Biological function of metallothionein

BIOCHEMICAL

MEDICINE

12,

Biological I. Synthesis

and

R. W. CHEN,

95-105 (1975)

Function Degradation

of Metallothionein. of Rat Liver

P. D. WHANGER,

AND

Metallothionein’

P. H. WESWIG

Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331 Received July 1, 1974

Metallothionein (MT), a protein unique in its high sulfhydryl and metal content, was first identified and characterized from equine kidney by Vallee and co-workers over a decade ago (l-3). Since then the protein has been found in many tissues of several species of animals (4- 12). Although found in low concentrations in tissues of animals, metal exposure apparently will increase its tissue concentration (6, 12- 15). As an example, the concentration of MT in tissues of normal rats was found to be about 0.1 and 0.4 mg per g tissue in liver and kidney, respectively, but increased to 4.4 and 4.1 mg per g of respective tissues after Cd exposure (15). The biological role for this protein, however, has not yet been fully established. Among the several roles suggested by Pulido et al. (3), a role in detoxification of heavy metals such as Cd and Hg has gained support from several laboratories (6,9, 13, 15, 16). Alternatively, other workers have suggested that MT may be involved in metabolism of some essential trace metals. Webb (12) proposed that the protein may be involved in the control of Zn metabolism comparable to the manner in which ferritin controls Fe metabolism. On the other hand, Evans and co-workers believe that MT is involved in intestinal Cu absorption (17, I8), and that synthesis of defective MT may be involved in the development of Wilson’s disease (19). However, no concrete evidence is available to support any of the proposed biological roles. Therefore, this study was conducted to investigate the synthesis and degradation of Cdstimulated MT in order to obtain further information which might help establish its biological role. METHODS Male Long-Evans rats raised on Purina Laboratory Chow were used to study the synthesis and degradation of liver MT employing r-‘“C-cystine and Sephadex G-75 chroma’ Supported by Public Health Service Research Grant Number ES 00529 from the National Institute of Environmental Health Sciences. Published with the approval of the Oregon State Agricultural Experiment Station as Technical Paper No. 3794. 95 Copyright CI 1975 by Academic Press, Inc. All rights of reproduction m any form reserved.

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WESWIG

tography. The G-75 columns were packed and standardized with proteins of known molecular weights (MW) as indicated previously (20). In the initial experiment, the time course of the incorporation of ‘“C-cystine into MT was studied. Since a preliminary experiment indicated that the incorporation of ‘“C-cystine in MT was very low in unexposed rats but was greatly enhanced by an injection of Cd, rats (2.50-280 g) were injected subcutaneously with CdCl, (26.7 pmole/kg body wt) in isotonic Na-acetate solution. Three days later, the rats (fasted 18 hr) were injected intraperitoneally with “C-cystine (New England Nuclear, Boston) at a dose of 2 ~Ci/lOO g body wt in isotonic NaCl solution (10 &i/ml, 250 &Ji/pmole cystine). At predetermined times after ‘“C-cystine injection. rats were killed. The liver was immediately removed, chilled in cold isotonic NaCl solution, blotted. and homogenized in a Potter-Elvehjem homogenizer with 2 vol of cold isotonic NaCl solution. The homogenate was then centrifuged at 10,000 g for IO min. The supernatant was further centrifuged at I 13,000 g for 60 min to obtain the soluble fraction. Ten milliliters of the soluble fraction were chromatographed at 4” on a Sephadex G-75 column. A I-ml aliquot from each elution fraction was counted for radioactivity (‘Q in a Packard Tri-Carb Liquid Scintillation Spectrometer (Model 3375). The radioactivity in the fractions corresponding to MT (V,/V, = 2) was summed and expressed as cpm/g liver. To determine the rate of degradation of MT, a group of rats (300-320 g) was injected with Cd and l*C-cystine, killed at predetermined times afterwards, and livers processed as described. In addition to Y?, Cd, and Zn content in MT fractions was also measured by atomic absorption spectrophotometry. Another group of rats was treated in the same way and was given an additional dose of Cd one day after “C injection to see if the second dose of Cd would effect the degradation rate of MT. Using a ‘YXabeling period of 2 hr, the time course of the stimulatory effect of Cd on MT synthesis was also investigated. In addition, the effect of other metals (Hg, Ag, Zn, Cu) on the synthesis of MT at time periods of 5 hr and three days after metal treatments were investigated. To verify that Sephadex G-75 chromatography employed was an adequate separation technique, the MT fractions obtained from the livers of rats injected with Cd and “‘C-cystine in the above studies were pooled, concentrated by ultrafiltration on a UM-2 Diaflo membrane, and further purified by DEAE-cellulose chromatography. Sulfhydryl content of the purified MT was determined with p-hydroxymercuribenzoate (PMB (2 1)). Glassware used in all experiments was acid washed and double distilled water was used for making buffers, reagents. and for rinsing glassware. Appropriate blanks were used for all determinations.

RESULTS

Sephadex G-75 chromatography of liver soluble fraction from rats injected with 14C-cystine revealed three 14C peaks (Fig. 1). The first peak was eluted with the void volume and contained proteins of MW larger than 70,000. The second peak, corresponding to a MW of 10,000 (VJV, = 2), contained proteins with a high sulfhydryl content, presumably MT. The third peak was eluted at low MW region (V,/I’, = 3) representing cystine and/or its low MW derivatives since free 14C-cystine was eluted at the same position. Addition of Cd and Zn to ‘“C-cystine solution did not effect its elution pattern. In the liver soluble fraction of normal rats (without Cd injection), only a small amount of 14C (1.3% of total) was found in association with MT (Fig. la). It was, however, greatly increased (5.0% of total) by Cd injection (Fig. 1b), which repre-

METALLOTHIONEIN

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WESWIG

sented a fourfold increase of cystine incorporation into MT. Therefore, Cd injection was used to facilitate the study on the synthesis and degradation of MT in the present work. The incorporation of 14C into MT and the large MW proteins reached a maximum level around 2 hr after 14C-cystine injection (Fig. 2). A peak in radioactivity of the low MW fraction was reached 2 hr after injection, and then declined very rapidly up to 12 hr afterwards. Therefore, a labeling period of 2 hr was chosen in the following experiments to study the incorporation of ‘“C-cystine into MT. The stimulatory effect of Cd injection on the incorporation of I%-cystine into MT appeared to be long-lasting with two time peaks, occurring 5 hr and six days after Cd injection (Fig. 3). This apparent abnormal pattern has been observed in repeated experiments. After the injection of a single dose of Cd, the amount of Cd in the MT fraction increased with time and reached a maximum level four days after the injection. It then remained at that level for the remainder of the experiment which was 11 days (Fig. 3). Interestingly, Cd injection also caused accumulation of Zn in the MT fraction. The amount of Zn associated with MT increased with time after Cd injection and reached the highest level four days after Cd injection. In contrast to Cd, however, its concentration decreased thereafter with time. In the study on the degradation of MT, rat liver MT was found to have a half-life (t&) of approximately 4.2 days (Fig. 4a) which is comparable to most liver proteins (22). The f+ of MT obtained from rats given a second dose of Cd one day after I% injection was 4.9 days (Fig. 4b). Interestingly, while the amount of Cd in the liver MT fraction remained constant, Zn disappeared from this fraction at a rate similar to that of I’C (Fig. 4). The initial rise in Cd, Zn, and 14C in MT fraction in Fig. 4b was probably caused by the second dose of Cd. In addition to Cd, other metals such as Zn, Cu, and Hg can also stimulate the synthesis of MT, as demonstrated by the increased incorporation of ‘C-cystine into it (Table 1). Ag, under the conditions used in the experiment, did not have this effect, however, administration by other routes may lead to different results. DEAE-cellulose chromatography of concentrated MT solution obtained by Sephadex G-75 chromatography from livers of rats treated with Cd and 14C-cystine revealed two MT peaks (MTP, and MTP, in Fig. 5). The nature of these peaks as MT species was indicated by their high &&&, ratio, and their high Cd, Zn, and *“C content. Measurement of sulfhydryl groups with PMB also indicated high sulfydryl content (13.9 and 14.0 -SH/lO,OOO MW for MTP, and MTP,, respectively). The major difference between these peaks appeared to be their total metal content, 3.8 and 5.4 g-atoms/IO,000 MW for MTP, and

METALLOTHIONEIN

I

4

9 Hours

After

12 “C-Cystine

99

16

20

24

/

Injection

FIG. 2. Incorporation of “C into rat liver soluble proteins at various hours after “C-cystine injection (see text for methods).

Days After

Cd Injection

FIG. 3. The time course of the stimulatory effect of Cd on the accumulation of Zn, and the incorporation of ‘?C into metahothionein. Rats were injected with a dose of Cd (26.7 pmole CdCl,/kg body wt, s.c.). At various times after Cd injection, one rat, having been fasted for 18 hrs, was injected with a dose of 14C-cystine (2 &i/l00 g body wt, ip). Two hours later the rat was killed, MT isolated by Sephadex G-75 chromatography (see Fig. I) and the amount of ‘“C, Cd, and Zn in MT measured.

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II ‘4c 1)---33 Cd

O---u

Zn

A----A

,/‘-----o-----0

0

OH ,

? 2nd

Cd

\

I

2

4

6

Days

8

After

14C-Cystine

4

2

6

e

Injection

liver metallothionein from rats not FIG. 4. Degradation of Cd-stimulated, ‘T-labeled given (A) or given (B) an additional dose of Cd one day following “C-cystine injection. Two groups of rats were injected with a dose of Cd (26.7 pmofe CdCl,/kg body wt, s.c.). Three days later, all rats were injected with a dose of “C-cystine injection, one group of the rats (B) was injected with a second dose of Cd. After the “C-cystine injection, one rat from each group was killed on alternate days and the amount of “‘C, Cd, and Zn determined in liver MT.

MTP,, respectively. Only trace amounts of Cu were found in either peak. More than 90% of l*C, Cd, and Zn put on the DEAE-cellulose column was eluted with these two MT peaks. DISCUSSION

The present study suggests that a number of factors could affect the synthesis and degradation of rat liver MT. The incorporation of ‘*C-cystine into MT was normally low but was greatly increased by a single injection of Cd, which is in agreement with data of other workers (14, 23). Since inhibitors of protein synthesis have been shown to prevent the

101

METALLOTHIONEIN TABLE 1 EFFECT OF METALS ON SYNTHESIS OF METALLOTHIONEIN (MT) ‘T in MT, cpm/g liver Metal”

Dose

None Cd’ Hg2 42 Cd* Zn2 CU’

(Na-acetate) 26.7 pmoleikg 26.7 pmole/kg 26.7 pmole/kg 106.8 pmole/kg 106.8 pmole/kg 106.8 pmole/kg

5 hr body body body body body body

wt wt wt wt wt wt

1426 7526 2588 1352 6353 3913 5693

Three

days

4815 3512 1436 3387 1946

(LRats were injected subcutaneously with various metal salts in isotonic acetate solution. Five hours or three days later, the rats were injected intraperitoneahy with ‘“C-cystine at a dose of 2 yCi/lOO g body wt. Two hours after l*C injection the rats were killed and the amounts of “C in the liver MT fraction measured. b Metals were given as: (1) chloride; (2) acetate; (3) nitrate.

stimulatory effect of Cd on the incorporation of cystine into MT (12), this element apparently stimulates the de novo synthesis of this protein. The stimulatory effect of Cd on MT synthesis appeared to be longlasting, resulting in the production of two peaks with time. This is not explainable at present. However, the accumulation of Zn in the MT fraction caused by Cd injection might be involved since this metal can also stimulate MT synthesis (Table 1). The first time peak may be due mainly to Cd whereas the second one may be due primarily to Zn. The inducibility of various MT species by metals are under study in this laboratory. Another possibility is that one of the MT species may be more readily inducible than another one. At 0 to 5 hr after Cd injection some Cd was found in association with proteins eluted with void volume. One day after Cd injection, however, almost all the Cd in the soluble fraction was associated with MT. An involvement of MT in Cd metabolism is clearly indicated. However, MT may be more importantly involved in the metabolism of Zn, since high levels of Zn, an essential element, have been found in association with this protein (3,4,6,12,24). With regard to the degradation of rat liver MT, a tj of 4.2 days was found. The & of MT obtained from the livers of rats given a second dose of Cd was 4.9 days, insignificantly different from the above value although it possessed twice the Cd content. This indicated that MT was not substantially stabilized by Cd. This is in contrast to that reported for the stabilizing effect of iron on ferritin (27). However, since our results (24) indicate that MT is involved in Zn metabolism, this element rather than Cd may stabilize the protein. Since Cd remained constant in liver

102

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METALLOTHIONEIN

103

MT fraction (Fig. 4) and since Hg (23) and Cd (26) have not been found in association with MT in plasma, the disappearance of ld’c from the MT fraction with time appears to represent its true degradation rate and not merely its mobilization from the liver. Interestingly, Zn disappeared from the MT fraction at a rate similar to that of “C (Fig. 4), while Cd remained constant from 4 up to 11 days after Cd injection. This provides further evidence for the involvement of MT in Zn metabolism. The persistence of Cd in the liver MT fraction up to 19 days after Cd injection was observed in another experiment, confirming a similar observation made by Nordberg et al. (13). This indicates the fallacy of previous assumptions that MT had a very long t& based on the persistence of Cd in tissues. The present study indicates that the ti of MT does not differ significantly from other liver proteins (22). The question then arises as to how Cd in the MT fraction remained constant while MT was degraded. Several possibilities are under investigation in our laboratory. First, Cd may bind to newly synthesized MT and thus did not disappear with the degraded protein moiety. That is, when the protein moiety was degraded, the released Cd might immediately stimulate MT synthesis and again become bound to the newly synthesized protein. This possibility was supported by the long-lasting stimulatory effect of Cd on MT synthesis. Secondly, Cd could be attached to a MT species which did not become labeled with j4C and has a slow turnover rate, whereas Zn was bound to another one that was labeled and was degraded as indicated. This possibility is small however, since a second dose of Cd did not significantly stabilize the t+ of MT even though MT has a much higher affinity for Cd than for Zn (2). The involvement of MT in Zn metabolism was further indicated by the observation that Zn injections stimulated the incorporation of ‘Ccystine into MT. Subcutaneous injection of Cu and Hg also stimulated the synthesis of rat liver MT (Table 1). Since Cd resulted in two incorporation peaks with time (Fig. 3) two arbitrary periods after injection were selected for the other metals. Although the time patterns could in all probability be different for each metal, which was not the purpose of this study, the present results do indicate other metals can stimulate the synthesis of MT. The percentage of total Zn in the soluble fraction which is associated with MT increased with time after Cd exposure (data not shown), providing additional evidence for the critical involvement of MT in Zn metabolism. Consequently, we would like to propose that MT is a Zn storage protein in which the levels can be influenced by other elements such as Cd, Hg, and Cu. Upon DEAE-cellulose chromatography, the concentrated MT solution obtained by Sephadex G-75 chromatography from the livers of rats

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treated with Cd and 14C-cystine was separated into two major peaks. Both peaks contained large amounts of Cd, Zn, and sulfhydryl groups and had high A,,,,/Azso ratios, indicating that they were MT species. More than one species of MT have been reported in several previous studies (2,5,28,29,30). Although the analysis indicated high sulfhydryl content, the values were not as high as might be expected for MT. Similar values for MT were obtained by another worker (I 1) using PMB, which may be suggestive this is not a good reagent for measuring sulfhydryl groups of MT. The metals, particularly Cd, may have to be removed first before accurate results can be obtained with this compound. Since more than 90% of ‘C, Cd, and Zn put on the DEAEcellulose column was eluted with the two MT peaks, it was, for practical purposes, sufficiently separated from other proteins in the soluble fraction to justify the conclusions drawn in the present study. SUMMARY

The synthesis and degradation of rat liver metallothionein (MT) were investigated using 14C-cystine as precursor, Cd to stimulate the synthesis of this protein, and Sephadex G-75 chromatography as an isolation method. Incorporation of 14C-cystine into MT was normally low, but was greatly stimulated by S.C. Cd injection which also caused a temporary, but great, accumulation of Zn in MT fraction. A biological half-life of 4.3 days was found for liver MT. A second dose of Cd given after ip ‘-‘C-cystine injection prolonged only slightly the half-life (4.9 days). While Cd in the MT fraction remained at a constant level, Zn disappeared from this fraction at a rate similar to that of I-‘C-labeled MT, indicating the involvement of MT in Zn metabolism. This was further indicated by the stimulatory effect of S.C. Zn injection on the incorporation of ‘“C-cystine into MT. This stimulatory effect was also shared by S.C. injection of Cu and Hg. Further purification of MT fraction obtained by Sephadex G-75 chromatography on a DEAE-cellulose column revealed two MT peaks which contained more than 90% of the 14C, Cd and Zn put on the column, and high sulfhydryl groups (13.9 and 14.0 -SH/lO,OOO MW, respectively). This verified that ‘C-cystine was indeed incorporated into MT and that Sephadex G-75 chromatography was an adequate separation technique for isolation of MT for the present study. ACKNOWLEDGMENT

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