ABCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Copper-Chelatin:
R. PREMAKUMAR, Department
170,
(1975)
Isolation from Various Sources1
D. R. WINGE,2
of Biochemistry,
~~8-%8
Duke
R. D. WILEY,
University Received
Medical February
AND
Center,
Durham,
Eucaryotic
K. V. RAJAGOPALAN North
Carolina
27710
14, 1975
Low molecular weight copper-containing proteins have been isolated from a variety of eucaryotic sources after exposure of organisms to high levels of copper. The proteins identified in rat kidney, rabbit liver, chicken liver, and in yeast resemble one another and display strong similarity to Cu-chelatin, the protein previously isolated from livers of copper-treated rats. In addition to having similar solubility properties in media containing organic solvents, all these proteins have approximate molecular weights of 6000, display similar absorption spectra and contain high amounts of cysteine, averaging about 15 mol%. Electrophoresis on polyacrylamide gels reveals identical mobility. The major portion of the copper in all proteins is diamagnetic but can be released as eprdetectable Cu*+-EDTA, in the presence of EDTA, by sulthydryl reagents and oxidizing compounds. In yeast, the copper-stimulated formation of Cu-chelatin is dependent on de novo protein synthesis. These results show that in all eucaryotic organisms examined, Cu-chelatin is formed in response to exposure to high levels of copper.
In the preceding papers, the isolation of Cu-chelatin from rat liver cytosol was reported (1, 2). As isolated, the low molecular weight copper-binding protein has a copper content of 4.2%, predominantly in an epr-silent form. Formation of the protein is induced by administered copper by a mechanism which appears to involve transcriptional control (2). Whether chelatin exerts a physiological role in copper storage, absorption and assimilation under nontoxic conditions is yet to be investigated. It has been suggested, for example, that the absorption of copper through the intestinal mucosa is mediated by a copperbinding protein (3, 4). As a prelude to the elucidation of the possible physiological role of Cu-chelatin we have screened a variety of eucaryotic sources for the presence of Cu-chelatin. In this paper we report the presence of Cu-chelatin in yeast,
mung bean, chicken and rabbit livers and rat kidney. Presumptive evidence for its presence in human liver is also presented. MATERIALS
1 This work was supported by Grant No. GM00091 from the United States Public Health Service. z Supported by Predoctoral Traineeship, Grant No. GM-00233 from the National Institutes of Health.
METHODS
3 The abbreviations used are: ClHgBzO,parochloromercuribenzene; EDTA, (ethylenedinitrilo)tetraacetic acid; Nbst, 5,5’-dithiobis(2-nitrobenzoic acid). 278
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
Sephadex G-75, G-50 and G-25 were purchased from Pharmacia. Spectrophotometric grade acetone was obtained from Matheson, Coleman and Bell. DNase and RNase were purchased from Worthingten, ClHgBz03-sulfonic acid and cycloheximide from Sigma, and Nbs, from Aldrich. Animals. Male Sprague-Dawley rats weighing 200-250 g, male New Zealand rabbits weighing about 2.6 kg and Hubbard-Shaver chickens weighing 1.5 kg were maintained under normal laboratory conditions. Copper was administered once daily to the animals by subcutaneous injection at a dose of 2.5 mg (as CuCL) per kg body weight. Rats and chickens received two injections while the rabbits were given three doses. Animals were killed 24 h after the last injection. Yeast. Saccharomyces cerevisiae var. ellipsoideus ATCC-560 was grown for 24 h at 34°C in Fernbach
DISTRIBUTION
OF
flasks on a rotating platform in a medium described by Gesswagner et al. (5). Copper was added at a final concentration of 0.1 mr+i as CuSOI. The cells were harvested by centrifugation at 15,OOOg for 10 min and washed three times with water. A 30% suspension of the wet yeast cells in 0.01 M potassium phosphate, pH 7.8, was subjected twice to a pressure of 20,000 psi in a French press to rupture the cells. The volume of the homogenate was adjusted with buffer to correspond to a 20% suspension of cells by wet weight. This crude homogenate served as the starting point for the isolation of yeast Cu-chelatin. Mung bean. Mung bean seeds (Phaseolus aureus) were obtained from a local store and were germinated at 30°C in the dark for 48 h. The seedlings were grown under controlled conditions for 7 days in trays containing the salt solution described by Arnon and Hoagland (6) at one-half strength. Copper concentrations up to 10-j M CuS04 were used in the growth medium. The shoots and leaves were blended in five volumes of 0.01 M potassium phosphate, pH 7.8. Human liver. Livers of patients who had died of coronary thrombosis were obtained at autopsy. The liver tissue was processed in the same manner as described for other tissues. Centrifugations were carried out in refrigerated Sorvall centrifuges, while preparative ultracentrifugation was performed in a Spinco Model L ultracentrifuge. Concentration of samples was achieved by lyophilization in a VirTis lyophilizer. Absorption spectra were recorded with a Cary Model 14 spectrophotometer. Acrylamide-gel electrophoresis at pH 8.9 was carried out according to the method of Jovin et al. (7). Amino acid analysis was performed with a Beckman Model 120C analyzer. Performic acid oxidation for cysteine determination was carried out according to Hirs (8). Protein was determined by the method of Lowry et al. (9) with bovine serum albumin as the standard. Copper analysis was performed on a Perkin-Elmer 107 atomic absorption spectrometer equipped with a heated graphite atomizer (HGA2000). Epr spectroscopy was performed with a Varian E9HF spectometer equipped with a 9.5-GHz microwave bridge assembly. All epr recordings were performed on samples at -100°C at a modulation frequency of 100 KHz. RESULTS
Uptake of Injected Copper by Rat Tissues Cu-chelatin was originally isolated as the component accumulating in rat liver as a consequence of injection of high levels of CuCl, into the animals (1). The striking increase in copper content of livers of copper-injected rats is evident from the data
279
COPPER-CHELATIN
in Table I which also shows that, among all the other tissues examined, kidney was the only tissue showing increased uptake of the metal. It was thus apparent that, besides liver, kidney was the only tissue in which significant induction of Cu-chelatin could be a possible response to copper administration. Presence of Cu-Chelatin in Rat Kidney and Other Systems After exposure of the organisms to excess copper, kidney from rats, livers from chickens and rabbits, and seedlings of mung bean were examined for the presence of Cu-chelatin. Homogenates were prepared in five volumes of 0.01 M potassium phosphate, pH 7.8. All homogenates were centrifuged at 27,000g for 10 min, followed by centrifugation of the resultant supernatant fluids at 100,OOOgfor 60 min to obtain nonparticulate preparations. The high speed supernatant fractions were maintained at 60°C for 10 min, and the resulting coagulated material was removed by centrifugation at 27,000g for 15 min. The clear supernatant fluid in each case retained a large proportion of the excess copper. These soluble extracts were concentrated by lyophilization and chromatographed on Sephadex G-75 (85 X 4 cm) equilibrated and eluted with 0.01 M potassium phosphate, pH 7.8. Figure 1 shows the elution profiles of the soluble
UPTAKE
OF COPPER
Tissue
Liver Kidney Brain Heart Spleen Lung
TABLE I BY VARIOUS Control (CLg of Cu/g)
4.8 8.9 2.2 6.2 5.7 4.1
RAT TISSUES” Cu-treated (PLP of Cu/g)
63.7 20.5 2.3 6.7 4.3 4.2
a Tissues were excised from rats given repeated intraperitoneal injections of CuCl*. Twenty percent homogenates were prepared with 0.1 M potassium phosphate at pH 7.8. Copper was analyzed in ashed aliquots of the crude homogenates.
ET AL.
PREMAKUMAR
YEAS
001
J’ p.&
’ A40
20
60
60 Tube
NumWr
FIG. 1. Sephadex G-75 elution profiles of the soluble protein extracts of yeast, rabbit liver, chicken liver and rat kidney. The extracts were prepared as described in the text. The column (85 x 4 cm) was operated at 4°C and eluted with 0.01 M potassium phosphate, pH 7.8, at a flow rate of 25 ml/h. The elution profiles of absorbance at 280 nm (-) and copper content (- - -) are indicated.
I-d-B-i
Ic 4.0B ‘;
-
0.3 7 I , -L. -02 $
Pp
3.0-
:: s 2.011 2 4 IO-
3 -
I 20
FIG. Column
2. Sephadex conditions
40
60
Tube
80
?
loo Number
G-75 elution profile of the are the same as in Fig. 1.
protein fractions from yeast, rabbit liver, chicken liver and rat kidney. Analysis of the eluate fractions revealed that each extract contained a major copper-containing component eluting with an apparent molecular weight of 8000. In each case, only a minor copper peak was observed in this region in preparations obtained from organisms not exposed to copper. A coppercontaining fraction of similar molecular weight was observed when the soluble protein extract of a mixture of leaves and
0.1
heat-stable
protein
extract
of mung
bean.
shoots of Phaseolus aureus was chromatographed on Sephadex G-75 (Fig. 2). This material was absent from an extract of the roots of copper-treated mung bean seedlings. In each case, the chelatinlike component was divided into two fractions, the leading half of the peak being labeled a! and the trailing half p (Figs. 1 and 2). The CYand /3 fractions were subjected to acetone fractionation and the precipitates formed at O40, 40-60 and 60-80% acetone (v/v) were
DISTRIBUTION
OF
collected by centrifugation at 27,000g for 5 min. In all cases there was selective enrichment of copper in the 60-80% acetone fraction. In subsequent studies the latter material was used for further analyses of the copper-containing component from each source. The above purification procedure, which had earlier been used for isolation of rat liver Cu-chelatin with a copper content of 2.5%, yielded preparations of similar purity from rabbit liver, chicken liver and yeast. An additional step in the purification of the copper-protein from yeast was necessitated by the presence of contaminating nucleic acid breakdown products. The sample obtained by acetone fractionation was made 5 mM with MgCl, and incubated for
COPPER-CHELATIN
283
successive 30-min periods with 20 and 60 units of DNase and RNase, respectively, per mg of sample. The solution was then chromatographed on a 30 x 2.4 cm column of Sephadex G-25 eluted with 0.01 M potassium phosphate, pH 7.8, to remove the resultant nucleotides. This treatment had no effect on the properties of the Cu-chelatin itself. Cu-chelatin samples from various sources were examined by electrophoresis on polyacrylamide gels at pH 8.9 and staining with Coomassie blue. The results of electrophoresis of the copper protein preparations from yeast, rabbit liver, chicken liver and rat kidney are shown in Fig. 3. Major protein bands with mobilities identical to that of purified rat liver Cu-chelatin
FIG. 3. Polyacrylamide-gel electrophoresis of copper-protein preparations from various sources. Electrophoresis of samples (50 pg) on 7.5% polyacrylamide gels was performed at 2.5 mA per gel. Migration was toward the anode (bottom). The gels were stained with Coomassie blue and destained in a vat. Gel A contained rat liver Cu-chelatin. The other gels contained copper proteins from: B, yeast; C, rabbit liver; D, chicken liver; and E, rat kidney.
282
PREMAKUMAR
(Fig. 3A) were observed in all samples, justifying the conclusion that in all cases the proteins giving rise to this band are quite similar to Cu-chelatin. The rabbit liver preparation showing two stained bands (Fig. 3C) had a copper content exceeding 3%. The two protein bands from unstained gels were extracted by homogenization of the gel segments. Reelectrophoresis of the extracted proteins revealed that the cathodal protein component yielded both. bands whereas the anoda1 component contained only the single anodal band, indicating that the two protein components are related, with the anoda1 component arising from the other. The only staining seen with the mung bean preparation was a broad band migrating with the tracking dye, bromophenol blue. The mung bean sample, besides having a low &/protein ratio, also exhibited an intense brown color presumably due to contaminating polyphenols. Properties
of Cu-Chelatin Sources
from Various
Purified Cu-chelatin from chicken liver, rabbit liver and yeast exhibited only a slight yellowish hue at copper concentrations of 1 mM, and no absorption bands were observed in the visible spectral region. Ultraviolet absorption spectroscopy of the samples revealed spectra similar to that of rat liver Cu-chelatin (Fig. 4), reflecting metal-protein interaction. Extraction of the Cu-chelatin from all the five sources mentioned earlier with chloroform and ethanol according to the procedure of Tsuchihashi (10) resulted in the recovery of the chelatin in the ethanol phase. Chromatography of the Cu-chelatins of chicken liver and yeast on Sephadex G-50 (180 x 1.5 cm) by elution with 0.01 M potassium phosphate, pH 7.8, revealed the presence of the two copper-containing species exhibiting similar ultraviolet absorption and epr properties. Identical behavior of rat liver Cu-chelatin was previously shown to result from reversible aggregation (1). The molecular weights of the various Cu-chelatins were estimated by gel filtration on Sephadex G-50 (60 x 1.5 cm) with ovalbumin, carbonic anhydrase,
ET
AL.
220
240
260
280
300
“Ill
FIG. 4. Ultraviolet absorption spectra of Cu-chelatin from rat liver (---), yeast (....), chicken liver (- - -), and rabbit liver (-.-.) at a concentration of 100 pg/ml in 0.01 M potassium phosphate, pH 7.8.
myoglobin and cytochrome c as standards. Analogous to rat liver Cu-chelatin, the major, lower molecular weight copper proteins from yeast, chicken and rabbit showed approximate molecular weights of 8000. The most distinctive feature in the amino acid composition of rat liver Cuchelatin is the cysteine content of 14.6 mol% (1). Table II shows that the amino acid composition of yeast Cu-chelatin is quite similar to that of rat liver Cu-chelatin. Acid hydrolysates of performic acidoxidized Cu-chelatins from chicken and rabbit livers were found to contain 16.2 and 12.8 mol%, respectively, of cysteic acid. Since the chicken liver Cu-chelatin preparation was inhomogenous, the aggregate form of chelatin obtained after elution from Sephadex G-50 was used for this purpose. These results show that the cysteine contents of chicken and rabbit Cu-chelatins are quite similar to those of the proteins from rat liver and yeast. Epr Properties Examination of the Cu-chelatins by epr spectroscopy, after either Sephadex G-75 chromatography or acetone fractionation, revealed that the metal was primarily in
DISTRIBUTION TABLE AMINO
ACID
Residue
LYS His Arg ASP Thr Ser Glu Pro GUY Ala Cysb Val Met Ile Leu Tyr Phe
II
COMPOSITION OF CU-CHELATIN RAT LIVER AND YEASTY
Yeast
OF
Cu-chelatin (mol%)
FROM
Rat liver Cuchelatin (mol%)
8.8 1.5 3.6 11.1 4.4 5.4 16.3 ’ 4.2 12.0 6.5 13.0 4.1 0.4 2.4 3.2 1.0 2.0
D Values represent duplicate analyses drolysates. b Determined as cysteic acid.
13.1 1.1 2.7 9.8 5.1 4.2 11.1 3.6 8.0 6.0 14.6 5.2 1.6 4.3 6.3 1.3 2.1 of 24-h hy-
the diamagnetic state. Table III shows the percentage of the total copper in the native proteins from the various sources in the paramagnetic state. These data were obtained by recording the epr signal of the protein in 10 mM EDTA before and after incubation for 10 min at 100°C. It was previously demonstrated that such treatment releases all of the copper from rat liver Cuchelatin as Cu2+-EDTA. As can be seen in Table III, native chicken liver Cu-chelatin shows no paramagnetism, whereas the chelatin from other sources shows varying extents of muted paramagnetism. The slight paramagnetism in Cu-chelatins from rabbit liver, rat liver and yeast could possibly be due to labilization of the copper from its interaction with the protein in the course of lyophilization. Incubation of the chicken liver chelatin in 10 mM EDTA at 100°C anaerobically did not cause the signal augmentation observed after aerobic incubation (Fig. 5). Anaerobically, the conversion to paramagnetism could be accomplished by incu-
283
COPPER-CHELATIN
bating the protein in EDTA with 1 mM ClHgBzO-sulfonic acid (Fig. 6). Sulthydryl reagents (ClHgBzO-sulfonic acid and I%&, oxidizing reagents [Fe(CN)63and (NH4)&Osl and metal ions (Ag+ and Hg? were found to release copper in the case of the proteins from rabbit, chicken and yeast. The epr behavior of these proteins thus closely resembles that of rat liver Cuchelatin in this property as well. Presence
of Cu-Chelatin
in Human
Liver
Two post-mortem human livers were processed by the standard procedure for identification of Cu-chelatin. The heattreated supernatant fractions were chromatographed on Sephadex G-75 under conditions similar to those described earlier. As can be seen from the elution profile (Fig. 7), there is nonidentity in the copper and zinc elution patterns in the region of Cu-chelatin. The predominant metal in this region is zinc, with only trace levels of cadmium present. The elution profile of cadmium showed congruence with that of zinc. The difference in the elution profiles of copper and zinc was reproducible and was seen in extracts from both human liver samples. Examination of liver extracts from rats administered cadmium reveals that cadmium and zinc elute in congruence in conjunction with metallothiTABLE EPR-DETECTABLE
III
COPPER IN CU-CHELATIN VARIOUS SOIJRCES~
Source
FROM
Percent native magnetism
Rat liver Rabbit liver Chicken liver Rat kidney Yeast Mung bean a Values represent signal amplitudes proteins in 10 mM EDTA as percent of tudes of corresponding samples incubated at 100°C. The following epr conditions Modulation amplitude, 10 G; microwave mW; temperature, -100°C; microwave 9.105 GHz; time constant, 1.0 s; scan Gimin.
para-
17 12 0 24 13 36 of native the amplifor 10 min were used: power, 20 frequency, rate, 250
284
ET AL.
PREMAKUMAR
I
I
2600
2800
I 3ooo GAUSS
3200
3400
FIG. 5. Epr spectroscopy of chicken liver Cu-chelatin. All samples contained Cu-chelatin corresponding to 0.36 mM copper in 10 mM EDTA. Spectrum A is of the sample in EDTA. Spectrum B is the recording after the sample was incubated anaerobically for 5 min at 100°C. Spectrum C is the trace after readmission of air and further incubation for 5 min at 100°C. The spectra were obtained under the following epr conditions: Temperature, -100°C; modulation amplitude, 10 G; microwave power, 20 mW; microwave frequency, 9.105 GHz; time constant, 1.0 a; receiver gain, 5000; and scan rate 250 g/mm.
I 2600
I 2800
I 3000 GAUSS
1 3200
3400
FIG. 6. Effect of ClHgBzO-sulfonic acid on the epr signal of chicken liver Cu-chelatin. The same sample was used as that in Fig. 5 but at a concentration of 0.18 mM copper. Spectrum A is of the protein in 10 mM EDTA recorded anaerobically. Spectrum B is of the protein in 10 mM EDTA mixed with 1 mM
onein, whereas the copper of Cu-chelatin reproducibly elutes at a slightly later volume compared to zinc. Fractions corresponding to (Y and /3 (Fig. 7) were pooled, concentrated and subjected to acetone fractionation. The 60-80% fractions which contained both metal components were electrophoresed on polyacrylamide gels. Figure 8 shows the Coomassie blue-staining pattern for the (Y and p human samples and comparative gels containing rat liver Cu-chelatin and Cd-thionein. According to the manner in which the (Y and p samples were pooled, the Zncomponent was primarily in the LYfraction and the Cu-component largely in the /3 fraction. As can be seen in Fig. 8, the Q! fraction showed a band, less prominent in ClHgBzO-sulfonic tions were identical
acid anaerobically. Epr condito those given in Fig. 5.
DISTRIBUTION
OF
285
COPPER-CHELATIN
I
I-
140 630 r.
8
04-
'I 20 g
03-
B a
02-
IO
Ol20
40
60
SO
Tube
100
Number
120
140
FIG. 7. Sephadex G-75 elution profile of the heat-stable protein Chromatographic conditions are similar to those described in Fig. absorbance at 280 nm (-), copper content (- - -) and zinc content
FIG.
human fraction;
160
fraction of human liver. 1. The elution profiles of (-.-.) are indicated.
8. Polyacrylamide-gel electrophoresis of acetone-fractionated a and /3 fractions of liver metal proteins. Conditions were identical to those described in Fig. 3. A, (Y B, p fraction; C, rat liver Cu-chelatin; and D, rat liver Cd-thionein.
286
PREMAKUMAR
ET
the /3 fraction, which has a mobility corresponding to metallothionein. Similarly, the p fraction showed enrichment in a protein band that has the mobility corresponding to Cu-chelatin. Epr spectroscopy of the copper-rich fraction from human liver revealed that none of the copper was present in the paramagnetic form (Fig. 9). One intriguing aspect is that aerobic incubation at 100°C did not release the copper from the human protein. However, ClHgBzO-sulfonic acid or AgN03 was effective in displacing the protein-bound copper.
synthesis. For this, the effect of cycloheximide, an inhibitor of protein synthesis, on the content of the protein in yeast in the stationary phase was studied. Copper sulfate (0.1 mM final concentration) was added to the medium, and the culture was incubated at 34°C on a rotating platform. One hour before harvesting, 13Hllysine (25 pCi/liter) was added to all flasks. One culture was pretreated with cycloheximide (10 pg/ml) for 1 h prior to addition of the copper. Cells were harvested 6 h after addition of copper and processed as described in Materials and Methods. The soluble protein fraction was chromatographed on Sephadex G-75 under the standard conditions described earlier. Analysis of the fraction corresponding to Cu-chelatin showed a fivefold increase in copper content and a 30-fold increase in [?Hllysine incorporation in cells exposed to the metal. Cycloheximide completely inhibited the copper-stimulated labeling, suggesting that the accumulation of Cu-
Formation of Cu-Chelatin in Yeast Yeast grown in the presence of 0.1 mM copper showed an appreciable quantity of Cu-chelatin (fractions llO-130), whereas in the absence of the added metal in the culture medium only a trace of the metalloprotein was detected (Fig. 10). It was of interest to determine whether formation of chelatin was dependent on de nouo protein
I
I
I
2600
2000
AL.
I 3ooo GAUSS
I
I
3200
3400
FIG. 9. Epr spectroscopy of copper protein from human liver. The fraction was used at 0.1 rnM copper concentration in 10 mM EDTA. A, sample in EDTA; and B, sample after aerobic incubation at 100°C for 10 min. Spectra C and D are of the protein after 2-min aerobic incubation with 10 mM AgNO, and 10 mM ClHgBzO-sulfonic acid, respectively.
DISTRIBUTION
40
OF
60
80 Tube
287
COPPER-CHELATIN
I00
I20
140
Number
FIG. 10. Sephadex G-75 column chromatography of the soluble protein fraction from yeast grown in the absence (A) and presence (B) of 0.1 mM CuSO,. Each sample represents the soluble protein fraction of a l-liter culture of yeast (about 18 g wet weight of cells) grown for 24 h at 34°C. Cu-chelatin eluted between fractions 110 and 130. Column conditions are as described in Fig. 1.
chelatin in yeast is dependent on protein synthesis. Cycloheximide had no effect on entry of copper into yeast cells but did prevent the increase in the Cu-chelatin fraction. The sensitivity of this induction to actinomycin D could not be tested due to the inefficient accumulation of the inhibitor in intact yeast cells. DISCUSSION
Low molecular weight copper proteins have been isolated from rat kidney, rabbit liver, chicken liver, yeast and mung beans after exposure of the organisms to copper. The copper proteins are similar to rat liver Cu-chelatin in the following properties: a) All of the proteins have molecular weights of approximately 8000, assuming a globular conformation; b) they are precipitated in the 6040% acetone fraction and are soluble in the ethanolic phase on Tsuchihashi extraction; c) all are stable on heating to 60°C; d) they exhibit similar ultraviolet absorption spectra; e) they show a cysteine content of about 15 mol%; f, all of them except the sample from mung bean are visualized as stained protein bands of identical mobility on polyacrylamide gel electrophoresis; and g) all of them contain
predominantly diamagnetic copper which can be released from the protein by sullhydry1 reagents and oxidizing compounds. Because of the degree of identity, it may be concluded that the protein isolated from each source is the idiotype of Cu-chelatin previously isolated from rat liver. This suggests that Cu-chelatin may have universal occurrence in all eucaryotic organisms. Whether the protein is present in procaryotic organisms is uncertain. It has not been possible to induce its formation in Escherichia coli. The present studies have indicated that in human liver two distinct metalloproteins are present, one containing zinc and cadmium while the other contains copper. The protein eluting earlier contains predominantly zinc with traces of cadmium. On acrylamide gels it shows a protein band with electrophoretic mobility similar to that of Cd-thionein. Pulido et al. have reported the purification of metallothionein from human renal cortex containing primarily cadmium and zinc (11). The Cu-component of human liver has an electrophoretic mobility identical to that of rat liver Cu-chelatin. Furthermore, the copper is present on the protein in a
288
PREMAKUMAR
diamagnetic form and is released by sulfiydry1 and oxidizing reagents as is the copper on Cu-chelatin. These findings warrant the conclusion that, in human liver, both metallothionein and Cu-chelatin are present. The level of Cu-chelatin in yeast is determined by the copper concentration of the medium. However, yeast cells grown under identical conditions but in the presence of 0.01 mM CdC12, ZnCl, or HgClz do not appear to accumulate the related protein, metallothionein. This differential induction further illustrates the nonidentity of these two metalloproteins. As yet no definitive physiological role for Cu-chelatin has been established. Because of the widespread occurrence of Cuchelatin, it may be speculated that the protein exerts a significant role in copper homeostasis and possibly in other biological functions. ACKNOWLEDGMENTS We thank Dr. Randall Alberte for assistance growing mung beans and Dr. Howard Steinman assistance in amino acid analysis.
in for
ET
AL. REFERENCES
1. WINGE, D. R., PREMAKUMAR, R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. B&hem. Biophys. 170, 253-266. 2. PREMAKUMAR, R., WINGE, D. R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 267-2’71. 3. STARCHER, B. C. (1969) J. Nutr. 97,321-326. 4. EVANS, G. W., MAJORS, P. F., AND CORNATZER, W. E. (1970) Biochem. Biophys. Res. Commun. 40, 1142-1147. D., ALTMANN, H., SZILVINYI, A. 5. GESSWAGNER, V., AND KAINDL, K. (1968) Znt. J. Appl. Ra&at. Zsotop. 19, 152-153. 6. ARNON, D. I., AND HOAGLAND, D. R. (1940) Soil Sci. 50, 463-485. A., AND NAUGHTON, M. I. JOVIN, T., CHRAMBACH, A. (1964) Anal. Biochem. 9, 351-369. 8. HIRS, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, p. 59, Academic Press, New York. 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem 193, 265-275. M. (1923) Biochem. 2. 140, 63LO. TSUCHIHASHI, 112. 11. PULIDO, R., KAGI, J. H. R., AND VALLEE, B. L. (1966) Biochemistry 5, 1768-1777.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Copper-Chelatin:
R. PREMAKUMAR, Department
170,
(1975)
Isolation from Various Sources1
D. R. WINGE,2
of Biochemistry,
~~8-%8
Duke
R. D. WILEY,
University Received
Medical February
AND
Center,
Durham,
Eucaryotic
K. V. RAJAGOPALAN North
Carolina
27710
14, 1975
Low molecular weight copper-containing proteins have been isolated from a variety of eucaryotic sources after exposure of organisms to high levels of copper. The proteins identified in rat kidney, rabbit liver, chicken liver, and in yeast resemble one another and display strong similarity to Cu-chelatin, the protein previously isolated from livers of copper-treated rats. In addition to having similar solubility properties in media containing organic solvents, all these proteins have approximate molecular weights of 6000, display similar absorption spectra and contain high amounts of cysteine, averaging about 15 mol%. Electrophoresis on polyacrylamide gels reveals identical mobility. The major portion of the copper in all proteins is diamagnetic but can be released as eprdetectable Cu*+-EDTA, in the presence of EDTA, by sulthydryl reagents and oxidizing compounds. In yeast, the copper-stimulated formation of Cu-chelatin is dependent on de novo protein synthesis. These results show that in all eucaryotic organisms examined, Cu-chelatin is formed in response to exposure to high levels of copper.
In the preceding papers, the isolation of Cu-chelatin from rat liver cytosol was reported (1, 2). As isolated, the low molecular weight copper-binding protein has a copper content of 4.2%, predominantly in an epr-silent form. Formation of the protein is induced by administered copper by a mechanism which appears to involve transcriptional control (2). Whether chelatin exerts a physiological role in copper storage, absorption and assimilation under nontoxic conditions is yet to be investigated. It has been suggested, for example, that the absorption of copper through the intestinal mucosa is mediated by a copperbinding protein (3, 4). As a prelude to the elucidation of the possible physiological role of Cu-chelatin we have screened a variety of eucaryotic sources for the presence of Cu-chelatin. In this paper we report the presence of Cu-chelatin in yeast,
mung bean, chicken and rabbit livers and rat kidney. Presumptive evidence for its presence in human liver is also presented. MATERIALS
1 This work was supported by Grant No. GM00091 from the United States Public Health Service. z Supported by Predoctoral Traineeship, Grant No. GM-00233 from the National Institutes of Health.
METHODS
3 The abbreviations used are: ClHgBzO,parochloromercuribenzene; EDTA, (ethylenedinitrilo)tetraacetic acid; Nbst, 5,5’-dithiobis(2-nitrobenzoic acid). 278
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
Sephadex G-75, G-50 and G-25 were purchased from Pharmacia. Spectrophotometric grade acetone was obtained from Matheson, Coleman and Bell. DNase and RNase were purchased from Worthingten, ClHgBz03-sulfonic acid and cycloheximide from Sigma, and Nbs, from Aldrich. Animals. Male Sprague-Dawley rats weighing 200-250 g, male New Zealand rabbits weighing about 2.6 kg and Hubbard-Shaver chickens weighing 1.5 kg were maintained under normal laboratory conditions. Copper was administered once daily to the animals by subcutaneous injection at a dose of 2.5 mg (as CuCL) per kg body weight. Rats and chickens received two injections while the rabbits were given three doses. Animals were killed 24 h after the last injection. Yeast. Saccharomyces cerevisiae var. ellipsoideus ATCC-560 was grown for 24 h at 34°C in Fernbach
DISTRIBUTION
OF
flasks on a rotating platform in a medium described by Gesswagner et al. (5). Copper was added at a final concentration of 0.1 mr+i as CuSOI. The cells were harvested by centrifugation at 15,OOOg for 10 min and washed three times with water. A 30% suspension of the wet yeast cells in 0.01 M potassium phosphate, pH 7.8, was subjected twice to a pressure of 20,000 psi in a French press to rupture the cells. The volume of the homogenate was adjusted with buffer to correspond to a 20% suspension of cells by wet weight. This crude homogenate served as the starting point for the isolation of yeast Cu-chelatin. Mung bean. Mung bean seeds (Phaseolus aureus) were obtained from a local store and were germinated at 30°C in the dark for 48 h. The seedlings were grown under controlled conditions for 7 days in trays containing the salt solution described by Arnon and Hoagland (6) at one-half strength. Copper concentrations up to 10-j M CuS04 were used in the growth medium. The shoots and leaves were blended in five volumes of 0.01 M potassium phosphate, pH 7.8. Human liver. Livers of patients who had died of coronary thrombosis were obtained at autopsy. The liver tissue was processed in the same manner as described for other tissues. Centrifugations were carried out in refrigerated Sorvall centrifuges, while preparative ultracentrifugation was performed in a Spinco Model L ultracentrifuge. Concentration of samples was achieved by lyophilization in a VirTis lyophilizer. Absorption spectra were recorded with a Cary Model 14 spectrophotometer. Acrylamide-gel electrophoresis at pH 8.9 was carried out according to the method of Jovin et al. (7). Amino acid analysis was performed with a Beckman Model 120C analyzer. Performic acid oxidation for cysteine determination was carried out according to Hirs (8). Protein was determined by the method of Lowry et al. (9) with bovine serum albumin as the standard. Copper analysis was performed on a Perkin-Elmer 107 atomic absorption spectrometer equipped with a heated graphite atomizer (HGA2000). Epr spectroscopy was performed with a Varian E9HF spectometer equipped with a 9.5-GHz microwave bridge assembly. All epr recordings were performed on samples at -100°C at a modulation frequency of 100 KHz. RESULTS
Uptake of Injected Copper by Rat Tissues Cu-chelatin was originally isolated as the component accumulating in rat liver as a consequence of injection of high levels of CuCl, into the animals (1). The striking increase in copper content of livers of copper-injected rats is evident from the data
279
COPPER-CHELATIN
in Table I which also shows that, among all the other tissues examined, kidney was the only tissue showing increased uptake of the metal. It was thus apparent that, besides liver, kidney was the only tissue in which significant induction of Cu-chelatin could be a possible response to copper administration. Presence of Cu-Chelatin in Rat Kidney and Other Systems After exposure of the organisms to excess copper, kidney from rats, livers from chickens and rabbits, and seedlings of mung bean were examined for the presence of Cu-chelatin. Homogenates were prepared in five volumes of 0.01 M potassium phosphate, pH 7.8. All homogenates were centrifuged at 27,000g for 10 min, followed by centrifugation of the resultant supernatant fluids at 100,OOOgfor 60 min to obtain nonparticulate preparations. The high speed supernatant fractions were maintained at 60°C for 10 min, and the resulting coagulated material was removed by centrifugation at 27,000g for 15 min. The clear supernatant fluid in each case retained a large proportion of the excess copper. These soluble extracts were concentrated by lyophilization and chromatographed on Sephadex G-75 (85 X 4 cm) equilibrated and eluted with 0.01 M potassium phosphate, pH 7.8. Figure 1 shows the elution profiles of the soluble
UPTAKE
OF COPPER
Tissue
Liver Kidney Brain Heart Spleen Lung
TABLE I BY VARIOUS Control (CLg of Cu/g)
4.8 8.9 2.2 6.2 5.7 4.1
RAT TISSUES” Cu-treated (PLP of Cu/g)
63.7 20.5 2.3 6.7 4.3 4.2
a Tissues were excised from rats given repeated intraperitoneal injections of CuCl*. Twenty percent homogenates were prepared with 0.1 M potassium phosphate at pH 7.8. Copper was analyzed in ashed aliquots of the crude homogenates.
ET AL.
PREMAKUMAR
YEAS
001
J’ p.&
’ A40
20
60
60 Tube
NumWr
FIG. 1. Sephadex G-75 elution profiles of the soluble protein extracts of yeast, rabbit liver, chicken liver and rat kidney. The extracts were prepared as described in the text. The column (85 x 4 cm) was operated at 4°C and eluted with 0.01 M potassium phosphate, pH 7.8, at a flow rate of 25 ml/h. The elution profiles of absorbance at 280 nm (-) and copper content (- - -) are indicated.
I-d-B-i
Ic 4.0B ‘;
-
0.3 7 I , -L. -02 $
Pp
3.0-
:: s 2.011 2 4 IO-
3 -
I 20
FIG. Column
2. Sephadex conditions
40
60
Tube
80
?
loo Number
G-75 elution profile of the are the same as in Fig. 1.
protein fractions from yeast, rabbit liver, chicken liver and rat kidney. Analysis of the eluate fractions revealed that each extract contained a major copper-containing component eluting with an apparent molecular weight of 8000. In each case, only a minor copper peak was observed in this region in preparations obtained from organisms not exposed to copper. A coppercontaining fraction of similar molecular weight was observed when the soluble protein extract of a mixture of leaves and
0.1
heat-stable
protein
extract
of mung
bean.
shoots of Phaseolus aureus was chromatographed on Sephadex G-75 (Fig. 2). This material was absent from an extract of the roots of copper-treated mung bean seedlings. In each case, the chelatinlike component was divided into two fractions, the leading half of the peak being labeled a! and the trailing half p (Figs. 1 and 2). The CYand /3 fractions were subjected to acetone fractionation and the precipitates formed at O40, 40-60 and 60-80% acetone (v/v) were
DISTRIBUTION
OF
collected by centrifugation at 27,000g for 5 min. In all cases there was selective enrichment of copper in the 60-80% acetone fraction. In subsequent studies the latter material was used for further analyses of the copper-containing component from each source. The above purification procedure, which had earlier been used for isolation of rat liver Cu-chelatin with a copper content of 2.5%, yielded preparations of similar purity from rabbit liver, chicken liver and yeast. An additional step in the purification of the copper-protein from yeast was necessitated by the presence of contaminating nucleic acid breakdown products. The sample obtained by acetone fractionation was made 5 mM with MgCl, and incubated for
COPPER-CHELATIN
283
successive 30-min periods with 20 and 60 units of DNase and RNase, respectively, per mg of sample. The solution was then chromatographed on a 30 x 2.4 cm column of Sephadex G-25 eluted with 0.01 M potassium phosphate, pH 7.8, to remove the resultant nucleotides. This treatment had no effect on the properties of the Cu-chelatin itself. Cu-chelatin samples from various sources were examined by electrophoresis on polyacrylamide gels at pH 8.9 and staining with Coomassie blue. The results of electrophoresis of the copper protein preparations from yeast, rabbit liver, chicken liver and rat kidney are shown in Fig. 3. Major protein bands with mobilities identical to that of purified rat liver Cu-chelatin
FIG. 3. Polyacrylamide-gel electrophoresis of copper-protein preparations from various sources. Electrophoresis of samples (50 pg) on 7.5% polyacrylamide gels was performed at 2.5 mA per gel. Migration was toward the anode (bottom). The gels were stained with Coomassie blue and destained in a vat. Gel A contained rat liver Cu-chelatin. The other gels contained copper proteins from: B, yeast; C, rabbit liver; D, chicken liver; and E, rat kidney.
282
PREMAKUMAR
(Fig. 3A) were observed in all samples, justifying the conclusion that in all cases the proteins giving rise to this band are quite similar to Cu-chelatin. The rabbit liver preparation showing two stained bands (Fig. 3C) had a copper content exceeding 3%. The two protein bands from unstained gels were extracted by homogenization of the gel segments. Reelectrophoresis of the extracted proteins revealed that the cathodal protein component yielded both. bands whereas the anoda1 component contained only the single anodal band, indicating that the two protein components are related, with the anoda1 component arising from the other. The only staining seen with the mung bean preparation was a broad band migrating with the tracking dye, bromophenol blue. The mung bean sample, besides having a low &/protein ratio, also exhibited an intense brown color presumably due to contaminating polyphenols. Properties
of Cu-Chelatin Sources
from Various
Purified Cu-chelatin from chicken liver, rabbit liver and yeast exhibited only a slight yellowish hue at copper concentrations of 1 mM, and no absorption bands were observed in the visible spectral region. Ultraviolet absorption spectroscopy of the samples revealed spectra similar to that of rat liver Cu-chelatin (Fig. 4), reflecting metal-protein interaction. Extraction of the Cu-chelatin from all the five sources mentioned earlier with chloroform and ethanol according to the procedure of Tsuchihashi (10) resulted in the recovery of the chelatin in the ethanol phase. Chromatography of the Cu-chelatins of chicken liver and yeast on Sephadex G-50 (180 x 1.5 cm) by elution with 0.01 M potassium phosphate, pH 7.8, revealed the presence of the two copper-containing species exhibiting similar ultraviolet absorption and epr properties. Identical behavior of rat liver Cu-chelatin was previously shown to result from reversible aggregation (1). The molecular weights of the various Cu-chelatins were estimated by gel filtration on Sephadex G-50 (60 x 1.5 cm) with ovalbumin, carbonic anhydrase,
ET
AL.
220
240
260
280
300
“Ill
FIG. 4. Ultraviolet absorption spectra of Cu-chelatin from rat liver (---), yeast (....), chicken liver (- - -), and rabbit liver (-.-.) at a concentration of 100 pg/ml in 0.01 M potassium phosphate, pH 7.8.
myoglobin and cytochrome c as standards. Analogous to rat liver Cu-chelatin, the major, lower molecular weight copper proteins from yeast, chicken and rabbit showed approximate molecular weights of 8000. The most distinctive feature in the amino acid composition of rat liver Cuchelatin is the cysteine content of 14.6 mol% (1). Table II shows that the amino acid composition of yeast Cu-chelatin is quite similar to that of rat liver Cu-chelatin. Acid hydrolysates of performic acidoxidized Cu-chelatins from chicken and rabbit livers were found to contain 16.2 and 12.8 mol%, respectively, of cysteic acid. Since the chicken liver Cu-chelatin preparation was inhomogenous, the aggregate form of chelatin obtained after elution from Sephadex G-50 was used for this purpose. These results show that the cysteine contents of chicken and rabbit Cu-chelatins are quite similar to those of the proteins from rat liver and yeast. Epr Properties Examination of the Cu-chelatins by epr spectroscopy, after either Sephadex G-75 chromatography or acetone fractionation, revealed that the metal was primarily in
DISTRIBUTION TABLE AMINO
ACID
Residue
LYS His Arg ASP Thr Ser Glu Pro GUY Ala Cysb Val Met Ile Leu Tyr Phe
II
COMPOSITION OF CU-CHELATIN RAT LIVER AND YEASTY
Yeast
OF
Cu-chelatin (mol%)
FROM
Rat liver Cuchelatin (mol%)
8.8 1.5 3.6 11.1 4.4 5.4 16.3 ’ 4.2 12.0 6.5 13.0 4.1 0.4 2.4 3.2 1.0 2.0
D Values represent duplicate analyses drolysates. b Determined as cysteic acid.
13.1 1.1 2.7 9.8 5.1 4.2 11.1 3.6 8.0 6.0 14.6 5.2 1.6 4.3 6.3 1.3 2.1 of 24-h hy-
the diamagnetic state. Table III shows the percentage of the total copper in the native proteins from the various sources in the paramagnetic state. These data were obtained by recording the epr signal of the protein in 10 mM EDTA before and after incubation for 10 min at 100°C. It was previously demonstrated that such treatment releases all of the copper from rat liver Cuchelatin as Cu2+-EDTA. As can be seen in Table III, native chicken liver Cu-chelatin shows no paramagnetism, whereas the chelatin from other sources shows varying extents of muted paramagnetism. The slight paramagnetism in Cu-chelatins from rabbit liver, rat liver and yeast could possibly be due to labilization of the copper from its interaction with the protein in the course of lyophilization. Incubation of the chicken liver chelatin in 10 mM EDTA at 100°C anaerobically did not cause the signal augmentation observed after aerobic incubation (Fig. 5). Anaerobically, the conversion to paramagnetism could be accomplished by incu-
283
COPPER-CHELATIN
bating the protein in EDTA with 1 mM ClHgBzO-sulfonic acid (Fig. 6). Sulthydryl reagents (ClHgBzO-sulfonic acid and I%&, oxidizing reagents [Fe(CN)63and (NH4)&Osl and metal ions (Ag+ and Hg? were found to release copper in the case of the proteins from rabbit, chicken and yeast. The epr behavior of these proteins thus closely resembles that of rat liver Cuchelatin in this property as well. Presence
of Cu-Chelatin
in Human
Liver
Two post-mortem human livers were processed by the standard procedure for identification of Cu-chelatin. The heattreated supernatant fractions were chromatographed on Sephadex G-75 under conditions similar to those described earlier. As can be seen from the elution profile (Fig. 7), there is nonidentity in the copper and zinc elution patterns in the region of Cu-chelatin. The predominant metal in this region is zinc, with only trace levels of cadmium present. The elution profile of cadmium showed congruence with that of zinc. The difference in the elution profiles of copper and zinc was reproducible and was seen in extracts from both human liver samples. Examination of liver extracts from rats administered cadmium reveals that cadmium and zinc elute in congruence in conjunction with metallothiTABLE EPR-DETECTABLE
III
COPPER IN CU-CHELATIN VARIOUS SOIJRCES~
Source
FROM
Percent native magnetism
Rat liver Rabbit liver Chicken liver Rat kidney Yeast Mung bean a Values represent signal amplitudes proteins in 10 mM EDTA as percent of tudes of corresponding samples incubated at 100°C. The following epr conditions Modulation amplitude, 10 G; microwave mW; temperature, -100°C; microwave 9.105 GHz; time constant, 1.0 s; scan Gimin.
para-
17 12 0 24 13 36 of native the amplifor 10 min were used: power, 20 frequency, rate, 250
284
ET AL.
PREMAKUMAR
I
I
2600
2800
I 3ooo GAUSS
3200
3400
FIG. 5. Epr spectroscopy of chicken liver Cu-chelatin. All samples contained Cu-chelatin corresponding to 0.36 mM copper in 10 mM EDTA. Spectrum A is of the sample in EDTA. Spectrum B is the recording after the sample was incubated anaerobically for 5 min at 100°C. Spectrum C is the trace after readmission of air and further incubation for 5 min at 100°C. The spectra were obtained under the following epr conditions: Temperature, -100°C; modulation amplitude, 10 G; microwave power, 20 mW; microwave frequency, 9.105 GHz; time constant, 1.0 a; receiver gain, 5000; and scan rate 250 g/mm.
I 2600
I 2800
I 3000 GAUSS
1 3200
3400
FIG. 6. Effect of ClHgBzO-sulfonic acid on the epr signal of chicken liver Cu-chelatin. The same sample was used as that in Fig. 5 but at a concentration of 0.18 mM copper. Spectrum A is of the protein in 10 mM EDTA recorded anaerobically. Spectrum B is of the protein in 10 mM EDTA mixed with 1 mM
onein, whereas the copper of Cu-chelatin reproducibly elutes at a slightly later volume compared to zinc. Fractions corresponding to (Y and /3 (Fig. 7) were pooled, concentrated and subjected to acetone fractionation. The 60-80% fractions which contained both metal components were electrophoresed on polyacrylamide gels. Figure 8 shows the Coomassie blue-staining pattern for the (Y and p human samples and comparative gels containing rat liver Cu-chelatin and Cd-thionein. According to the manner in which the (Y and p samples were pooled, the Zncomponent was primarily in the LYfraction and the Cu-component largely in the /3 fraction. As can be seen in Fig. 8, the Q! fraction showed a band, less prominent in ClHgBzO-sulfonic tions were identical
acid anaerobically. Epr condito those given in Fig. 5.
DISTRIBUTION
OF
285
COPPER-CHELATIN
I
I-
140 630 r.
8
04-
'I 20 g
03-
B a
02-
IO
Ol20
40
60
SO
Tube
100
Number
120
140
FIG. 7. Sephadex G-75 elution profile of the heat-stable protein Chromatographic conditions are similar to those described in Fig. absorbance at 280 nm (-), copper content (- - -) and zinc content
FIG.
human fraction;
160
fraction of human liver. 1. The elution profiles of (-.-.) are indicated.
8. Polyacrylamide-gel electrophoresis of acetone-fractionated a and /3 fractions of liver metal proteins. Conditions were identical to those described in Fig. 3. A, (Y B, p fraction; C, rat liver Cu-chelatin; and D, rat liver Cd-thionein.
286
PREMAKUMAR
ET
the /3 fraction, which has a mobility corresponding to metallothionein. Similarly, the p fraction showed enrichment in a protein band that has the mobility corresponding to Cu-chelatin. Epr spectroscopy of the copper-rich fraction from human liver revealed that none of the copper was present in the paramagnetic form (Fig. 9). One intriguing aspect is that aerobic incubation at 100°C did not release the copper from the human protein. However, ClHgBzO-sulfonic acid or AgN03 was effective in displacing the protein-bound copper.
synthesis. For this, the effect of cycloheximide, an inhibitor of protein synthesis, on the content of the protein in yeast in the stationary phase was studied. Copper sulfate (0.1 mM final concentration) was added to the medium, and the culture was incubated at 34°C on a rotating platform. One hour before harvesting, 13Hllysine (25 pCi/liter) was added to all flasks. One culture was pretreated with cycloheximide (10 pg/ml) for 1 h prior to addition of the copper. Cells were harvested 6 h after addition of copper and processed as described in Materials and Methods. The soluble protein fraction was chromatographed on Sephadex G-75 under the standard conditions described earlier. Analysis of the fraction corresponding to Cu-chelatin showed a fivefold increase in copper content and a 30-fold increase in [?Hllysine incorporation in cells exposed to the metal. Cycloheximide completely inhibited the copper-stimulated labeling, suggesting that the accumulation of Cu-
Formation of Cu-Chelatin in Yeast Yeast grown in the presence of 0.1 mM copper showed an appreciable quantity of Cu-chelatin (fractions llO-130), whereas in the absence of the added metal in the culture medium only a trace of the metalloprotein was detected (Fig. 10). It was of interest to determine whether formation of chelatin was dependent on de nouo protein
I
I
I
2600
2000
AL.
I 3ooo GAUSS
I
I
3200
3400
FIG. 9. Epr spectroscopy of copper protein from human liver. The fraction was used at 0.1 rnM copper concentration in 10 mM EDTA. A, sample in EDTA; and B, sample after aerobic incubation at 100°C for 10 min. Spectra C and D are of the protein after 2-min aerobic incubation with 10 mM AgNO, and 10 mM ClHgBzO-sulfonic acid, respectively.
DISTRIBUTION
40
OF
60
80 Tube
287
COPPER-CHELATIN
I00
I20
140
Number
FIG. 10. Sephadex G-75 column chromatography of the soluble protein fraction from yeast grown in the absence (A) and presence (B) of 0.1 mM CuSO,. Each sample represents the soluble protein fraction of a l-liter culture of yeast (about 18 g wet weight of cells) grown for 24 h at 34°C. Cu-chelatin eluted between fractions 110 and 130. Column conditions are as described in Fig. 1.
chelatin in yeast is dependent on protein synthesis. Cycloheximide had no effect on entry of copper into yeast cells but did prevent the increase in the Cu-chelatin fraction. The sensitivity of this induction to actinomycin D could not be tested due to the inefficient accumulation of the inhibitor in intact yeast cells. DISCUSSION
Low molecular weight copper proteins have been isolated from rat kidney, rabbit liver, chicken liver, yeast and mung beans after exposure of the organisms to copper. The copper proteins are similar to rat liver Cu-chelatin in the following properties: a) All of the proteins have molecular weights of approximately 8000, assuming a globular conformation; b) they are precipitated in the 6040% acetone fraction and are soluble in the ethanolic phase on Tsuchihashi extraction; c) all are stable on heating to 60°C; d) they exhibit similar ultraviolet absorption spectra; e) they show a cysteine content of about 15 mol%; f, all of them except the sample from mung bean are visualized as stained protein bands of identical mobility on polyacrylamide gel electrophoresis; and g) all of them contain
predominantly diamagnetic copper which can be released from the protein by sullhydry1 reagents and oxidizing compounds. Because of the degree of identity, it may be concluded that the protein isolated from each source is the idiotype of Cu-chelatin previously isolated from rat liver. This suggests that Cu-chelatin may have universal occurrence in all eucaryotic organisms. Whether the protein is present in procaryotic organisms is uncertain. It has not been possible to induce its formation in Escherichia coli. The present studies have indicated that in human liver two distinct metalloproteins are present, one containing zinc and cadmium while the other contains copper. The protein eluting earlier contains predominantly zinc with traces of cadmium. On acrylamide gels it shows a protein band with electrophoretic mobility similar to that of Cd-thionein. Pulido et al. have reported the purification of metallothionein from human renal cortex containing primarily cadmium and zinc (11). The Cu-component of human liver has an electrophoretic mobility identical to that of rat liver Cu-chelatin. Furthermore, the copper is present on the protein in a
288
PREMAKUMAR
diamagnetic form and is released by sulfiydry1 and oxidizing reagents as is the copper on Cu-chelatin. These findings warrant the conclusion that, in human liver, both metallothionein and Cu-chelatin are present. The level of Cu-chelatin in yeast is determined by the copper concentration of the medium. However, yeast cells grown under identical conditions but in the presence of 0.01 mM CdC12, ZnCl, or HgClz do not appear to accumulate the related protein, metallothionein. This differential induction further illustrates the nonidentity of these two metalloproteins. As yet no definitive physiological role for Cu-chelatin has been established. Because of the widespread occurrence of Cuchelatin, it may be speculated that the protein exerts a significant role in copper homeostasis and possibly in other biological functions. ACKNOWLEDGMENTS We thank Dr. Randall Alberte for assistance growing mung beans and Dr. Howard Steinman assistance in amino acid analysis.
in for
ET
AL. REFERENCES
1. WINGE, D. R., PREMAKUMAR, R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. B&hem. Biophys. 170, 253-266. 2. PREMAKUMAR, R., WINGE, D. R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 267-2’71. 3. STARCHER, B. C. (1969) J. Nutr. 97,321-326. 4. EVANS, G. W., MAJORS, P. F., AND CORNATZER, W. E. (1970) Biochem. Biophys. Res. Commun. 40, 1142-1147. D., ALTMANN, H., SZILVINYI, A. 5. GESSWAGNER, V., AND KAINDL, K. (1968) Znt. J. Appl. Ra&at. Zsotop. 19, 152-153. 6. ARNON, D. I., AND HOAGLAND, D. R. (1940) Soil Sci. 50, 463-485. A., AND NAUGHTON, M. I. JOVIN, T., CHRAMBACH, A. (1964) Anal. Biochem. 9, 351-369. 8. HIRS, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, p. 59, Academic Press, New York. 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem 193, 265-275. M. (1923) Biochem. 2. 140, 63LO. TSUCHIHASHI, 112. 11. PULIDO, R., KAGI, J. H. R., AND VALLEE, B. L. (1966) Biochemistry 5, 1768-1777.
