Isolation and partial characterization of native metallothionein in fetal rabbit liver

Life Sciences, Vol. 27, pp. 585-593 Printed in the U.S.A

Pergamon Press

ISOLATION AND PARTIAL CHARACTERIZATION OF NATIVE METALLOTHIONEIN IN FETAL RABBIT LIVER Michael P. Waalkes and John U. Bell Department of Pharmacology and Toxicology West Virginia University Medical Center Morgantown, West Virginia 26506 (Received in final form June 9, 1980) S,,~m~ry The endogenous zinc content of hepatic cytosol in the term fetal rabbit was found to be three-fold higher than corresponding maternal levels. Following gel-filtration, the bulk of this zinc was found to be associated with a peak, not detectable in the maternal cytosol, having a relative elution volumn similar to that of cadmium-induced metallothionein. In vitro addition of increasing amounts of cadmium to the fetal cytosol prior to gel-filtration caused progessive displacement of endogenous zinc from the peak, selective cadmium binding and eventual cadmium saturation. Anion-exchange chromatography disclosed two distinct peaks, corresponding to the two cadmium-containing peaks detected in the cytosol from cadmium-induced animals. SDS-polyacrylamide gel electrophoresis of purified protein also revealed the existance of two bands in both the fetus and induced adult. Results indicate the presence of endogenous metallothioneins in the hepatic cytosol of the normal term rabbit fetus which are similar to the metallothioneins which appear in the adult hepatic cytosol following exposure to cadmium. Although metallothionein (MT) was first detected more than twenty years ago (i), its exact biological function remains a matter of some controversy. This low molecular weight, cysteine-rich (2) protein has been found in several diverse species (3,4,5,6) and has been isolated from a variety of tissues (4,5, 6). MT has been ascribed various theoretical functions, including a protective role following exposure to selected heavy metals. This role is based primarily on the observations that certain heavy metals can induce the hepatic synthesis and thus the metal-binding capacity of MT (2,7,8,9). Recent reports have described the presence of relatively high levels of native MT in fetal hepatic tissues obtained from the human (i0), rat (ll) and sheep (12,13). In both the sheep and the rat fetus, MT appears to be the major zinc-containing component of the hepatic cytosol, suggesting a possible role as an intracellular storage site for this essential element in the fetus. In this study, we describe the presence of MT in the hepatic cytosol of the term rabbit fetus. Materials and Methods Male and female New Zealand White Rabbits (Hilltop Lab Animals, Inc., Scottdale, PA) were housed individually with free access to standard labor0024-3205/80/330585-09502.00/0 Copyright (c) 1980 Pergamon Press Ltd.

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atory food and water. Following at least one week of acclimatization in our animals quarters, virgin females (2.7 - 3.2 kg) were placed with proven males until mounting behavior was observed (day 0). Pregnancies were confirmed by manual palpation 14 days following mating. Dams were sacrificed with an intravenous overdose of pentobarbital between days 28 and 30 and fetuses were removed by cesarean section and sacrificed by decapitation. To obtain reference MT, adult male rabbits were administered a single subcutaneous injection of CdCI2 (2mg cadmium/kg) 48 hr prior to sacrifice. The procedure for gel-filtration of hepatic MT was essentially that of Probst et al (9) with minor modifications. Livers were removed, quickly weighed and homogenized at 4°C in buffer 'A' (0.02M Tris-HCl, pH 8.6; 0.005M 2-Mercaptoethanol; 0.25M Sucrose) using a Potter-Elvehjem glass homogenizer and motor-driven teflon pestle to yield a 20 percent (W/V) homogenate. The homogenate was then centrifuged for 90 min at 70,000g and the resulting supernatant was carefully removed and stored in 6.0 ml aliquots at -20°C. After thawing, samples were mixed with various amounts of CdCI 2 (0.5, 1.0, 2.0, 4.0, 6.0, 8.0 or 12.0 m o l e s ) in a constant volume of 0.4 ml for saturation curve analysis. For 'cadmium-unsaturated' samples 0.4 ml of distilled H20 was added to the supernatant. The appropriate supernatant was then recentrifuged for 45 min at 70,O00g and a 4.0 ml aliquot was loaded onto a 2.6 x 52-cm calibrated column of Sephacryl S-200 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) equilibrated with buffer 'B' (O.02M Tris-HCl pH 8.6; 0.05M NaCI and O.005M 2-Mercaptoethanol). Descending buffer flow was used at a flow rate of 124 ml/hr and 7.4 ml fractions were collected. For anion-exchange chromatography, supernatant samples were first applied to a column of Sephacryl S-200 equilibrated in O.02M Tris-HCl buffer (pH 8.6) at a flow rate of 124 ml/hr. Fractions with a relative elution volume of 1.9 to 2.3 (MT peak) were then pooled and loaded onto a 0.9 x 15-cm column of DEAE Sephadex A-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) equilibrated with 0.02M Tris-HCl buffer. Protein fractions were eluted from the column using a linear Tris-HCl gradient (0.02-0.50M) at a flow rate of 35 ml/hr. Conductance of the fractions was measured using a Radiometer type CDM 2d conductance meter. MT was purified using the method described by Sobocinski et al (14). Hepatic cytosol (70,000g x 90 min supernatant) was heated at 85°C for i0 min and then centrifuged at 27,000g for 15 min. Heat-soluble material was then subjected to selective acetone precipitation (0-40%, 40-60%, 60-80% acetone by volume). The material precipitating at an acetone concentration of 80 percent was reconstituted in distilled water and subjected either to SDS-polyacrylamide gel electrophoresis, by the method of Sobocinski et al (14), or to ultraviolet spectral analysis. Protein content was measured using the protein dye-binding method described by Bradford (15) using bovine serum albumin (Sigma Fraction V) as the standard. Metal analysis was performed using a Perkin-Elmer model 305-B atomic absorption spectrophotometer. The instrument was used in the standard flame mode (acetylene-air) and samples were analyzed by direct aspiration. Metal content was calculated using certified zinc and cadmium standards. Ultraviolet spectral analysis of purified MT (22 ~gm protein/ml) was carried out using a Hitachi model 100-80 computerized spectrophotometer in the double-beam mode. Absorbance was measured by scanning between 220 and 300 nm following the addition of increasing amounts of CdC12.

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587

Results The concentration of zinc in the hepatic cytosol of fetal, maternal and Cd-treated adult male rabbits is shown in Table I. The zinc content of the fetal cytosol was found to be 2.7 times higher than the zinc content of the maternal cytosol. When expressed in terms of cytosolic protein content this difference became more pronounced (i.e. 4.0 fold higher). TABLE i Levels of Zinc and Protein in Cytosol Obtained from Fetal, Maternal and Cadmium-Treated Adult Male Rabbit Liver

n

Fetal

7

Maternal

7

Cd-treated Adultb

3

Total Zinc (ng.atoms/ml) 220.8 ± 38.0 a

Protein (mg/ml)

Zinc (ng.atoms) Protein (mg)

12.24 ± 2.01

17.99 ± 1.43

7.8

18.24 ± 1.36

4.49 ± 0.33

197.0 ± 24.5

19.73 ± 5.20

10.60 ± 3.92

81.8 ±

aValues represent mean ± S D bTreatment consisted of a single administration wt sc) 48 hr prior to sacrifice

of CdCI 2 (2mg Cd/kg body

A number of recent reports have shown in the rat (ii) and the sheep fetus (12,13) that a large portion of the hepatic cytosolic zinc is associated with a protein having characteristics similar to cadmium and/or zinc induced hepatic 30E ~ 20

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FIG. 1 Sephacryl S-200 chromatographic profiles for endogenous zinc and cadmium following gel-filtration of 'cadmium-unsaturated' cytosols (see: Materials and Methods) prepared from fetal, maternal and cadmium-treated adult male rabbit livers. Brackets indicate a relative elution volume (Ve/Vo) of 1.9 to 2.3.

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Metallothionein in Fetal Rabbit Liver

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MT. Figure 1 shows representative metal elution patterns of liver cytosols obtained from term fetal, maternal and cadmium-treated adult male rabbits following gel-filtration using Sephacryl S-200. The major cadmium-containing peak in the pretreated adult, characterized by a relative elution volumn (Ve/Vo) of 1.9 to 2.3, corresponded to the major zinc-containing peak in the term fetus. This peak was not present in the maternal cytosol. For convenience this peak will henceforth be referred to as the MT (metallothionein) peak. In the fetus over 80

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FIG. 2 Sephacryl S-200 chromatographic profiles (left) for exogenous cad-mium (A) and endogenous zinc (B) following gel-filtration of cytosol obtained from control term fetal rabbit liver. Increasing amounts of CdCI 2 were added to the cytosol in vitro prior to gelfiltration. The panels on the right summarize the binding of exogeous cadmium (A) and endogenous zinc (B) to the MT (Ve/Vo 1.9 2.3) and LMW (Ve/Vo 2.4 - 2.9) peaks following addition of various amounts of cadmium.

i 8

1

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Metallothioneln in Fetal Rabbit Liver

589

70% of the cytosolic zinc was associated with the MT peak and this figure roughly corresponds to the maternal-fetal difference seen in cytosolic zinc levels. Indirect quantitation of MT levels can be obtained by assessing the metal binding capacity of the protein following cadmium saturation (9). Figure 2 shows such in vitro saturation analysis for fetal hepatic cytosol. Representative elution patterns for both cadmium (figure 2A) and zinc (figure 2 B ) a r e shown following the in vitro addition of varying amounts of CdCI 2 (0.0, 0.5, 2.0, 6.0 moles). At lower concentrations, cadmium bound almost exclusively to the MT peak; at higher concentrations, however, the metal began to bind to 'low molecular weight' material (LMW) with a relative elution volumn (Ve/Vo) of 2.4 to 2.9. Upon saturation of the MT peak, additional cadmium was found to be associated with the LMW peak. Although zinc was progessively displaced from

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FIG. 3 DEAE-Sephadex A-25 chromatography of metallothionein isolated by gelfiltration from cadmium-treated adult and control fetal rabbit hepatlc cyt0sol. Samples in 0.02M Trls-HCl (pH 8.6) were applied to the column which was washed with 75 ml of the same buffer at a flow rate of 35 m!/hr followed by elution with a linear gradient of 0.02-0.50M "Trls-HCl (pH 8.6). Conductance was measured in each fraction (7.5mi). the MT peak following the addition of cadmium, a small amount remained This 'residual' zinc component eventually stabilized at about 21.7% of the t o t a l binding capacity of the MT peak. At very high levels of cadmium(>__ 8.0 ~moles) there was apparently some loss of LMW material presumably due to precipitation. Following this in vitro titration of MT with CdCI2, 6.0 ~moles was chosen as the level of CdCI 2 required for the saturation and indirect quantitation of hepatlcMT. Gel-filtration elution patterns of the 'cadmium-saturated' cytosols (not shown) confirmed that the metal-blnding peak in the fetal cytosol corresponded to the MT peak in the Cd-pretreated adult cytosol and also conflrmedthe absence of a corresponding peak in the maternal cytosol. The total binding capacity of fetal cytosollc MT averaged 0.797 ~g.atoms (range 0.582 0.922; n=7) compared to the Cd-treated adult value of 0.788 ~g.atoms (range 0.675 - 0.847; n=3).

590

Metallothionein

in Fetal Rabbit Liver

Vol. 27, No. 7, 1980

When the gel-filtration MT peak from 'cadmium-unsaturated' cytosol was subjected to anion-exchange chromatography, two distinct peaks (designated MTA and MT-B) were revealed in both the fetus and Cd-pretreated adult (figure 3). The primary metal associated with the fetal peaks was zinc whereas the Cd-pretreated adult peaks contained both zinc and cadmium. The zinc distribution between the two peaks was similar in both the fetus and pretreated adult. MT purified by heat denaturation and selective acetone precipitation was subjected to SDS-polyacrylamide gel electrophoresis (figure 4). The protein from both the fetus and the Cd-pretreated adult split into two distinct bands (band I and band II) with similar electrophoretic migrational qualities.

FIG. 4 SDS-polyacrylamide gel electrophoresis of metallothionein(s) isolated from cadmium-treated adult and control fetal rabbit hepatic cytosols by a combination of heat denaturation and selective acetone precipitation. Protein (15 ~g) was applied to each gel (7%) and electrophoresis was performed at a constant voltage of lOOV for 0.5 hr followed by 2.5 hr at 175V. Gels were stained with Coomassie blue (0.1%) dissolved in 25% Isopropanol, 10% acetic acid and 10% trichloroacetic acid. Ultraviolet spectral analysis of purified MT from both the fetus and Cdtreated adult is shown in figure 5. In both cases, there was no absorption peak at 280 nm, characteristic of a protein lacking aromatic amino acids, yet there was an absorption peak around 250 nm, characteristic of metal-sulfur interactions. It should be noted that the 250 nm, absorbance peak in the fetal protein involves zinc-MT bonds whereas both zinc and cadmium are involved in the pretreated adult. When cadmium was added to the protein solution, there was a concomitant increase in the absorbance at 250 um suggesting an increase in metal-MT binding. Although the 250 n m a b s o r b a n c e peaks for the native proteins were qualitatively similar, the fetal protein displayed a g r e a t e r i n crease in absorption for a given addition of cadmium than was seen with the protein isolated from the Cd-pretreated adult.

Vol. 27, No. 7, 1980

Metallothionein

in Fetal Rabbit Liver

591

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WAVELENGTH (nm) FIG. 5 Ultraviolet absorption spectra of metallothionein(s) isolated from cadmium-treated adult and control fetal rabbit hepatic cytosols by a combination of heat denaturation and selective acetone precipitation. Spectra were obtained by scanning the appropriate protein solution in the absence and presence of added CdCI 2. Discussion Following gel-filtration of hepatic cytosol from adult male rabbits pretreated with cadmium chloride, cadmium was detected as a single peak with a relative elution volume of 1.9 :to 2.3. The peak was not present in maternal hepatic cytosol; however, the bulk of the zinc in the hepatic cytosol of the term rabbit fetus was also found to elute as a single peak having the same relative elution volume. In vitro addition of cadmium to the fetal cytosol revealed that at low levels, the metal bound almost exclusively to this peak and displaced the endogenous zinc. At saturating cadmium levels, approximately 20 percent of the endogenous zinc still remained. It has previously been reported that cadmium has a higher affinity for metallothionein than zinc (2) and that the multiple metal binding sites of metallothionein are not ~homogeneous (16,22). The gel-filtration data are consistant with the suggestion that the fetal zinc-binding protein is metallothionein. Anion-exchange chromatography disclosed that the fetal protein isolated by gel-filtration was in fact two proteins containing similar amounts of zinc. Once again, the elution pattern of the fetal proteins mirrored the elution pattern seen in the Cd-pretreated adult. The existance in the adult of two forms of metallothionein having similar molecular weights but different electrical charge properties has been well documented in the literature (4,7,14,17,18,19). The polymorphic nature of the fetal proteins and their similarity to Cd-induced metallothionein(s) were also confirmed by SDS-polyacrylamlde gel

592

Metallothionein in Fetal Rabbit Liver

Vol. 27, No. 7, 1980

electrophoresis following purification. The existance of two forms of metallothionein with differing electrophoretic mobilities has been previously demonstrated in the adult following exposure to a variety of metallothionein~nducing stimuli (4,8,14). Spectral analysis of the purified fetal proteins also strongly indicate that they are in fact metallothioneins. The characteristic lack of absorbance at 280 nm and pronounced absorbance at 250nm, indicative of metal-sulfur interactions, were observed with protein purified from fetal rabbit liver and from Cd-pretreated adult rabbit liver. Once again these findings are in accord with previous reports (2,6,7,18). A variety of functions have been proposed for hepatic metallothionein in the adult. One suggestion which has received considerable attention is that metallothionein functions in the detoxlfication of potentially toxic heavy metals (7,9). This role is based on the observation that metal-induced metallothionein is capable of sequestering the inducing metal; an observation that we have confirmed in this study. In addition, there have been reports which suggest that the presence of metallothionein will protect the animal against subsequet exposure to the metal (7,9). Unfortunately, since hepatic metallothionein levels have been shown to be either very low (19,20,21) or not detectable (8) in adult animals not exposed to inducing agents, it is tempting to suggest that this protective role only comes into play during certain pathological states. A number of recent studies have supported the existance of relatively high levels of native metallothionein(s) in the fetal hepatic cytosol of a variety of species. In both that rat fetus (ii) and the sheep fetus (12,13) hepatic metallothionein appears to be a major zinc-bindlng protein. Wong and Klaassen (23) have isolated and characterized metallothionein from the liver of i- to 4-dayold rats and found it to be a zinc-containing protein with characteristics similar to zinc-lnduced adult metallothionein. Their findings complement the present study which indicates that metallothionein is also present in the hepatic cytosol of the term rabbit fetus and that it is involved with the binding of endogenous zinc. Acknowledgements This work was supported by U.S. Department of Energy Contract EY-77-C-218087 and NIH Training Grant Number 5T32-GM07039. References i. 2. 3. 4.

M. Margoshes and B.L. Vallee, J. Amer. Chem. Soc. 79, 4813-4814 (1957). H.R. Kagl and B.L. Vallee, J. Biol. Chem. 236, 2435-2442 (1961). A.J. Zelazowski and J.K. Piotrowskl, Experlentia 33,1624-1625 (1977). R.W. Olafson, R.G. Sim and K.G. Boto, Comp. Biochem. Physiol. 623, 407-416 (1979). 5. Y. Kojima and H.R. Kagi, Trends in Biol. Sci. ~, 90-93 (1978). 6. V. Weser, H. Rupp, F. Donay, F. Linnemann, W. Voelter, W. Voetsch and G.Jung, Eur. J. Biochem. 39, 127-140 (1973). 7. A.P.Leber and T.S. Miya, Toxicol. Appl. Pharmacol. 37, 403-414 (1976). 8. D.R. Winge and K.V. Rajagopalan, Arch. Biochem. Biophys.153, 755-762 (1972). 9. G.S. Probst, W.F. Bousquet and T.S. Miya, Toxicol. Appl. Pharmacol. 39, 6169 (1977). i0. L. Ryden and H.F. Deutsh, J. Biol. Chem. 253, 519-524 (1978). 11. J.U. Bell, Toxlcol. Appl. Pharmacol. 48, 139-144 (1979). 12. I. Bremner, R.B. Williams and B.W. Young, Br. J. Nutr. 38, 87-92 (1977). 13. J.U. Bell, Toxicol. Letters 4, 407-411 (1979).

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Metallothionein in Fetal Rabbit Liver

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14. P.Z. Sobocinski, W.J. Canterbury, C.A. Mapes and R.E. Dinterman, Amer. J. Physiol. 234, E399-E406 (1978). 15. M.M. Bradford, Anal. Biochem. 72, 248-254 (1976). 16. J.U. Bell, Toxicol. Appl. Pharmcol. 50, 101-107 (1979). 17. M. Kimura, N. Otaki, S. Yoshiki, M. Suzuki, N. Horivchi and T. Suda, Arch. Biochem. Biophys. 165, 340-348 (1974). 18. D.R. Winge, R. Premakumar and K.V. Rajagopalan, Arch. Biochem. Biophys. 170, 242-252 (1975). 19. R.W. Chen, P.D. Whanger and P.H. Weswig, Biochem. Med. 12, 95-105 (1975). 20. A.Z. Shaikh and J.C. Smith, Chem. Biol. Interact, 15, 327-336 (1976). 21. K.S. Squibb and R.J. Cousins, Biochem. Biophys. Res. Comm. 75, 806-812 (1977). 22. D.R. Winge, R. Premakumar and K.V. Rajagopalan, Arch. Biochem. Biophys. 188, 466-475 (1978). 23. K.-L. Wong and C.D. Klaassen, J. Biol. Chem. 254, 12399-12403 (1979).