparations, sample 0815 and sample 0121,were incubated as described in the legend of Fig. 2. The Zn concentration was 18 pM. The reaction was monitored at 215 nm.
petition binding experiment with EDTA. The opposite cell chambers contained EDTA at a twofold molar excess relative to the Zn concentration of metallothionein. After 4 days of dialysis at 25”C, metal analysis was performed to determine the metal ion distribution in the membrane-separated chambers. Over 95% of the Cd remained associated with thionein with both isoforms I and II. Whereas only 12% of the Zn remained associated with isoform I, 38% of the Zn was bound to isoform II after the 4 days. The observed difference in Zn-binding affinity was substantiated by qualitatively similar results in an equilibrium dialysis of a second preparation of Cd,Zn-Th. DISCUSSION
It is well established that two major polymorphic forms of metallothionein exist in various tissues and cell lines, and that the isoforms differ to a limited extent
86
WINGE
AND MIKLOSSY TABLE
III
REACTIVITY OF Zn-THIONEIN ISOFORMS WITH EDTA k observed (X104 s’)
EDTA concentration (mM)
Sample Zn-Th 0126 Zn-Th 0204 Cd, Zn-Th 0815 Cd, Zn-Th 0121 Cd, Zn-Th 0729 Cd, Zn-Th 0522
Percentage
of Zn as Zn-Th
I
II
min
I
II
15 10 15 -
45 11.7 25.5 -
71 33.7 53.1
15 15 60
9.6 27.8 3.7
43.2 51.7 21.1
0.2 1.0 0.2 0.2
3.9
1.92
4.0 1.9
3.5 0.92
0.5 0.2 0.5 0.2 0.2
1.69 2.2 2.9
0.88 .73 1.2
Note. The pseudo-first-order rate constants were calculated from spectrophotometric data as described in the text. The percentages of Zn recovered in the Zn-thionein fraction after gel filtration of mixtures of the protein and EDTA incubated for the specified times are listed.
in their primary structure (13). Both forms are inducible by metal ions, although the extent of induction of the isoforms can vary. The ratio of form I to form II in the livers of adult rats induced with Zn2+ about 24 h prior to killing has been shown to vary from about 0.5 (5, 34) to about 1.5 (35). In the livers of partially hepatectomized adult rats, Ohtake and Koga (10) reported that only isoform II of Zn-Th was present. Thionein molecules induced by Cu2+also can be resolved into polymorphic forms (6, 14). Although these forms are
IO
12
14
I6 FRACTION
18
20
22
distinct from the isoforms of Cd,Zn-Th in metal content and their charge-to-mass ratio, we have shown by limited amino acid sequence analysis that isoform II of the thionein molecules induced by Cd and Cu are identical. The sequence of the first 25 amino acids of isoform II is identical to that reported by Kissling et aZ. (36). Although our sequence data is limited to only the NH2-terminal 25 residues, heterogeneity in M-Th is most pronounced at
24
NUMBER
FIG. 4. Gel filtration of the reaction mixture of Znthionein and EDTA. Zn-thionein isoform I (0) and II (A) of sample 0126 were incubated with EDTA as described in Fig. 2. After an incubation period of 15 min, the mixture was chromatographed on Sephadex G-25. The void volume of the column corresponded to fractions 12-15, whereas the included column voiume corresponded to fractions 17-21.
IO
I2
I4
I6 FRACTION
18
20
22
24
NUMBER
FIG. 5. Gel filtration of the reaction mixture of Cd,Zn-thionein with EDTA. Cd,Zn-Th isoform I (sample 0121) was incubated with 1 mM EDTA in 0.1 M Tris-Cl, pH 7.5, at 25°C. After 10 min, the mixture was chromatographed on Sephadex G-25. Each fraction was monitored for Cd (0) and Zn (A).
POLYMORPHIC
FORMS OF METALLOTHIONEIN
the NHz-terminal end. In equine renal MTh I and II, 4 of the 7 differences are clustered in the first 25 residues (13), whereas in mouse M-Th I and II 9 of the 14 differences are likewise located in the NHz-terminal 25 residues (37). So although the comparison between Cd-Th and Cd,Zn-Th was based on limited sequence analysis, the results suggest that isoform II of the two metallotheoneins are identical. It is, however, conceivable that a difference may exist in the COOH-terminal portion. Isoforms I and II of metallothionein are dissimilar in primary and tertiary structure shown by amino acid sequencing and Stokes radius determination. We were interested to determine whether these differences affect the metal-binding sites which may be of significance in the physiological functioning of metallothionein. Since the metabolic function of the protein has not been unequivically established, studies addressing differences between the isoforms are difficult to interpret. In the process of investigating the metallothionein-dependent reconstitution of apometalloenzymes, we observed that the two polymorphic forms of Zn-Th vary in their ability to reactivate apo-CA. Isoform I invariably restored greater esterase activity to apo-CA than did isoform II. This kinetic variation was also apparent when the reactivity of metallothionein with EDTA was studied. EDTA removed Zn from Znthionein isoform I at a greater rate compared to isoform II. These observations are true for Zn-Th as well as Cd,Zn-Th. Equilibrium dialysis of isoforms of Cd,ZnTh suggested that the Zn-binding affinity was less in isoform I compared to form II. In the Cd,Zn-Th molecules, Cd reacts only slowly with EDTA and is not transferred to apo-CA. The slow reactivity of Cd-Th with EDTA is well known (22, 32). The observed differences in reactivity with apo-CA and EDTA between the isoforms of Cd,Zn-Th are reproducible, but variations between preparations of Zn-Th were observed. The results with Cd,Zn-Th are very clear in indicating that the apparent Zn-binding affinities of the two isoforms differ. Both isoforms of Cd,Zn-Th had similar metal ion compositions. The
87
significance of the diversity in Zn-Th preparations remains obscure. It is conceivable that a cellular component exists that can modulate Zn-binding affinity or that variation occurred due to instability of Zn-Th. Further studies concerning those questions are in progress. Although the diversity in preparations of Zn-Th makes a comparison of isoforms from different preparations difficult, the relative difference between the two isoforms from the same purification is significant; since, within the same preparation, the two isoforms were exposed to identical conditions. Suzuki and Yamamura (18-20) have reported that the relative ratios of the two polymorphic forms of metallothionein induced with Zn,Cd or Cu change with time. In their experiments with Zn-Th, isoform II was always the most abundant form. In the time span from 6 h to 4 days, the ratio of isoform I to isoform II decreased. This ratio for the isoforms of Cd-induced thionein changes from approximately unity at 6 h to lower values up to 2 days and then the ratio increased above unity for times up to 7 days, The variation observed in isoform ratios may be related to a dissimilar, pattern of biosynthetic or degradation rates. Alternatively, it is conceivable that the two forms differ in metabolic functions, and that changes in the isoform ratio may be related to rates of reaction. The different Zn-binding affinities of the isoforms reported presently may be of significance in such physiological variations. ACKNOWLEDGMENTS The authors wish to thank Dr. G. R. Lee and Dr. R. E. Lynch for their support and discussions. REFERENCES 1. MARGOSHES, M., AND VALLEE, B. L. (1957) J. Amer. Chem. Sot. 79, 4813-4814. 2. NORDBERG, G. F., NORDBERG, M., PISCATOR, M., AND VESTERBERG, 0. (1972) B&hem, J. 126, 491-498. 3. SHAIKH, Z. A., AND LUCIS, 0. J. (1972) A&. Envirmz Health 24, 419-425. 4. WESER, U., RUPP, H., DONAY, F., LINNEMANN, F., VOELTER, W., VOETSCH, W., AND JUNG, G. (1973) Eur. J Biochem. 39, 127-140.
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WINGE
AND MIKLOSSY
5. WINGE, D. R., PREMAKUMAR, R., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 242-252. 6. BREMNER, I., AND YOUNG, B. W. (1976) B&hem J. 155, 631-635. 7. BREMNER, I., AND YOLJNG, B. W. (1976) Biochem .J. 157.517-520. 8. TSUNOO, H., KINO, K., NAKAJIMA, H., HATA, A., HUANG, I. Y., AND YOSHIDA, A. (1978) J. Biol. Chem 253, 4172-4174. 9. HIDALGO, H. A., KOPPA, V., AND BRYAN, S. E. (1978) Biochem J. 170,219-225. 10. OHTAKE, H., AND KOGA, M. (1979) B&hem. J. 183,683-690. 11. ZELAZOWSKI, A. J., AND PIOTROWSKI, J. K. (1980)
Biochim Biophys. Acta 625, 89-99. 12. KOJIMA, Y., BERGER, C., VALLEE, B. L., AND KAGI, J. H. R. (1976) Proc. Nat. Acad. Sci USA 73, 3413-3417. 13. KOJIMA, Y., AND KAGI, J. H. R. (1978) Trends
23. 24.
25. 26.
27. 28. 29. 30.
Biochem Sci 3,90-93. 14. WINGE, D. R., GELLER, B. L., AND GARVEY, J. (1981) Arch Biochem Biqphys. 208, 160-166. 15. OTVOS, J. D., AND ARMITAGE, I. M. (1979) J. Amer. Chem. Sot 101, 7734-7736. 16. OTVOS, J. D., AND ARMITAGE, I. M. (1980) Proc.
Nat. Acad. Sti USA 77,7094-7098.
-.. VASAK, M. (1980) J. Amer. Chem Sot. 102,3953-
17
3955. 18. SUZUKI, K. T., AND YAMAMURA, M. (1980) Toxicol. L&t. 6, 59-65. 19. SUZUKI, K. T., AND YAMAMURA, M. (1980) Biochem. Pharmacol. 29, 2407-2412. 20. SUZUKI, K. T., AND YAMAMURA, M. (1980) To&cot. Z.&t. 6,301-307. 21. UDOM, A. O., AND BRADY, F. 0. (1980) Biochem. J. 187, 329-335. 22. LI, T. Y., KRAKER, A. J., SHAW, C. F., AND PE-
31.
TERING, D. H. (1980) Proc. Nat. Acud Sci. USA 77, 6334-6338. GRASSETI, D. R., AND MURRAY, J. J., JR. (1967) Arch. Biochem Biophys. 119,41-49. HIRS, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 199-203, Academic Press, New York. HUNKAPILLER, M. W., AND HOOD, L. E. (1978) Biochemistry 17,2124-2133. LAI, C. Y. (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S., eds.), Vol. 47, pp. 369-373, Academic Press, New York. LINKSKOG, S. (1960) B&him. Biophys. Acta 39, 218-226. POCKER, Y., AND STONE, J. T. (1967) Biochemistry 6, 668-678. HUNT, J. B., RHEE, M. J., AND STORM, C. B. (1977) Anal. B&hem. 79, 614-617. LINDSKOG, S., HENDERSON, L. E., KANNAN, K., LILJAS, A., NYMAN, P. O., AND STRANDBERG, B. (1971) Enzymes 5, 587-665. LINDSKOG, S., AND MALMSTROM, B. G. (1962) J.
Biol. Chem 237,1129-1137. 32. KAGI, J. H. R., AND VALLEE, B. L. (1961) J. Biol
Chem 236,2435-2442. 33. GELLER, B. L., AND WINGE, D. R. (1981) Achiw. B&hem. Biophys., in press. 34. BREMNER, I., AND DAVIES, N. T. (1975) Biochem. J. 149, 733-738. 35. WONG, K. L., AND KLAASSEN, C. D. (1979) J. BioL Chem. 254, 12,399-12,403. 36. KISSLING, M. M., BERGER, C., KAGI, J. H. R., ANDERSON, R. D., AND WESER, U. (1979) Ezperientia Suppl. 34,181-185. 37. HUANG, I. Y., KIMURA, M., HATA, A., TSUNOO, H., AND YOSHIDA, A. (1981) J. Biochem 89,18391845.
Differences
in the Polymorphic
DENNIS
Forms
R. WINGE2 AND KATHY-ANNE
University of Utah Medical Center, Salt
Luke
of Metallothionein’ MIKLOSSY City, Utah 84132
Received on July 20, 1981, and in revised form October 16, 1981 Metallothionein induced in rat liver by metal ions can be resolved into two forms, referred to as isoforms I and II. These polymorphic forms differ in several properties. Sequence analysis of the CNBr-digested thionein isoforms revealed seven differences in the first 25 amino acids between isoforms I and II of Cd-induced thionein. The sequence of the first 25 amino acids in isoform II of Cu-induced thionein was identical to that of isoform II of Cd-induced thionein, suggesting that the same polymorphic forms are induced with different metal ions. Isoform II of Cd,Zn-thionein exhibited a reproducibly greater Stokes radius (1’7 A) by gel filtration compared to isoform I (16.2 A), whereas isoform II of Cu-thionein eluted with a Stokes radius of 16.2 A. The isoforms also differ in Zn-binding affinity. Isoform I of several preparations of Cd,Zn-thionein and Zn-thionein invariably reconstituted greater esterase activity in apo-carbonic anhydrase than to apodid isoform II. In Cd,Zn-thionein samples, only the Zn ‘+ ions were transferred carbonic anhydrase. Similarily, the Zn ‘+ ions in isoform I were more reactive with EDTA compared to isoform II. The greater reactivity of isoform I with EDTA was evident kinetically from spectrophotometric assays as well as thermodynamically from equilibrium dialysis experiments. The significance of these differences is unclear, but it is conceivable that the dissimilarities in the conformational and Zn-binding affinity of rat liver metallothionein may correlate with a functional distinction.
unclear whether the Cu-thionein isoforms differed structurally from the Cd,Zn-thionein isoforms. The significance of the polymorphism remains unknown. Recently, Suzuki and Yamamura (18-20) observed that the ratio of the two isometallothioneins changed with time after injection of the inducing metal ion regardless of whether the inducing ion was Cd, Zn, or Cu. These results suggest that the turnover of the two forms may be dissimilar which raises the possibility that they may also differ functionally. In the course of investigating the extent and significance of the thionein polymorphism, we observed that the two isomeric forms of Zn-thionein vary in their ability to reconstitute apo-carbonic anhydrase. Zn ion transfer from Zn-thionein to apometalloenzymes has been recently reported by Udom and Brady (21) and Li et al. (22). Their results have indicated that metallothionein may function in intracel-
Metallothionein is a protein of unknown function that was first described in 1957 by Margoshes and Vallee (1). The protein is capable of being induced in cultured cells or tissues by administration of various metal ions, e.g., Zn2+, Cd2+, and Cu2+ (2-11). Two major variants of equine metallothionein have been characterized that differ in sequence in 7 out of the 61 total amino acids (12, 13). Each structurally distinct polymorphic form of Cd,Zn-thionein can bind 7 metal ions (usually 5 Cd ions and 2 Zn ions), whereas the Cu-thionein isoforms bind 10 Cu ions (14). The metal ions are ligated in cysteine-metal ion clusters (15-17). The isoforms induced by Cd, Zn, Hg, and Ag appear to be similar, whereas the forms induced by Cu differ from those induced by these other metal ions in their electrophoretic mobility and the number of metal ions bound. It was 1 Supported by Grant AM 20207-04. ’ To whom correspondence should be addressed. 0003-9861/82/030080-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
80
POLYMORPHIC
FORMS OF METALLOTHIONEIN
lular Zn transfer. In the present study we have used this transfer reaction with apocarbonic anhydrase as an assay system to study the diversity between metallothionein isoforms. We report that the two forms of metallothionein appear to differ in their intrinsic Zn-binding affinity. This variation in the isometallothioneins may correlate with the difference in turnover of the two forms observed by Suzuki and Yamamura. MATERIALS
AND METHODS
Cd,Zn-thionein, Zn-thionein, and Cu-thionein were purified from the livers of rats injected subcutanteously with CdClz (3 mg Cd/kg body wt), ZnSOl (8 mg Zn/kg), or CuS04 (2.5 mg Cu/kg), respectively. The animals were Sprague-Dawley males from Simmonsen. The preparation of the metallothioneins was performed as described previously (14) with NP-saturated buffers. DEAE-cellulose chromatography of Cd,Zn-thionein or Zn-thionein was carried out in 10 mM Tris-Cl, pH 8.6, using an elution gradient of 0 to 25 mM NaCI. Elution of Cu-thionein was accomplished with a gradient of 2.5 to 25 mM potassium phosphate, pH ‘7. Metallothionein fractions located by metal analysis were concentrated by lyophilization and desalted by Sephadex G-25 column chromatography. Purity of the protein samples was assessed by polyacrylamide gel electrophoresis and amino acid analysis. Protein concentrations were determined by quantitative amino acid analysis on a Beckman 120 C analyzer. Metal analysis was carried out on a Perkin-Elmer Model 303 spectrometer. Sulfhydryl titrations were done with 2,2’-dithiodipyridine (Pys$ purchased from Sigma in 0.2 M sodium acetate, pH 4, containing 1% sodium dodecyl sulfate and 1 mM EDTA at 25°C. The final Psy, concentration was 128 PM, whereas metallothionein concentrations ranged from 1.5 to 2.5 pM. The absorbance at 343 nm was recorded after 60 min of incubation, and calculations were performed using a molar extinction coefficient of 7.06 X lo3 (23). Analytical gel filtration was done on a Sephadex G-75 superfine column (2.5 X 110 cm) in 50 mM potassium phosphate, pH 7.8, at 25°C. Protein standards and metallothionein samples were chromatographed in varying combinations. Blue Dextran and NaCl were added to mark the excluded and included volumes, respectively. Thionein samples were prepared for amino acid 3 Abbreviations used: Pys,, 2,2’-dithiodipyridine; Th, thionein; CA, carbonic anhydrase. (the two polymorphic forms of metallothionein (M-Th) are referred to as isoforms I and II); HPLC, high-performance liquid chromatography.
81
sequencing by CNBr digestion followed by carboxymethylation. Five milligrams of CNBr was added to 150-200 nmol of thionein in 70% formic acid. The mixtures were incubated for about 60 h at 25°C in the dark. The samples were then diluted and dried by lyophilization. Carboxymethylation in 6 M guanidine hydrochloride and 0.2 M Tris-Cl, pH 8.6, containing 2 mM EDTA, was performed according to Hirs (24). The carboxymethylated thioneins were desalted by gel filtration in 5 mM HCI, dried, and loaded in the sequencer cup with 50% acetic acid. Automated Edman degradations were carried out with a Beckman 89OB sequencer using a 0.1 M Quadrol program (12078). Polybrene was included in each sequencer run, and five pretreatment cycles were run in the presence of the dipeptide lysyl-glycine prior to loading thionein (25). Fractions recovered from each cycle were automatically converted to phenylthiohydantoins with a Sequemat P-6 converter. Phenylthiohydantoins were analyzed by HPLC using a Waters SISP-710A instrument and certain assignments were confirmed by amino acid analysis after back hydrolysis in 5.7 N HCl containing 0.1% SnC&
(26). Carbonic anhydrase was purified from bovine blood according to the procedure of Lindskog (27). The assay employed was the esterase activity toward p-nitrophenylacetate (2 mM final concentration) in 50 mM Tris-Cl, pH 7.5, containing 0.5 mM EDTA (28). Zinc was removed by dialysis of carbonic anhydrase in 0.2 M potassium phosphate, pH 7, containing 50 mM dipicolinic acid for 2 days followed by a 24-h dialysis in only the buffer (29). The resulting sample was 96% apo as determined by Zn analysis and enzymatic activity measurements. Protein concentrations were calculated from the molar extinction coefficient at 280 nm of 5.6 X lo4 (27). The apoprotein could be readily reactivated with ZnSO( as reported (29-31). All protein samples were stored at -80°C. The reaction of metallothionein with EDTA (Sigma) was monitored spectrophotometrically at 215 nm where Zn-thionein exhibits an absorption maximum (32). The reaction mixture contained concentrations of metallothionein ranging from 7 to 10 PM and 0.1 to 1 mM EDTA in 0.1 M Tris-Cl, pH 7.5. After various periods of incubation, the mixtures were chromatographed on Sephadex G-25 (1 X 26 cm) to separate Zn-thionein from Zn-EDTA. The elution buffer was 0.01 M Tris-Cl, pH 7.5. Equilibrium dialysis of metallothionein with EDTA was carried out in 0.1 M Tris-Cl, pH 7.5, at 25°C with a Spectrapor 3 dialysis membrane separating the two compartments. Equal volumes of Cd,Zn-thionein (25 PM Zn) and EDTA (50 pM) were initially in the separated compartments. The chambers were filled with liquid and sealed to exclude air. The partition of EDTA assayed as [14C]EDTA across the membrane was completed within 38 h.
82
WINGE
AND MIKLOSSY
RESULTS
Characterization Metallothionein
of the Isoform
of
Ion-exchange chromatography was used to resolve the two major polymorphic forms of Zn-thionein and of Cd,Zn-thionein. The same forms exist regardless of whether Cd or Zn is used as the inducing metal ion (5). Metallothionein-I eluted from DEAE-cellulose at a NaCl concentration of 4.5 mM, whereas form II eluted at a salt concentration of 15.5 mM. The metal ion content of the two polymorphic forms was similar. Both Cd,Zn-thionein I and II averaged 5 g atoms Cd/mol protein and 2 g atoms Zn/mol. Zn-thionein I and II contained over 5 g atoms Zn/mol. The lower metal content of the Zn-thioneins was a result of slight contaminants present in the sample. Rechromatography of such a sample on DEAE-cellulose gave a Zn-thionein with 6.0 to 6.5 g atoms Zn bound/m01 protein. The Cu content of the two Zn-thionein forms was less than 0.3 g atoms/mol, whereas that of the two Cd,Zn-thionein forms was less than 0.1 g atom Cu/mol. The polymorphic forms of both Zn-thionein and Cd,Zn-thionein had molar ratios of titratable sulfhydryls to metal ions of 2.8 to 3.1. The number of reduced thiols per molecule was between 18 to 20. The isoforms were chromatographed on a gel filtration column calibrated with proteins of known Stokes radii. Cd,Zn-thionein- eluted with a Kd of 0.55 f .003 (&SD, n = 5) which corresponded to a Stokes radius of 16.2 A, whereas form II eluted with a Kd of 0.535 f 0.005 (n = 4) which corresponded to a Stokes radius of 1’7 A. Chromatography of mixtures of Cd,Zn-thionein I and II with one isoform labeled with [14C]glycine resulted in separation of the two forms according to their Stokes radii. Since the two isoforms have the same number of amino acids (12), the difference in Stokes radius suggests that the two forms are slightly dissimilar in tertiary conformation. Cu-thionein was also resolved into two forms by ion-exchange chromatography, but the elution positions differed from
those of the two Cd,Zn-thioneins. Under the same conditions used for separation of the two isoforms of Cu-thionein, Cu-Th II eluted at a potassium phosphate concentration of 6 mM, whereas Cd,Zn-Th II eluted at a concentration of 4 mM. On analytical gel filtration, Cu-Th II exhibited an apparent Stokes radius of 16.2 A compared to the Stokes radius of 17 A for Cd,Zn-Th II. As described previously (14), Cu-thionein bound 10 g atoms Cu/mol protein. Since the metal ion content and ionexchange elution position of Cu-Th II differed from those of Cd,Zn-Th II, the question arose whether the Cu-thionein polypeptides were dissimilar from the Cd,Znthionein polypeptides. To resolve this question, the two isoforms of Cd,Zn-Th and Cu-Th II were digested with CNBr and subjected to automated Edman degradation. Kojima et al. (12) showed that CNBr digestion in formic acid released the acetylated N-terminal methionine. The remainder of the 61 amino acid polypeptide is therefore free to react with phenylisothiocyanate. Degradations were continued for 25 cycles. The sequences of Cd,Zn-Th II and Cu-Th II were found to be the same, whereas there were differences between Cd,Zn-Th isoforms I and II (Table I). The data suggest that the differing Stokes radii and chromatographic behavior did not reflect differing primary structures but instead another property associated with the different metals bound. There appear to be only two major polymorphic forms of thionein rat liver regardless of the metal ion used to induce the protein. Therefore, in our studies on the significance of the polymorphism, we chose to use the isoforms of Zn-Th and Cd,Zn-Th as models of thionein polymorphism. Metallothionein-dependent Reconstitution of Apo-Carbonic Anhydrase
Since the two isoforms of Cd,Zn-Th bind the same number of metal ions but fold in differing tertiary conformations, it is conceivable that the geometry of the metal binding sites of the isoforms may differ. One approach to investigate Zn binding in metallothionein is to test the ability of the
POLYMORPHIC
83
FORMS OF METALLOTHIONEIN TABLE
I
AUTOMATED SEQUENCE ANALYSIS OF ISOFORMS OF METALLOTHIONEIN Cd,Zn-Th Cycle
Residue
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
ASP Pro Asn CYS Ser CYS Ser Thr GUY GUY Ser CYS Thr CYS Ser Thr Ser CYS GUY CYS LYS Asn CYS LYS CYS
Cd,Zn-Th
I Yield 52 63 54 44 14 36 11 8 30 88 10 34 29 26 5 14 6 39 45 43 41 18 27 30 28
Cu-Th II
II
Residue
Yield
Residue
Yield
ASP Pro Asn CYS Ser CYS Ala Thr ASP GUY Ser CYS Ser CYS Ala GUY Ser CYS LYS CYS LYS Gln CYS LYS CYS
108 115 63 53 28 68 90 33 41 63 7 36 10 41 54 30 5 34 33 27 51 23 24 42 24
ASP Pro Asn CYS Ser CYS Ala Thr ASP GUY Ser CYS Ser CYS Ala GUY Ser CYS LYS CYS LYS Gln CYS LYS CYS
25 26 16 11 7 12 23 8 25 24 5 11 4 11 14 13 3 9 9 6 5 3 3 6 4
Note. Each sequence was repeated twice with carboxymethylated or carboxamidomethylated cyanogen bromide-treated thionein. PTH-amino acids were identified and quantitated by HPLC analysis. For each thionein isoform, first cycle yields of the samples in the polybrene-treated cup were about 20% of the quantities loaded.
isoforms to reactivate apocarbonic anhy- tween preparations, but this was not a drase (CA). The isoforms of Cu-thionein result of partially oxidized thionein molwere not included since anaerobically pre- ecule, since all samples had the same numpared Cu-Th does not reactivate apome- ber of titratable sulfhydryls per metal ion. talloenzymes (33). The reaction system in- The kinetics of the reconstitution of apovolved incubation of 6.6 PM of apo-CA with CA by the two forms of Zn-thionein (sammetallothionein in 0.1 M Tris-Cl, pH 7.5. ple 0126) at three different thionein conThe reconstitution observed with 5 eq centrations are shown in Fig. 1. The conZnSOJmol apo-CA was defined as 100%. trast in the ability of the two isoforms to Results shown in Table II with metalloreconstitute apo-CA is apparent throughthionein as the Zn donor are expressed as out the time of the three experiments. In the percentages of reactivation seen with similar kinetic experiments, a second ZnSO, after a 60-min incubation. Isoform preparation of Zn-thionein I (sample 0204) I of metallothionein invariably reacti- and Cd,Zn-thionein I from several prepavated apo-CA to a greater extent than did rations, restored greater esterase activity isoform II. This was true with both Zn-Th than did their corresponding isoforms II and Cd,Zn-Th. Diversity was apparent be- at all time points. Zn-thionein samples
84
WINGE TABLE
AND MIKLOSSY
II
RECONSTITUTION OF APO-CARBONIC ANHYDRASE Reconstitution of apo-CA (% ) Zn-Th/CA ratio
Sample Cd, Zn-Th 0807 Cd, Zn-Th 0121 Zn-Th 1112 Zn-Th 1119 Zn-Th 1106 Zn-Th 0106 Zn-Th 0204 Zn-Th 0126
I
II
5 5 1 2 1 5 2
28 25 86 105 83 72 28
14 13 30 63 30 41 21
2 5
29 85
15 42
J 8
Note. The reconstitution assay was performed as described in the text. The percentage reactivation by various preparations of metallothionein after a 60min incubation with apo-CA is listed. The concentration of metallothionein varied from equimolar concentrations of Zn as Zn-thionein and apo-CA to a fivefold molar Zn excess.
Ix
60. 40-
0
IO
20
30 TIME
showed greater variation than did Cd,Znthionein. This may occur due to a greater instability of Zn-thionein compared to Cd,Zn-thionein or to a variation in the occupancy of the metal-binding sites. The significant observation is that within a given preparation in which the isoforms of Zn-Th or Cd,Zn-Th were exposed to identical conditions, the two isoforms show dissimilar reactivities toward apo-CA. In the incubations of Cd,Zn-Th with apo-CA, no Cd was transferred to the enzyme, since additions of ZnSOl to the mixture after the incubation period reactivated apo-CA completely. Any Cd binding by apo-CA would prevent full reactivation. Reactivity
of Metallothionein
with EDTA
Since polymorphic form I of metallothionein reconstituted apo-CA more rapidly than did form II, we asked whether its Zn ions were also more readily removed with EDTA. Li et al. (22) showed that under pseudo-first-order conditions for EDTA, the reaction could be followed spectrophotometrically at 215 nm. The second part of the triphasic reaction was
40
50
60
70
(MIN.)
FIG. 1. Reactivation of apo-carbonic anhydrase by Zn-thionein. APO-CA (6.6 GM) in 0.1 M Tris-Cl, pH 7.5, was incubated with different concentrations of Zn-thionein (sample 0126) isoforms I (0) and II (A). Concentrations used include 1 eq Zn/mol apoCA, twofold and fivefold molar excess.
monitored in the first 20 min because the initial phase of the reaction was already completed during mixing. In the second phase of the reaction, isoform I reacted more rapidly with EDTA than did isoform II. This was true with Zn-thioneins (Fig. 2) and Cd,Zn-thioneins (Fig. 3). Psuedofirst-order rate constants were calculated from the plots (Table III). Differences seen between metallothionein preparations in the reconstitution of e&erase activity are also apparent in the reactivity with EDTA. At intervals, samples of the mixtures of metallothionein and EDTA were subjected to gel filtration on Sephadex G-25 to separate the two components. Fractions were analyzed for Zn ion concentration. Metallothionein eluted in the excluded volume of the column, whereas Zn-EDTA eluted in the internal volume (Fig. 4). Results for several preparations show that the greater
85
POLYMORPHIC FORMS OF METALLOTHIONEIN 0.5
Cd, Zn-Th
(08151
Cd,Zn-Th
(Ol2l)
0.3I
04
FIG. 2. Kinetics of the reactivity
of Zn-thionein with EDTA. Zn-thionein isoforms I (0) and II (A) of two preparations, sample 0126 (---) and sample 0204 (- - -), were incubated at 25°C in 0.1 M Tris-Cl, pH 7.5, containing 0.2 mM EDTA. The Zn concentration was 46 pM. The reaction was monitored at 215
rate of loss of Zn by isoform I compared to isoform II correlated with a greater retention of Zn by isoform II on gel filtration (Table III). Results with Cd,Zn-Th are more reproducible than those with Zn-Th as was observed in the reconstitution studies with apo-CA. The difference between isoforms of Zn-thionein (sample 0204), which was slight in the spectrophotometric data, was greater in the gel filtration experiments. In experiments with Cd,Znthionein, there was little Cd exchange between thionein and EDTA. This was demonstrated by observing only a slight absorbance change at 254 nm which is the wavelength maximum of Cd-mercaptides (32) and, secondly, by recovering over 85% of the Cd in an incubation as Cd-thionein (Fig. 5). To determine whether the isoform variation in reactivity with apo-carbonic anhydrase and EDTA was due to thermodynamic differences in Zn-binding affinity, Cd,Zn-thionein I and II were placed in sealed equilibrium dialysis cells for a com-
1
I
0 2 4
,
6
8
1
I
IO
12
I
14
I
I
I
I6
18
20
8
1
22 24
MINUTES
FIG. 3. Kinetics of the reactivity of Cd,Zn-thionein with EDTA. Cd,Zn-Th isoforms I (0) and II (A) of two preparations, sample 0815 and sample 0121,were incubated as described in the legend of Fig. 2. The Zn concentration was 18 pM. The reaction was monitored at 215 nm.
petition binding experiment with EDTA. The opposite cell chambers contained EDTA at a twofold molar excess relative to the Zn concentration of metallothionein. After 4 days of dialysis at 25”C, metal analysis was performed to determine the metal ion distribution in the membrane-separated chambers. Over 95% of the Cd remained associated with thionein with both isoforms I and II. Whereas only 12% of the Zn remained associated with isoform I, 38% of the Zn was bound to isoform II after the 4 days. The observed difference in Zn-binding affinity was substantiated by qualitatively similar results in an equilibrium dialysis of a second preparation of Cd,Zn-Th. DISCUSSION
It is well established that two major polymorphic forms of metallothionein exist in various tissues and cell lines, and that the isoforms differ to a limited extent
86
WINGE
AND MIKLOSSY TABLE
III
REACTIVITY OF Zn-THIONEIN ISOFORMS WITH EDTA k observed (X104 s’)
EDTA concentration (mM)
Sample Zn-Th 0126 Zn-Th 0204 Cd, Zn-Th 0815 Cd, Zn-Th 0121 Cd, Zn-Th 0729 Cd, Zn-Th 0522
Percentage
of Zn as Zn-Th
I
II
min
I
II
15 10 15 -
45 11.7 25.5 -
71 33.7 53.1
15 15 60
9.6 27.8 3.7
43.2 51.7 21.1
0.2 1.0 0.2 0.2
3.9
1.92
4.0 1.9
3.5 0.92
0.5 0.2 0.5 0.2 0.2
1.69 2.2 2.9
0.88 .73 1.2
Note. The pseudo-first-order rate constants were calculated from spectrophotometric data as described in the text. The percentages of Zn recovered in the Zn-thionein fraction after gel filtration of mixtures of the protein and EDTA incubated for the specified times are listed.
in their primary structure (13). Both forms are inducible by metal ions, although the extent of induction of the isoforms can vary. The ratio of form I to form II in the livers of adult rats induced with Zn2+ about 24 h prior to killing has been shown to vary from about 0.5 (5, 34) to about 1.5 (35). In the livers of partially hepatectomized adult rats, Ohtake and Koga (10) reported that only isoform II of Zn-Th was present. Thionein molecules induced by Cu2+also can be resolved into polymorphic forms (6, 14). Although these forms are
IO
12
14
I6 FRACTION
18
20
22
distinct from the isoforms of Cd,Zn-Th in metal content and their charge-to-mass ratio, we have shown by limited amino acid sequence analysis that isoform II of the thionein molecules induced by Cd and Cu are identical. The sequence of the first 25 amino acids of isoform II is identical to that reported by Kissling et aZ. (36). Although our sequence data is limited to only the NH2-terminal 25 residues, heterogeneity in M-Th is most pronounced at
24
NUMBER
FIG. 4. Gel filtration of the reaction mixture of Znthionein and EDTA. Zn-thionein isoform I (0) and II (A) of sample 0126 were incubated with EDTA as described in Fig. 2. After an incubation period of 15 min, the mixture was chromatographed on Sephadex G-25. The void volume of the column corresponded to fractions 12-15, whereas the included column voiume corresponded to fractions 17-21.
IO
I2
I4
I6 FRACTION
18
20
22
24
NUMBER
FIG. 5. Gel filtration of the reaction mixture of Cd,Zn-thionein with EDTA. Cd,Zn-Th isoform I (sample 0121) was incubated with 1 mM EDTA in 0.1 M Tris-Cl, pH 7.5, at 25°C. After 10 min, the mixture was chromatographed on Sephadex G-25. Each fraction was monitored for Cd (0) and Zn (A).
POLYMORPHIC
FORMS OF METALLOTHIONEIN
the NHz-terminal end. In equine renal MTh I and II, 4 of the 7 differences are clustered in the first 25 residues (13), whereas in mouse M-Th I and II 9 of the 14 differences are likewise located in the NHz-terminal 25 residues (37). So although the comparison between Cd-Th and Cd,Zn-Th was based on limited sequence analysis, the results suggest that isoform II of the two metallotheoneins are identical. It is, however, conceivable that a difference may exist in the COOH-terminal portion. Isoforms I and II of metallothionein are dissimilar in primary and tertiary structure shown by amino acid sequencing and Stokes radius determination. We were interested to determine whether these differences affect the metal-binding sites which may be of significance in the physiological functioning of metallothionein. Since the metabolic function of the protein has not been unequivically established, studies addressing differences between the isoforms are difficult to interpret. In the process of investigating the metallothionein-dependent reconstitution of apometalloenzymes, we observed that the two polymorphic forms of Zn-Th vary in their ability to reactivate apo-CA. Isoform I invariably restored greater esterase activity to apo-CA than did isoform II. This kinetic variation was also apparent when the reactivity of metallothionein with EDTA was studied. EDTA removed Zn from Znthionein isoform I at a greater rate compared to isoform II. These observations are true for Zn-Th as well as Cd,Zn-Th. Equilibrium dialysis of isoforms of Cd,ZnTh suggested that the Zn-binding affinity was less in isoform I compared to form II. In the Cd,Zn-Th molecules, Cd reacts only slowly with EDTA and is not transferred to apo-CA. The slow reactivity of Cd-Th with EDTA is well known (22, 32). The observed differences in reactivity with apo-CA and EDTA between the isoforms of Cd,Zn-Th are reproducible, but variations between preparations of Zn-Th were observed. The results with Cd,Zn-Th are very clear in indicating that the apparent Zn-binding affinities of the two isoforms differ. Both isoforms of Cd,Zn-Th had similar metal ion compositions. The
87
significance of the diversity in Zn-Th preparations remains obscure. It is conceivable that a cellular component exists that can modulate Zn-binding affinity or that variation occurred due to instability of Zn-Th. Further studies concerning those questions are in progress. Although the diversity in preparations of Zn-Th makes a comparison of isoforms from different preparations difficult, the relative difference between the two isoforms from the same purification is significant; since, within the same preparation, the two isoforms were exposed to identical conditions. Suzuki and Yamamura (18-20) have reported that the relative ratios of the two polymorphic forms of metallothionein induced with Zn,Cd or Cu change with time. In their experiments with Zn-Th, isoform II was always the most abundant form. In the time span from 6 h to 4 days, the ratio of isoform I to isoform II decreased. This ratio for the isoforms of Cd-induced thionein changes from approximately unity at 6 h to lower values up to 2 days and then the ratio increased above unity for times up to 7 days, The variation observed in isoform ratios may be related to a dissimilar, pattern of biosynthetic or degradation rates. Alternatively, it is conceivable that the two forms differ in metabolic functions, and that changes in the isoform ratio may be related to rates of reaction. The different Zn-binding affinities of the isoforms reported presently may be of significance in such physiological variations. ACKNOWLEDGMENTS The authors wish to thank Dr. G. R. Lee and Dr. R. E. Lynch for their support and discussions. REFERENCES 1. MARGOSHES, M., AND VALLEE, B. L. (1957) J. Amer. Chem. Sot. 79, 4813-4814. 2. NORDBERG, G. F., NORDBERG, M., PISCATOR, M., AND VESTERBERG, 0. (1972) B&hem, J. 126, 491-498. 3. SHAIKH, Z. A., AND LUCIS, 0. J. (1972) A&. Envirmz Health 24, 419-425. 4. WESER, U., RUPP, H., DONAY, F., LINNEMANN, F., VOELTER, W., VOETSCH, W., AND JUNG, G. (1973) Eur. J Biochem. 39, 127-140.
88
WINGE
AND MIKLOSSY
5. WINGE, D. R., PREMAKUMAR, R., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 242-252. 6. BREMNER, I., AND YOUNG, B. W. (1976) B&hem J. 155, 631-635. 7. BREMNER, I., AND YOLJNG, B. W. (1976) Biochem .J. 157.517-520. 8. TSUNOO, H., KINO, K., NAKAJIMA, H., HATA, A., HUANG, I. Y., AND YOSHIDA, A. (1978) J. Biol. Chem 253, 4172-4174. 9. HIDALGO, H. A., KOPPA, V., AND BRYAN, S. E. (1978) Biochem J. 170,219-225. 10. OHTAKE, H., AND KOGA, M. (1979) B&hem. J. 183,683-690. 11. ZELAZOWSKI, A. J., AND PIOTROWSKI, J. K. (1980)
Biochim Biophys. Acta 625, 89-99. 12. KOJIMA, Y., BERGER, C., VALLEE, B. L., AND KAGI, J. H. R. (1976) Proc. Nat. Acad. Sci USA 73, 3413-3417. 13. KOJIMA, Y., AND KAGI, J. H. R. (1978) Trends
23. 24.
25. 26.
27. 28. 29. 30.
Biochem Sci 3,90-93. 14. WINGE, D. R., GELLER, B. L., AND GARVEY, J. (1981) Arch Biochem Biqphys. 208, 160-166. 15. OTVOS, J. D., AND ARMITAGE, I. M. (1979) J. Amer. Chem. Sot 101, 7734-7736. 16. OTVOS, J. D., AND ARMITAGE, I. M. (1980) Proc.
Nat. Acad. Sti USA 77,7094-7098.
-.. VASAK, M. (1980) J. Amer. Chem Sot. 102,3953-
17
3955. 18. SUZUKI, K. T., AND YAMAMURA, M. (1980) Toxicol. L&t. 6, 59-65. 19. SUZUKI, K. T., AND YAMAMURA, M. (1980) Biochem. Pharmacol. 29, 2407-2412. 20. SUZUKI, K. T., AND YAMAMURA, M. (1980) To&cot. Z.&t. 6,301-307. 21. UDOM, A. O., AND BRADY, F. 0. (1980) Biochem. J. 187, 329-335. 22. LI, T. Y., KRAKER, A. J., SHAW, C. F., AND PE-
31.
TERING, D. H. (1980) Proc. Nat. Acud Sci. USA 77, 6334-6338. GRASSETI, D. R., AND MURRAY, J. J., JR. (1967) Arch. Biochem Biophys. 119,41-49. HIRS, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 199-203, Academic Press, New York. HUNKAPILLER, M. W., AND HOOD, L. E. (1978) Biochemistry 17,2124-2133. LAI, C. Y. (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S., eds.), Vol. 47, pp. 369-373, Academic Press, New York. LINKSKOG, S. (1960) B&him. Biophys. Acta 39, 218-226. POCKER, Y., AND STONE, J. T. (1967) Biochemistry 6, 668-678. HUNT, J. B., RHEE, M. J., AND STORM, C. B. (1977) Anal. B&hem. 79, 614-617. LINDSKOG, S., HENDERSON, L. E., KANNAN, K., LILJAS, A., NYMAN, P. O., AND STRANDBERG, B. (1971) Enzymes 5, 587-665. LINDSKOG, S., AND MALMSTROM, B. G. (1962) J.
Biol. Chem 237,1129-1137. 32. KAGI, J. H. R., AND VALLEE, B. L. (1961) J. Biol
Chem 236,2435-2442. 33. GELLER, B. L., AND WINGE, D. R. (1981) Achiw. B&hem. Biophys., in press. 34. BREMNER, I., AND DAVIES, N. T. (1975) Biochem. J. 149, 733-738. 35. WONG, K. L., AND KLAASSEN, C. D. (1979) J. BioL Chem. 254, 12,399-12,403. 36. KISSLING, M. M., BERGER, C., KAGI, J. H. R., ANDERSON, R. D., AND WESER, U. (1979) Ezperientia Suppl. 34,181-185. 37. HUANG, I. Y., KIMURA, M., HATA, A., TSUNOO, H., AND YOSHIDA, A. (1981) J. Biochem 89,18391845.