ABCHIVES
OF
BIOCHEMISTRY
AND
Copper-Induced R. PREMAKUMAR,
Department
of Biochemistry,
BIOPHYSICS
170,
Synthesis DENNIS
Duke
267-277
(1975)
of Copper-Chelatin
R. WINGEZ, RAJAGOPALAN University
Received
Medical February
RALPH
Center,
in Rat Liver1
D. WILEY,
Durham,
North
AND
K. V.
Carolina
27710
14, 1975
Intraperitoneal injection of CuClz into rats leads to the accumulation of Cu-chelatin, a low molecular weight copper-containing protein which is not detectable in significant quantities in livers of control rats even after in vitro addition of copper to liver homogenates. In control rat liver preparations, the Sephadex G-75 eluate fraction corresponding to Cu-chelatin fails to bind copper under conditions in which the apochelatin is reconsitituted with the metal. A marked increase in the incorporation of [4,5-3H]lysine into Cuchelatin is observed in experiments where the labeled amino acid is used for pulselabeling 5.5 h after copper injection. Administration of cycloheximide or actinomycin D to rats prior to exposure to copper results in marked inhibition of the incorporation of the radioactive lysine into Cu-chelatin without preventing hepatic accumulation of the metal. Under these conditions, the extraneous copper is found to be associated with the major protein components, with no detectable increase in the Cu-chelatin fraction. The incorporation of [5-3H]orotic acid into liver RNA is elevated in Cu-treated rats. The Cu-stimulated labeling of RNA is abolished by the pretreatment of the animals with actinomycin D. These results provide evidence for the induction by copper of the synthesis of Cu-chelatin by a mechanism involving transcriptional control.
The low molecular weight copper-containing protein which accumulates in livers of rats exposed to high levels of this metal (l-3) has been purified and, because of its distinctive nature, termed Cu-chelatin (1). If the protein is a normal component of liver its content in cont,rol livers is too low to permit isolation and characterization. Because of the apparent dependence of the formation of the protein on de nouo biosynthesis it was of interest to investigate the mode of accumulation of Cuchelatin. Further impetus for such a study was provided by the fact that Cu-chelatin is distinct from metallothionein, the low molecular weight protein that is expressed in large amounts when cadmium rather than copper is the injected toxicant.
It could be hypothesized that Cu-chelatin exists in the tissues as a metal-free apoprotein and becomes observable on the administration of copper because of its Cuchelating ability. A modification of this postulate would be that in the absence of abnormal levels of copper, the hepatic content of the protein is very low due to rapid degradation of metal-free apochelatin and that accumulation is the result of stabilization through Cu binding. Alternatively, the accumulation of Cu-chelatin could reflect a true induction, dependent on control at the transcriptional stage. This article presents evidence for the need for RNA synthesis as well as protein synthesis as prerequisites for the formation and accumulation of Cu-chelatin in rat liver. A preliminary account of a portion of this work has appeared (4).
’ This work was supported by Grant No. GM 00091 from the United States Public Health Service. ’ Supported by Predoctoral Traineeship Grant No. GM 00233 from the National Institutes of Health. Present address: Department of Biochemistry, Universit6 de GenBve, Case 78, 1211 Gen&ve 8, Switzerland.
MATERIALS Materials. Ci/mmol), Ci/mmol), 267
Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
AND
METHODS
[4,5-3H]lysine (specific activity, 55 [5-3H]orotic acid (specific activity, 11.1 Protosol and Aquasol were purchased
268
PREMAKUMAR
from New England Nuclear. c[35S]cystine dihydrochloride (specific activity, 400 mCi/mmol) was obtained from AmershamSearle. Scintillation grade PP03 and POPOP were obtained from Packard and AmershamBearle, respectively. Cycloheximide and actinomycin D were obtained from Sigma Chemical Co. Sephadex G-75 was purchased from Pharmacia. Animals. Male Sprague-Dawley rats maintained on a commercial stock diet and weighing 200-250 g were used in the experiments. All injections were made intraperitoneally in 0.9% NaCl. CuCl, was administered at a dose of 2.5 mg of Cu per kg body weight. Isolation of liver CLchelatin. Livers from Cuinjected rats were excised and rinsed. Homogenates of the livers in four volumes of 0.01 M potassium phosphate buffer, pH 7.8, were prepared with a Thomas tissue grinder. The soluble protein fraction was prepared by centrifugation of the homogenate at 27,000g for 10 min and then at 100,OOOg for 1 h. Unless specifically stated, the 100,OOOg supernatant fraction was incubated in a 60°C water-bath for 10 min followed by centrifugation at 12,OOQg for 10 min to remove the coagulated protein. The resultant supernatant fluid was concentrated about 25-fold by lyophilization and chromatographed on Sephadex G75, with 0.01 M potassium phosphate buffer, pH 7.8, for elution. Copper was quantitated by atomic absorption spectroscopy utilizing a Perkin-Elmer Model 107 spectrometer equipped with a heated graphite atomizer, HGA-2000. Polyacrylamide-gel electrophoresis was carried out according to Jovin et al. (5). Digestion of gel slices containing [3H]lysine-labeled protein was accomplished with 0.3 ml of 30% H,O, at 60°C for several hours in sealed vials. The solubilized slices were mixed with scintillation cocktail and counted in a refrigerated Nuclear Chicago Mark I scintillation counter. The scintillation cocktail used for digested gel segments was a 2 to 1 mixture of toluene and Triton X-100 respectively, containing 5 g of PPO and 0.1 g of POPOP per liter. With all other samples, Aquasol was the scintillation fluid used. From quantitative amino acid analysis (11, the protein content of chelatin solutions was determined to be 60% of the value obtained by the method of Lowry et al. (6) when bovine serum albumin was used as standard. Measurement of [3H]lysine incorporation in liver protein. Aliquots of homogenates of livers from rats injected with [3H]lysine were treated with 10% trichloroacetic acid and the precipitates washed exhaustively with 5% trichloroacetic acid. The final precipitate was solubilized by hydrolysis with 1 ml of Protosol at 60°C. After complete solubilization, 15 ml of Aquasol scintillation cocktail were added. The 8 Abbreviations used: PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(!&phenyloxazolyl]]benzene.
ET
AL..
samples were kept at 40°C in the dark for 16 h before counting in a refrigerated Nuclear Chicago Mark I scintillation counter. The data were corrected for background and quenching. Aliquots of eluates from Sephadex G-75 were counted directly in the Aquasol scintillation fluid. Measurement of [3Hlorotic acid incorporation. Portions of 25% homogenates of livers from experimental animals were taken for extraction of the total RNA according to the method of Schneider (7). Nuclei were isolated according to the procedure of Wang (8) and the nuclear RNA was extracted by the hot sodium dodecyl sulfate/phenol method (9). Cytoplasmic RNA was extracted similarly from the postmitochondrial supernatant fraction. All samples were counted directly in Aquasol. RESULTS
Formation
of Cu-Chelatin on Copper Injection Sephadex G-75 elution profiles of the soluble protein fractions prepared as described above from livers of control and Cu-injected animals are shown in Fig. 1. It can be seen that there is only a minor copper peak in the control preparation in the fractions loo-125 (Fig. 1A) corresponding to the major portion of copper contained in the preparation derived from Cuinjected animals (Fig. 1B). The increased copper content in the chelatin region of Fig. 1B is reflected in the enhanced protein content of the fraction. After acetone fractionation, the protein content of the chelatin fraction (60-80% acetone fraction) from copper-injected animals was over five-fold greater than that from control animals. Since Cu-chelatin was detected by analyzing for copper, the possibility existed that the protein was normally present in the liver as apoprotein. To test this, the unheated 100,OOOg supernatant fraction from control livers was incubated with 0.1 mM CuCIZ for 60 min at 4”C, concentrated and then chromatographed on Sephadex G-75. The profile of copper elution (Fig. 2) was highly polydisperse, and most of the metal was associated with the major protein species with negligible binding occurring in the expected Cu-chelatin region (fractions 100-110). Also, incubation of the Sephadex G-75 eluate (fractions 100-110) from control preparations with 0.1 mM CuC12, followed by chromatography on Sephadex G-75, did not lead to any in-
INDUCTION
OF
COPPER
269
CHELATIN
FIG. 1. Sephadex G-75 chromatography of liver preparations from control and coppertreated rats. Three rata were used in each group. The experimental group received 3 mg of copper per kg body weight as CuC&. Six hours after the administration of copper, the rats were killed and the livers excised and processed as described in Materials and Methods. After heat treatment the supernatant fluid was concentrated by lyophilization and chromatographed on a Sephadex G-75 column (85 x 4 cm) equilibrated and eluted with 0.01 M potassium phosphate buffer, pH 7.8. Fractions of 6.5 ml were collected. Absorbance at 280 nm (O- - -0) and copper content (0-O) were monitored. A and B represent the elution profiles of control and experimental preparations, respectively.
FIG. 2. Attempted in vitro reconstitution of Cu-chelatin from control liver extracts. 100,OOOg supematant fraction from livers of three rats was divided into two equal parts, which was made 0.1 mM with respect to copper. The two samples were incubated for 60 4°C. They were then concentrated and chromatographed on Sephadex G-75 under the tions described in Fig. 1. Absorbance at 280 nm (O---O) and copper contents of the (0-O) and Cu-treated sample (0. . .O) were determined.
crease in copper content in this region. Nor did the concentrate of this fraction bind copper under conditions used for the reconstitution of the apochelatin as described in the preceding paper (1). These results indicate that the appearance of substantial levels of Cu-chelatin in livers of rats exposed
The one of min at condicontrol
to copper does not occur by chelation metal to preexisting apoprotein.
of the
Time Course of Hepatic Copper Uptake Cu-injected rats were killed at different intervals after the time of injection. The livers were processed as described earlier
270
PREMARUMAR
0
4
6
12
16
20
24
HOUR5 AFTER INJECTION
FIG. 3. Time course of copper uptake by rat liver. Each point represents the average result from four rats. SEM values were less than 210% and are not shown. The animals were injected with copper (3 mg per kg) as CuCl,. At the time points indicated, the rata were killed, and liver homogenates were prepared as described earlier. One milliliter of each of the homogenates from the different time periods was ashed for 16 h at 480°C. The ash was digested with 1 ml of a mixture of HCl and HNOI (1:3). Total liver copper was determined in the dissolved ash (0-O). Copper was analyzed in the 100,OOOg heat supernatant fraction (O- - -0) directly.
and the copper content of the heat-treated 100,OOOg supernatant fraction determined. As described in the preceding paper, the two major copper components observed after such heat treatment are superoxide dismutase and Cu-chelatin. Since the levels of superoxide dismutase and its metal content are unaffected by copper injections, the increase in copper in the heattreated supernatant fraction is proportional to the Cu-chelatin content. Figure 3 shows that the copper level in the supernatant fluid and the total copper uptake by the liver attain half-maximal levels in about 6 h. These findings revealed that a 6-h induction period was sufficient for studying the mode of Cu-chelatin formation by the experimental technique described below. Effect of Cycloheximide and Actinomycin D on Cu-Chelatin Formation In order to investigate the mechanism of the accumulation of Cu-chelatin, the effects of cycloheximide, an inhibitor of protein synthesis, and of actinomycin D, an inhibitor of RNA synthesis, on the content
ET
AL.
of the protein in rat liver were studied. At the same time the effects of these antibiotics on the incorporation of [3H]lysine into total liver protein and more specifically into Cu-chelatin were investigated by pulse-labeling with 13H]lysine for 30 min before the termination of the induction period. Table I shows that copper administration resulted in a 32% stimulation in the incorporation of 13Hllysine into acid-precipitable protein. The incorporation of the label was inhibited about 55% by pretreatment with cycloheximide, whereas in the presence of actinomycin D the Cu-stimulated increase in lysine incorporation was not observed. To determine the extent of [3Hllysine incorporation specifically into Cu-chelatin the homogenates were treated as described earlier for the isolation of Cu-chelatin. The Sephadex G-75 elution profiles are shown in Fig. 4. In can be seen from Fig. 4B that there was a dramatic and selective increase in 13Hllysine incorporation in the Cu-chelatin region in Cu-injected animals as compared to the labeling pattern in samples derived from control animals (Fig. 4A). The Cu-induced stimulation in labeling was clearly indicative of new protein TABLE
I
INCORPORATIONOF [3HlL~~~~~ INTO TOTAL LIVER PROTEINS Group Control Cu-treated Cycloheximide, Actinomycin
Cu-treated D, Cu-treated
[3Hllysine incorporation (cpmlg of liver) 103,300 136,000 61,600 103,700
” ? t 2
8,000 9,800 667 6,333
a Each entry is the mean lr SEM for three rats. All animals received 13H]lysine (20 PCi) injected intraperitoneally 30 min before killing. Copper was administered intraperitoneally as CuCl, at a dose of 3 mg per kg body weight. Cycloheximide or actinomycin D (1.5 mg per kg body weight) was injected into the respective groups 30 min before copper administration and twice thereafter at 2-h intervals. Control animals received injections of 0.9% NaCl at the same frequency. The animals were killed 6 h after the injection of copper, and liver homogenates were prepared as described in the methods section. Samples were processed for measurement of radioactivity as described in the text.
FIG. 4. Effect of copper on [3H11ysine incorporation into Cu-chelatin. Each group consisted of three rats. The injection doses and experimental protocol were the same as described in Table I. The homogenates were processed to obtain heat-treated extracts. The resulting supernatant fluids were concentrated and chromatographed on Sephadex G-75 columns (85 x 4 cm) equilibrated and eluted with 0.01 M potassium phosphate buffer, pH 7.8. Fractions of 6.5 ml were collected. The elution profiles shown represent control (A), Cu-treated (B), cycloheximideand Cu-treated (0, and actinomycin D- and Cu-treated (D) groups.
272
PREMAKUMAR
formation and, as such, was inhibited by pretreatment with cycloheximide (Fig. 40. Actinomycin D also had a profound effect, as seen in Fig. 4D, in preventing the incorporation of 13H]lysine into the Cu-chelatin fraction, suggesting that the synthesis of only that component was dependent on the formation of new mRNA. This is clear from the fact that actinomycin D did not significantly inhibit the labeling of other protein fractions. Neither actinomycin D nor cycloheximide affected uptake of copper by the liver, as seen from Table II. The copper levels in the 27,000g supernatant fractions derived from livers of actinomycin D- and cycloheximide-treated animals were the same as those of preparations from animals injected with copper only. However, in the heat-treated 100,OOOg supernatant fluid, the copper content of which reflect the level of Cu-chelatin, the preparations from antibiotic-treated animals showed significantly lower amounts of the metal than those observed in the case of Cu-treated controls. In summary, these experiments have shown that the formation of Cu-chelatin is totally dependent on de nouo protein synTABLE EFFECT
II
OF CYCLOHEXIMIDE AND ACTINOMYCIN HEPATIC COPPER UPTAKES
D ON
Total copper in Total copper in 27,OOOg superna100,OOOg postheat supernatant +g)
Group
tant &g)
Control Cu-treated Cycloheximide before Cu Actinomycin before Cu Actinomycin after 0.1
D D
28 k 4 191 k 17 160’
20 ? 1 149 t 20 40*
173 k 20
46 t 5
-c
115 + 13
a The results presented are mean t SEM for three rats. The injection doses and experimental protocol were the same as described in Table I. Copper was analyzed in the liver homogenate following 27,OOOg centrifugation and also after heat treatment of the 100,OOOg supernatant fraction. * Values for one surviving animal. c Not determined.
ET
AL.
thesis, as evidenced by the fact that accumulation of this protein as well as incorporation of r3Hllysine into the Sephadex G-75 eluate fraction containing Cu-chelatin was nearly completely abolished by prior injection of cycloheximide into the animals. The similar effect of antinomycin D is presumptive evidence for a requirement for RNA synthesis as a prelude to the formation of Cu-chelatin. The latter conclusion is corroborated by the observation that actinomycin D has little effect on the formation of Cu-chelatin if administered 1 h after injection of copper. It is of further interest that actinomycin D had considerably less inhibitory effect on the incorporation of 13Hllysine into other protein fractions (Fig. 4D). It is evident that actinomytin-resistant incorporation resulted from translation of preexistent stable mRNA molecules coding for a variety of proteins. Since the uptake of copper by the liver was unaffected by treatment with cycloheximide or actinomycin D, it was of interest to determine the distribution of the metal in liver extracts from animals pretreated with either antibiotic. For this purpose the unheated 100,OOOg supernatant fractions were concentrated and chromatographed on Sephadex G-75. Figure 5 shows the elution profiles of pooled supernatant fractions from the actinomycin D group as well as the unheated supernatant fractions from the group injected with copper only. In the latter group, the metal was predomiassociated with Cu-chelatin; nantly whereas, in the actinomycin D group, the metal was associated primarily with the major protein fractions. Figure 6 shows elution profiles for the heated and unheated 100,OOOg supernatant fractions from the cycloheximide-treated group. Again it is apparent that the copper is bound to the major protein species which are coagulated by heat. The incorporation of 13Hllysine into Cuchelatin was substantiated by analyzing the radioactivity in polyacrylamide-gel slices after electrophoresis of Cu-chelatin purified through acetone fractionation. Gels were sliced into 14 segments of 0.4 cm each, digested, and counted as described in Materials and Methods. The major
INDUCTION
50
60
70
OF
COPPER
80 90 100 TUBE NUMBERS
273
CHELATIN
110
ml
130
FIG. 5. Copper distribution after pretreatment with actinomycin D. The experimental conditions were the same as described in Table I except that the 100,OOOg supernatant fraction was not subjected to the 60°C beat treatment. The unheated supernatant fractions from the Cutreated and actinomycin D groups having the same initial total copper content of 150 pg were chromatographed on Sephadex G-75. The absorption at 280 nm (O---O), and copper contents of the eluates from the copper group (O..,O) and actinomycin D group (0-O) were determined.
20
40
So
SC TUBE WMSER
120
I40
160
FIG. 6. Effect of heat treatment on copper distribution in the cycloheximide group. The experimental conditions were the same as described in Table I, except that the 100,OOOg supernatant fluid was divided into two equal parts, one of which was subjected to the heat treatment. The two were then separately chromatographed on Sephadex G-75 under conditions as in Fig. 1. Absorbance at 280 nm (-1 and copper concentration (- - -1 of the eluates were determined. A and B represent the elution profiles of the unheated and heated samples, respectively.
r3H]lysine peak coincided with the segment containing the Cu-chelatin band. [3H]0rotic Acid Incorporation into RNA In view of the suggestive evidence for the dependence of Cu-chelatin formation on RNA synthesis, the effect of copper ad-
ministration on [3H]orotic acid incorporation into rapidly labeled hepatic RNA in control and actinomycin D-treated rats was investigated. The labeled erotic acid was administered 10 min after injection of CuCl, or saline, and the animals were killed 20 or 40 min thereafter. The livers were excised and isolation of RNA was carried out as described in Materials and Methods. Total cellular RNA was extracted from an aliquot of the 25% homogenate of liver. As can be seen in Table III, copper administration led to an 11% increase in [3Hlorotic acid incorporation into total cellular RNA. The incorporation was inhibited about 64% in the group pretreated with actinomycin D. The incorporation of the isotope into both cytoplasmic and nuclear RNA was substantially enhanced by copper adminstration. The 20min [3H]orotic acid pulse resulted primarily in nuclear RNA labeling whereas, after the 40-min pulse, a significant increase in the ratio of cytoplasmic to nuclear labeled RNA could be observed. Turnover of Cu-Chelatin in the Rat It was of interest to determine the fate of the accumulated hepatic copper and of the induced Cu-chelatin. For this purpose, a single intraperitoneal injection of copper was administered to animals. Animals were killed at various times ranging from 12-180 h after the injection, and the copper
274
PREMAKUMAR
ET
content of crude homogenates of the liver and kidney and the soluble protein fraction from the liver was measured (Fig. 7). In both intact liver and kidney, as well as in the cellular supernatant fractions, the accumulated copper content was depleted by more than 50% at the end of 48 h.
In a second experiment, rats were injetted with a single dose of CuClz and 3 h later with a solution containing [3Hllysine (20 /AN and [35Slcystine (20 /LX). The animals were killed either 15, 40 or 88 h from the time of copper administration. Cu-chelatin was purified from each group
TABLE INCORPORATION Group
AL.
III
OF [3H10~~~~~
ACID
Pulsing time (min)
INTO RNA”
3H RNA Cellular
Control Cu-treated Actinomycin Cu-treated
20 20 40 20
D,
49,000 55,300
of liver)
Nuclear
-+ 2,980 2 1,730
26,000 + 31,200 + 23,300 ” 9,300 k
18,000
(cpmlg
r 1,710
Cytoplasmic 3,240 3,500 1,860 1,470
6,100 7,100 13,600 5,700
2 2 5 k
207 507 1,030 606
LI Each group consisted of three rats, and the results presented are the mean r SEM for each group. Animals in all groups received [3H]orotic acid by intraperitoneal injection. Copper (3 mg per kg) as CuCl, was given to the animals in groups 2 and 3,lO min prior to the injection of erotic acid. Actinomycin D (1.5 mg per kg) was administered to rats in group 3,l h prior to copper injection. The animals were sacrificed 20 or 40 min after the erotic acid pulse. The incorporation of the label into different fractions of liver RNA was determined as described in the text.
30 t 0 \ :
20
P
t
IO
0 =& I
I
I
I
20
40
60
00
HOURS
AFTER
I
I
I
I
I
100
120
140
160
100
INJECTION
FIG. 7. Fate of copper in rat liver and kidney following a single intraperitoneal injection of CuCl%. Animals in groups of three were given copper at a dose of 3 mg per kg. At the time points indicated, the rata were killed, and tissue homogenates were prepared as described in Materials and Methods. Analysis of copper in the homogenates was according to the procedure described in Fig. 3. Each point represent the average result from three rats. The curves represent the following: (-•-) hepatic copper content, (-A-) renal copper content, and (O- - -0) total copper in the 100,OOOg supernatant fraction from liver.
INDUCTION
OF
COPPER
and from a control group by Sephadex G-75 column chromatography and acetone fractionation. The acetone-fractionated protein was analyzed for protein, copper content and radioactivity (Table IV). The turnover of protein and copper occurred in parallel, with the levels being 50% depleted by 40 h. DISCUSSION
Copper-chelatin is detected in rat liver cytosol following intraperitoneal administration of copper. The holoprotein is not detectable in control animals to any appreciable content. That this accumulation of Cu-chelatin is not due to the binding of copper to preexisting apoprotein is shown by the following lines of evidence. (a) Addition of copper to the 100,OOOg supernatant fractions of control livers does not lead to enhanced content of Cu-chelatin. (b) The Sephadex G-75 fraction from the control preparations corresponding to the Cu-chelatin region does not bind copper under conditions used for the reconstitution of apochelatin with the metal. (c) Treatment of animals with actinomytin D results in inhibition of the formation of Cu-chelatin on subsequent exposure to copper, though the hepatic uptake of the TABLE TURNOVER
Group
Control Cu-treated &-treated Cu-treated
IV
OF CU-CHELATIN
IN RAT
Hours Proafter la- tein be1 in- (mg) jection
Copper (Pi?)
1.2 6.0 3.3 1.8
2 54 27 12
15 15 40 88
LIVER”
3H ‘zgy
1,500 4,300 2,200 500
93 ‘zP$
4.300 13,800 6,600 1,300
’ Groups of three rats received single intraperitoneal injections of CuClz (3 mg of &/kg) and 3 h later were given an injection containing [3H]lysine (20 PCil and [Yllcystine (20 &il. Control animals received an injection of 0.9% NaCl instead of copper. Animals were killed at various times after the second injection. Cu-chelatin was purified as described earlier by Sephadex G-75 chromatography and acetone fractionation. The parameters listed below were analyzed as described in Materials and Methods.
CHELATIN
275
metal is not impaired. Under these conditions liver copper is bound nonspecifically to major protein species. The lack of accumulation of Cu-chelatin in liver under these in uivo conditions may be ascribed to the absence of significant amounts of functional apochelatin. Administration of copper stimulates the incorporation of r3Hllysine into Cu-chelatin without appreciably affecting the extent of labeling of other proteins. The increased labeling of Cu-chelatin is abolished by pretreatment of the animals with either cycloheximide or actinomycin D. The observed inhibition is not a consequence of interference with hepatic copper uptake. The evidence derived from use of actinomycin D indicates that large-scale synthesis of Cu-chelatin occurs through induction of the synthesis of mRNA specifically coding for Cu-chelatin. Since actinomycin D has been reported to exert anomalous effects (10-121, additional experimental verification was required for the RNA synthesis mechanism. Such evidence has come from observation that incorporation of 13Hlorotic acid into RNA in pulse-labeling experiments is significantly enhanced immediately following copper injection. This effect is seen in total cellular RNA as well as nuclear and cytoplasmic RNA. Pulse-labeling for 40 min suggested that with time the rapidly labeled RNA is released into the cytoplasm from the nucleus. The evidence presented above, i.e., the virtual absence of Cu-chelatin from control tissues, the absence of measurable apoprotein content, the stimulation by copper of lY3Hllysine incorporation, the enhancement of [3Hlorotic acid incorporation, and the inhibition by actinomycin D of the effects of copper, when taken together permit the conclusion that induction of Cu-chelatin is under transcriptional control. The mechanism could involve either stimulation of specific mRNA synthesis or stabilization by copper of the mRNA which is otherwise degraded rapidly. The latter mechanism, however, seems unlikely from the results of our experiments using actinomycin D. It cannot be stated at this time whether Cuchelatin is the only protein induced by
276
PREMAKUMAR
copper administration in the rat liver and whether the enhancement in [3H]orotic acid labeling represents only mRNA for the Cu-chelatin. The de nouo synthesis of chelatin appears to be a more general phenomenon. As discussed in the following paper (13), formation of Cu-chelatin in yeast is dependent upon exposure of the cells to copper and requires de nouo protein synthesis as demonstrated by the strong inhibitory effect of cycloheximide. It is uncertain whether the induction in yeast is also under transcriptional control as in rat liver. Our results on the inducible nature of Cu-chelatin conflict with the conclusion of Bloomer and Sourkes (2) that the protein is constitutive. It might be pointed out, however, that they did not use either cycloheximide or actinomycin D in their experiments to test the inducibility of the protein. Bremner and Davies (3) reported that injection of cycloheximide simultaneously with CuS04 into rats led to a 90% reduction in labeling of the rat liver copperbinding protein. The results presented in this report are in agreement with the observation of Bremner and Davies. The ability of metal ions to regulate the synthesis of metalloproteins is known. The ferritin content of mammalian tissues is regulated by iron through a translational mechansim (14,151. One postulated mechanism suggests that binding of iron to nascent apoferritin polypeptides on the free polysomes relieves an inhibition of apoferritin synthesis (16). As discussed in the first paper in this series (17), cadmium administration to rats results in the accumulation of metallothionein. Squibb and Cousins (18) reported that the induction was blocked by pretreatment of the animals with either cycloheximide or actinomycin D. Davies et al. (19) and Webb (20) also found that cycloheximide inhibited the metal-induced formation of thionein. In support of a transcriptional mechanism, Weser et al. (21, 22) reported that cadmium and zinc stimulated the incorporation of [6-14C]orotate into nuclear RNA. The content of Cu-chelatin formed in rat liver after a single subcutaneous injection of CuClz is depleted 50% in less than 40 h.
ET
AL.
In contrast, Shaikh and Lucis (23) found that 2 weeks after a single subcutaneous injection of CdCl, into rats, hepatic Cd, Znthionein was present at a concentration unchanged from that seen 1 day after the injection. The half-time for Cd, Zn-thionein turnover in the rat presumably exceeds 100 days (24). The significance of the widely different turnover times for Cu-chelatin and the thionein remains to be investigated. While no biological role for Cu-chelatin has been defined, these studies suggest that the protein can function to sequester free copper ions which are potentially cytotoxic. The rapid induction response to high levels of the metal would permit the protein to minimize the disruption of cellular functions by free copper ions. Finally, another significant feature of the copper-dependent induction of Cu-chelatin is apparent from the data in Table I. The marked enhancement in the incorporation of [3H]lysine into newly synthesized protein in livers of Cu-treated animals must result, to a large extent, from the synthesis of Cu-chelatin. This system therefore represents one of the few instances in which the rate of synthesis of a single protein is altered to such an extent in response to an extraneous stress as to make it the predominant single product of hepatic protein synthesis under these circumstances. REFERENCES 1. WINGE, D. R., PREMAKUMAR, R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 253-266. 2. BLOOMER, L. C., AND SOURKES, T. L. (1973) Biochem. Med. 8, 78-91. 3. BREMNER, I., AND DAVIES, N. T. (1974) B&hem. Sot. Trans. 2, 425-427. 4. PREMAKUMAR, R., WINGE, D. R., ANDRAJAGOPALAN, K. V. (1974) Fed. Proc. 33, 1385. 5. JOVIN, T., CHRAMBACH, A., AND NAUGHTON, M. A. (1964) Anal. Biochem. 9, 351-369. 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. SCHNEIDER, W. C. (1957) in Methods in Enzymoiogy (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 680, Academic Press, New York. 8. WANG, T. Y. (1967) in Methods in Enzymology
INDUCTION
9.
10. 11.
12. 13.
14.
15.
16.
OF
(Grossman, L., and Moldave, K. eds.), Vol. 12, p. 620, Academic Press, New York. GIRARD, M. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, p. 581, Academic Press, New York. REVEL, M., HIATT, H. H., AND REVEL, J. P. (1964) Science 146, 1311-1313. HONIG, G. R., AND RABINOVITZ, M. (1965) Science 149, 1504-1505. Acs, G., REICH, E., AND VALANJU, S. (1963) Biochim. Biophys. Actu 76, 68-79. PREMAKUMAR, R., WINGE, D. R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 000, 000-000. MUNRO, H. N., AND DRYSDALE, J. W. (1970)Fed. Proc. 29, 1469-1473. MILLAR, J. A., CUMMING, R. L. C., SMITH, J. A., AND GOLDBERG, A. (1970) Biochem. J. 119, 643-649. CRICHTON, R. R. (1971) N. Engl. J. Med. 284,
COPPER
CHELATIN
277
1413-1422. 17. WINGE, D. R., PREMAKUMAR, R., ANDRAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 000, 000-000. 18. SQUIBB, K. S., AND COUSINS, R. J. (1974) Enuiron. Physiol. Biochem. 4, 24. 19. DAVIES, N. T., BREMNER, I., AND MILLS, C. F. (1973) Biochem. Sot. Trans. 1, 985-988. 20. WEBB, M. (1972) Biochem. Pharmacol. 21,27512765. 21. WESER, U., RUPP, H., DONAY, F., LINNEMANN, F., VOELTER, W., VOETSCH, W., AND JUNG, G. (1973) Eur. J. Biochem. 39, 127-140. 22. WESER, U., AND HUBNER, L. (1970) FEBS Lett. 10, 169-174. 23. SHAIKH, Z. A., AND LUCIS, 0. J. (1972) Environ. Health 24, 419-429. 24. FRIBERG, L., PISCATOR, M., AND NORDBERG, G. F. (1971) Cadmium in the Environment, p. 44, C. R. C. Press, Cleveland, OH.
OF
BIOCHEMISTRY
AND
Copper-Induced R. PREMAKUMAR,
Department
of Biochemistry,
BIOPHYSICS
170,
Synthesis DENNIS
Duke
267-277
(1975)
of Copper-Chelatin
R. WINGEZ, RAJAGOPALAN University
Received
Medical February
RALPH
Center,
in Rat Liver1
D. WILEY,
Durham,
North
AND
K. V.
Carolina
27710
14, 1975
Intraperitoneal injection of CuClz into rats leads to the accumulation of Cu-chelatin, a low molecular weight copper-containing protein which is not detectable in significant quantities in livers of control rats even after in vitro addition of copper to liver homogenates. In control rat liver preparations, the Sephadex G-75 eluate fraction corresponding to Cu-chelatin fails to bind copper under conditions in which the apochelatin is reconsitituted with the metal. A marked increase in the incorporation of [4,5-3H]lysine into Cuchelatin is observed in experiments where the labeled amino acid is used for pulselabeling 5.5 h after copper injection. Administration of cycloheximide or actinomycin D to rats prior to exposure to copper results in marked inhibition of the incorporation of the radioactive lysine into Cu-chelatin without preventing hepatic accumulation of the metal. Under these conditions, the extraneous copper is found to be associated with the major protein components, with no detectable increase in the Cu-chelatin fraction. The incorporation of [5-3H]orotic acid into liver RNA is elevated in Cu-treated rats. The Cu-stimulated labeling of RNA is abolished by the pretreatment of the animals with actinomycin D. These results provide evidence for the induction by copper of the synthesis of Cu-chelatin by a mechanism involving transcriptional control.
The low molecular weight copper-containing protein which accumulates in livers of rats exposed to high levels of this metal (l-3) has been purified and, because of its distinctive nature, termed Cu-chelatin (1). If the protein is a normal component of liver its content in cont,rol livers is too low to permit isolation and characterization. Because of the apparent dependence of the formation of the protein on de nouo biosynthesis it was of interest to investigate the mode of accumulation of Cuchelatin. Further impetus for such a study was provided by the fact that Cu-chelatin is distinct from metallothionein, the low molecular weight protein that is expressed in large amounts when cadmium rather than copper is the injected toxicant.
It could be hypothesized that Cu-chelatin exists in the tissues as a metal-free apoprotein and becomes observable on the administration of copper because of its Cuchelating ability. A modification of this postulate would be that in the absence of abnormal levels of copper, the hepatic content of the protein is very low due to rapid degradation of metal-free apochelatin and that accumulation is the result of stabilization through Cu binding. Alternatively, the accumulation of Cu-chelatin could reflect a true induction, dependent on control at the transcriptional stage. This article presents evidence for the need for RNA synthesis as well as protein synthesis as prerequisites for the formation and accumulation of Cu-chelatin in rat liver. A preliminary account of a portion of this work has appeared (4).
’ This work was supported by Grant No. GM 00091 from the United States Public Health Service. ’ Supported by Predoctoral Traineeship Grant No. GM 00233 from the National Institutes of Health. Present address: Department of Biochemistry, Universit6 de GenBve, Case 78, 1211 Gen&ve 8, Switzerland.
MATERIALS Materials. Ci/mmol), Ci/mmol), 267
Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
AND
METHODS
[4,5-3H]lysine (specific activity, 55 [5-3H]orotic acid (specific activity, 11.1 Protosol and Aquasol were purchased
268
PREMAKUMAR
from New England Nuclear. c[35S]cystine dihydrochloride (specific activity, 400 mCi/mmol) was obtained from AmershamSearle. Scintillation grade PP03 and POPOP were obtained from Packard and AmershamBearle, respectively. Cycloheximide and actinomycin D were obtained from Sigma Chemical Co. Sephadex G-75 was purchased from Pharmacia. Animals. Male Sprague-Dawley rats maintained on a commercial stock diet and weighing 200-250 g were used in the experiments. All injections were made intraperitoneally in 0.9% NaCl. CuCl, was administered at a dose of 2.5 mg of Cu per kg body weight. Isolation of liver CLchelatin. Livers from Cuinjected rats were excised and rinsed. Homogenates of the livers in four volumes of 0.01 M potassium phosphate buffer, pH 7.8, were prepared with a Thomas tissue grinder. The soluble protein fraction was prepared by centrifugation of the homogenate at 27,000g for 10 min and then at 100,OOOg for 1 h. Unless specifically stated, the 100,OOOg supernatant fraction was incubated in a 60°C water-bath for 10 min followed by centrifugation at 12,OOQg for 10 min to remove the coagulated protein. The resultant supernatant fluid was concentrated about 25-fold by lyophilization and chromatographed on Sephadex G75, with 0.01 M potassium phosphate buffer, pH 7.8, for elution. Copper was quantitated by atomic absorption spectroscopy utilizing a Perkin-Elmer Model 107 spectrometer equipped with a heated graphite atomizer, HGA-2000. Polyacrylamide-gel electrophoresis was carried out according to Jovin et al. (5). Digestion of gel slices containing [3H]lysine-labeled protein was accomplished with 0.3 ml of 30% H,O, at 60°C for several hours in sealed vials. The solubilized slices were mixed with scintillation cocktail and counted in a refrigerated Nuclear Chicago Mark I scintillation counter. The scintillation cocktail used for digested gel segments was a 2 to 1 mixture of toluene and Triton X-100 respectively, containing 5 g of PPO and 0.1 g of POPOP per liter. With all other samples, Aquasol was the scintillation fluid used. From quantitative amino acid analysis (11, the protein content of chelatin solutions was determined to be 60% of the value obtained by the method of Lowry et al. (6) when bovine serum albumin was used as standard. Measurement of [3H]lysine incorporation in liver protein. Aliquots of homogenates of livers from rats injected with [3H]lysine were treated with 10% trichloroacetic acid and the precipitates washed exhaustively with 5% trichloroacetic acid. The final precipitate was solubilized by hydrolysis with 1 ml of Protosol at 60°C. After complete solubilization, 15 ml of Aquasol scintillation cocktail were added. The 8 Abbreviations used: PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(!&phenyloxazolyl]]benzene.
ET
AL..
samples were kept at 40°C in the dark for 16 h before counting in a refrigerated Nuclear Chicago Mark I scintillation counter. The data were corrected for background and quenching. Aliquots of eluates from Sephadex G-75 were counted directly in the Aquasol scintillation fluid. Measurement of [3Hlorotic acid incorporation. Portions of 25% homogenates of livers from experimental animals were taken for extraction of the total RNA according to the method of Schneider (7). Nuclei were isolated according to the procedure of Wang (8) and the nuclear RNA was extracted by the hot sodium dodecyl sulfate/phenol method (9). Cytoplasmic RNA was extracted similarly from the postmitochondrial supernatant fraction. All samples were counted directly in Aquasol. RESULTS
Formation
of Cu-Chelatin on Copper Injection Sephadex G-75 elution profiles of the soluble protein fractions prepared as described above from livers of control and Cu-injected animals are shown in Fig. 1. It can be seen that there is only a minor copper peak in the control preparation in the fractions loo-125 (Fig. 1A) corresponding to the major portion of copper contained in the preparation derived from Cuinjected animals (Fig. 1B). The increased copper content in the chelatin region of Fig. 1B is reflected in the enhanced protein content of the fraction. After acetone fractionation, the protein content of the chelatin fraction (60-80% acetone fraction) from copper-injected animals was over five-fold greater than that from control animals. Since Cu-chelatin was detected by analyzing for copper, the possibility existed that the protein was normally present in the liver as apoprotein. To test this, the unheated 100,OOOg supernatant fraction from control livers was incubated with 0.1 mM CuCIZ for 60 min at 4”C, concentrated and then chromatographed on Sephadex G-75. The profile of copper elution (Fig. 2) was highly polydisperse, and most of the metal was associated with the major protein species with negligible binding occurring in the expected Cu-chelatin region (fractions 100-110). Also, incubation of the Sephadex G-75 eluate (fractions 100-110) from control preparations with 0.1 mM CuC12, followed by chromatography on Sephadex G-75, did not lead to any in-
INDUCTION
OF
COPPER
269
CHELATIN
FIG. 1. Sephadex G-75 chromatography of liver preparations from control and coppertreated rats. Three rata were used in each group. The experimental group received 3 mg of copper per kg body weight as CuC&. Six hours after the administration of copper, the rats were killed and the livers excised and processed as described in Materials and Methods. After heat treatment the supernatant fluid was concentrated by lyophilization and chromatographed on a Sephadex G-75 column (85 x 4 cm) equilibrated and eluted with 0.01 M potassium phosphate buffer, pH 7.8. Fractions of 6.5 ml were collected. Absorbance at 280 nm (O- - -0) and copper content (0-O) were monitored. A and B represent the elution profiles of control and experimental preparations, respectively.
FIG. 2. Attempted in vitro reconstitution of Cu-chelatin from control liver extracts. 100,OOOg supematant fraction from livers of three rats was divided into two equal parts, which was made 0.1 mM with respect to copper. The two samples were incubated for 60 4°C. They were then concentrated and chromatographed on Sephadex G-75 under the tions described in Fig. 1. Absorbance at 280 nm (O---O) and copper contents of the (0-O) and Cu-treated sample (0. . .O) were determined.
crease in copper content in this region. Nor did the concentrate of this fraction bind copper under conditions used for the reconstitution of the apochelatin as described in the preceding paper (1). These results indicate that the appearance of substantial levels of Cu-chelatin in livers of rats exposed
The one of min at condicontrol
to copper does not occur by chelation metal to preexisting apoprotein.
of the
Time Course of Hepatic Copper Uptake Cu-injected rats were killed at different intervals after the time of injection. The livers were processed as described earlier
270
PREMARUMAR
0
4
6
12
16
20
24
HOUR5 AFTER INJECTION
FIG. 3. Time course of copper uptake by rat liver. Each point represents the average result from four rats. SEM values were less than 210% and are not shown. The animals were injected with copper (3 mg per kg) as CuCl,. At the time points indicated, the rata were killed, and liver homogenates were prepared as described earlier. One milliliter of each of the homogenates from the different time periods was ashed for 16 h at 480°C. The ash was digested with 1 ml of a mixture of HCl and HNOI (1:3). Total liver copper was determined in the dissolved ash (0-O). Copper was analyzed in the 100,OOOg heat supernatant fraction (O- - -0) directly.
and the copper content of the heat-treated 100,OOOg supernatant fraction determined. As described in the preceding paper, the two major copper components observed after such heat treatment are superoxide dismutase and Cu-chelatin. Since the levels of superoxide dismutase and its metal content are unaffected by copper injections, the increase in copper in the heattreated supernatant fraction is proportional to the Cu-chelatin content. Figure 3 shows that the copper level in the supernatant fluid and the total copper uptake by the liver attain half-maximal levels in about 6 h. These findings revealed that a 6-h induction period was sufficient for studying the mode of Cu-chelatin formation by the experimental technique described below. Effect of Cycloheximide and Actinomycin D on Cu-Chelatin Formation In order to investigate the mechanism of the accumulation of Cu-chelatin, the effects of cycloheximide, an inhibitor of protein synthesis, and of actinomycin D, an inhibitor of RNA synthesis, on the content
ET
AL.
of the protein in rat liver were studied. At the same time the effects of these antibiotics on the incorporation of [3H]lysine into total liver protein and more specifically into Cu-chelatin were investigated by pulse-labeling with 13H]lysine for 30 min before the termination of the induction period. Table I shows that copper administration resulted in a 32% stimulation in the incorporation of 13Hllysine into acid-precipitable protein. The incorporation of the label was inhibited about 55% by pretreatment with cycloheximide, whereas in the presence of actinomycin D the Cu-stimulated increase in lysine incorporation was not observed. To determine the extent of [3Hllysine incorporation specifically into Cu-chelatin the homogenates were treated as described earlier for the isolation of Cu-chelatin. The Sephadex G-75 elution profiles are shown in Fig. 4. In can be seen from Fig. 4B that there was a dramatic and selective increase in 13Hllysine incorporation in the Cu-chelatin region in Cu-injected animals as compared to the labeling pattern in samples derived from control animals (Fig. 4A). The Cu-induced stimulation in labeling was clearly indicative of new protein TABLE
I
INCORPORATIONOF [3HlL~~~~~ INTO TOTAL LIVER PROTEINS Group Control Cu-treated Cycloheximide, Actinomycin
Cu-treated D, Cu-treated
[3Hllysine incorporation (cpmlg of liver) 103,300 136,000 61,600 103,700
” ? t 2
8,000 9,800 667 6,333
a Each entry is the mean lr SEM for three rats. All animals received 13H]lysine (20 PCi) injected intraperitoneally 30 min before killing. Copper was administered intraperitoneally as CuCl, at a dose of 3 mg per kg body weight. Cycloheximide or actinomycin D (1.5 mg per kg body weight) was injected into the respective groups 30 min before copper administration and twice thereafter at 2-h intervals. Control animals received injections of 0.9% NaCl at the same frequency. The animals were killed 6 h after the injection of copper, and liver homogenates were prepared as described in the methods section. Samples were processed for measurement of radioactivity as described in the text.
FIG. 4. Effect of copper on [3H11ysine incorporation into Cu-chelatin. Each group consisted of three rats. The injection doses and experimental protocol were the same as described in Table I. The homogenates were processed to obtain heat-treated extracts. The resulting supernatant fluids were concentrated and chromatographed on Sephadex G-75 columns (85 x 4 cm) equilibrated and eluted with 0.01 M potassium phosphate buffer, pH 7.8. Fractions of 6.5 ml were collected. The elution profiles shown represent control (A), Cu-treated (B), cycloheximideand Cu-treated (0, and actinomycin D- and Cu-treated (D) groups.
272
PREMAKUMAR
formation and, as such, was inhibited by pretreatment with cycloheximide (Fig. 40. Actinomycin D also had a profound effect, as seen in Fig. 4D, in preventing the incorporation of 13H]lysine into the Cu-chelatin fraction, suggesting that the synthesis of only that component was dependent on the formation of new mRNA. This is clear from the fact that actinomycin D did not significantly inhibit the labeling of other protein fractions. Neither actinomycin D nor cycloheximide affected uptake of copper by the liver, as seen from Table II. The copper levels in the 27,000g supernatant fractions derived from livers of actinomycin D- and cycloheximide-treated animals were the same as those of preparations from animals injected with copper only. However, in the heat-treated 100,OOOg supernatant fluid, the copper content of which reflect the level of Cu-chelatin, the preparations from antibiotic-treated animals showed significantly lower amounts of the metal than those observed in the case of Cu-treated controls. In summary, these experiments have shown that the formation of Cu-chelatin is totally dependent on de nouo protein synTABLE EFFECT
II
OF CYCLOHEXIMIDE AND ACTINOMYCIN HEPATIC COPPER UPTAKES
D ON
Total copper in Total copper in 27,OOOg superna100,OOOg postheat supernatant +g)
Group
tant &g)
Control Cu-treated Cycloheximide before Cu Actinomycin before Cu Actinomycin after 0.1
D D
28 k 4 191 k 17 160’
20 ? 1 149 t 20 40*
173 k 20
46 t 5
-c
115 + 13
a The results presented are mean t SEM for three rats. The injection doses and experimental protocol were the same as described in Table I. Copper was analyzed in the liver homogenate following 27,OOOg centrifugation and also after heat treatment of the 100,OOOg supernatant fraction. * Values for one surviving animal. c Not determined.
ET
AL.
thesis, as evidenced by the fact that accumulation of this protein as well as incorporation of r3Hllysine into the Sephadex G-75 eluate fraction containing Cu-chelatin was nearly completely abolished by prior injection of cycloheximide into the animals. The similar effect of antinomycin D is presumptive evidence for a requirement for RNA synthesis as a prelude to the formation of Cu-chelatin. The latter conclusion is corroborated by the observation that actinomycin D has little effect on the formation of Cu-chelatin if administered 1 h after injection of copper. It is of further interest that actinomycin D had considerably less inhibitory effect on the incorporation of 13Hllysine into other protein fractions (Fig. 4D). It is evident that actinomytin-resistant incorporation resulted from translation of preexistent stable mRNA molecules coding for a variety of proteins. Since the uptake of copper by the liver was unaffected by treatment with cycloheximide or actinomycin D, it was of interest to determine the distribution of the metal in liver extracts from animals pretreated with either antibiotic. For this purpose the unheated 100,OOOg supernatant fractions were concentrated and chromatographed on Sephadex G-75. Figure 5 shows the elution profiles of pooled supernatant fractions from the actinomycin D group as well as the unheated supernatant fractions from the group injected with copper only. In the latter group, the metal was predomiassociated with Cu-chelatin; nantly whereas, in the actinomycin D group, the metal was associated primarily with the major protein fractions. Figure 6 shows elution profiles for the heated and unheated 100,OOOg supernatant fractions from the cycloheximide-treated group. Again it is apparent that the copper is bound to the major protein species which are coagulated by heat. The incorporation of 13Hllysine into Cuchelatin was substantiated by analyzing the radioactivity in polyacrylamide-gel slices after electrophoresis of Cu-chelatin purified through acetone fractionation. Gels were sliced into 14 segments of 0.4 cm each, digested, and counted as described in Materials and Methods. The major
INDUCTION
50
60
70
OF
COPPER
80 90 100 TUBE NUMBERS
273
CHELATIN
110
ml
130
FIG. 5. Copper distribution after pretreatment with actinomycin D. The experimental conditions were the same as described in Table I except that the 100,OOOg supernatant fraction was not subjected to the 60°C beat treatment. The unheated supernatant fractions from the Cutreated and actinomycin D groups having the same initial total copper content of 150 pg were chromatographed on Sephadex G-75. The absorption at 280 nm (O---O), and copper contents of the eluates from the copper group (O..,O) and actinomycin D group (0-O) were determined.
20
40
So
SC TUBE WMSER
120
I40
160
FIG. 6. Effect of heat treatment on copper distribution in the cycloheximide group. The experimental conditions were the same as described in Table I, except that the 100,OOOg supernatant fluid was divided into two equal parts, one of which was subjected to the heat treatment. The two were then separately chromatographed on Sephadex G-75 under conditions as in Fig. 1. Absorbance at 280 nm (-1 and copper concentration (- - -1 of the eluates were determined. A and B represent the elution profiles of the unheated and heated samples, respectively.
r3H]lysine peak coincided with the segment containing the Cu-chelatin band. [3H]0rotic Acid Incorporation into RNA In view of the suggestive evidence for the dependence of Cu-chelatin formation on RNA synthesis, the effect of copper ad-
ministration on [3H]orotic acid incorporation into rapidly labeled hepatic RNA in control and actinomycin D-treated rats was investigated. The labeled erotic acid was administered 10 min after injection of CuCl, or saline, and the animals were killed 20 or 40 min thereafter. The livers were excised and isolation of RNA was carried out as described in Materials and Methods. Total cellular RNA was extracted from an aliquot of the 25% homogenate of liver. As can be seen in Table III, copper administration led to an 11% increase in [3Hlorotic acid incorporation into total cellular RNA. The incorporation was inhibited about 64% in the group pretreated with actinomycin D. The incorporation of the isotope into both cytoplasmic and nuclear RNA was substantially enhanced by copper adminstration. The 20min [3H]orotic acid pulse resulted primarily in nuclear RNA labeling whereas, after the 40-min pulse, a significant increase in the ratio of cytoplasmic to nuclear labeled RNA could be observed. Turnover of Cu-Chelatin in the Rat It was of interest to determine the fate of the accumulated hepatic copper and of the induced Cu-chelatin. For this purpose, a single intraperitoneal injection of copper was administered to animals. Animals were killed at various times ranging from 12-180 h after the injection, and the copper
274
PREMAKUMAR
ET
content of crude homogenates of the liver and kidney and the soluble protein fraction from the liver was measured (Fig. 7). In both intact liver and kidney, as well as in the cellular supernatant fractions, the accumulated copper content was depleted by more than 50% at the end of 48 h.
In a second experiment, rats were injetted with a single dose of CuClz and 3 h later with a solution containing [3Hllysine (20 /AN and [35Slcystine (20 /LX). The animals were killed either 15, 40 or 88 h from the time of copper administration. Cu-chelatin was purified from each group
TABLE INCORPORATION Group
AL.
III
OF [3H10~~~~~
ACID
Pulsing time (min)
INTO RNA”
3H RNA Cellular
Control Cu-treated Actinomycin Cu-treated
20 20 40 20
D,
49,000 55,300
of liver)
Nuclear
-+ 2,980 2 1,730
26,000 + 31,200 + 23,300 ” 9,300 k
18,000
(cpmlg
r 1,710
Cytoplasmic 3,240 3,500 1,860 1,470
6,100 7,100 13,600 5,700
2 2 5 k
207 507 1,030 606
LI Each group consisted of three rats, and the results presented are the mean r SEM for each group. Animals in all groups received [3H]orotic acid by intraperitoneal injection. Copper (3 mg per kg) as CuCl, was given to the animals in groups 2 and 3,lO min prior to the injection of erotic acid. Actinomycin D (1.5 mg per kg) was administered to rats in group 3,l h prior to copper injection. The animals were sacrificed 20 or 40 min after the erotic acid pulse. The incorporation of the label into different fractions of liver RNA was determined as described in the text.
30 t 0 \ :
20
P
t
IO
0 =& I
I
I
I
20
40
60
00
HOURS
AFTER
I
I
I
I
I
100
120
140
160
100
INJECTION
FIG. 7. Fate of copper in rat liver and kidney following a single intraperitoneal injection of CuCl%. Animals in groups of three were given copper at a dose of 3 mg per kg. At the time points indicated, the rata were killed, and tissue homogenates were prepared as described in Materials and Methods. Analysis of copper in the homogenates was according to the procedure described in Fig. 3. Each point represent the average result from three rats. The curves represent the following: (-•-) hepatic copper content, (-A-) renal copper content, and (O- - -0) total copper in the 100,OOOg supernatant fraction from liver.
INDUCTION
OF
COPPER
and from a control group by Sephadex G-75 column chromatography and acetone fractionation. The acetone-fractionated protein was analyzed for protein, copper content and radioactivity (Table IV). The turnover of protein and copper occurred in parallel, with the levels being 50% depleted by 40 h. DISCUSSION
Copper-chelatin is detected in rat liver cytosol following intraperitoneal administration of copper. The holoprotein is not detectable in control animals to any appreciable content. That this accumulation of Cu-chelatin is not due to the binding of copper to preexisting apoprotein is shown by the following lines of evidence. (a) Addition of copper to the 100,OOOg supernatant fractions of control livers does not lead to enhanced content of Cu-chelatin. (b) The Sephadex G-75 fraction from the control preparations corresponding to the Cu-chelatin region does not bind copper under conditions used for the reconstitution of apochelatin with the metal. (c) Treatment of animals with actinomytin D results in inhibition of the formation of Cu-chelatin on subsequent exposure to copper, though the hepatic uptake of the TABLE TURNOVER
Group
Control Cu-treated &-treated Cu-treated
IV
OF CU-CHELATIN
IN RAT
Hours Proafter la- tein be1 in- (mg) jection
Copper (Pi?)
1.2 6.0 3.3 1.8
2 54 27 12
15 15 40 88
LIVER”
3H ‘zgy
1,500 4,300 2,200 500
93 ‘zP$
4.300 13,800 6,600 1,300
’ Groups of three rats received single intraperitoneal injections of CuClz (3 mg of &/kg) and 3 h later were given an injection containing [3H]lysine (20 PCil and [Yllcystine (20 &il. Control animals received an injection of 0.9% NaCl instead of copper. Animals were killed at various times after the second injection. Cu-chelatin was purified as described earlier by Sephadex G-75 chromatography and acetone fractionation. The parameters listed below were analyzed as described in Materials and Methods.
CHELATIN
275
metal is not impaired. Under these conditions liver copper is bound nonspecifically to major protein species. The lack of accumulation of Cu-chelatin in liver under these in uivo conditions may be ascribed to the absence of significant amounts of functional apochelatin. Administration of copper stimulates the incorporation of r3Hllysine into Cu-chelatin without appreciably affecting the extent of labeling of other proteins. The increased labeling of Cu-chelatin is abolished by pretreatment of the animals with either cycloheximide or actinomycin D. The observed inhibition is not a consequence of interference with hepatic copper uptake. The evidence derived from use of actinomycin D indicates that large-scale synthesis of Cu-chelatin occurs through induction of the synthesis of mRNA specifically coding for Cu-chelatin. Since actinomycin D has been reported to exert anomalous effects (10-121, additional experimental verification was required for the RNA synthesis mechanism. Such evidence has come from observation that incorporation of 13Hlorotic acid into RNA in pulse-labeling experiments is significantly enhanced immediately following copper injection. This effect is seen in total cellular RNA as well as nuclear and cytoplasmic RNA. Pulse-labeling for 40 min suggested that with time the rapidly labeled RNA is released into the cytoplasm from the nucleus. The evidence presented above, i.e., the virtual absence of Cu-chelatin from control tissues, the absence of measurable apoprotein content, the stimulation by copper of lY3Hllysine incorporation, the enhancement of [3Hlorotic acid incorporation, and the inhibition by actinomycin D of the effects of copper, when taken together permit the conclusion that induction of Cu-chelatin is under transcriptional control. The mechanism could involve either stimulation of specific mRNA synthesis or stabilization by copper of the mRNA which is otherwise degraded rapidly. The latter mechanism, however, seems unlikely from the results of our experiments using actinomycin D. It cannot be stated at this time whether Cuchelatin is the only protein induced by
276
PREMAKUMAR
copper administration in the rat liver and whether the enhancement in [3H]orotic acid labeling represents only mRNA for the Cu-chelatin. The de nouo synthesis of chelatin appears to be a more general phenomenon. As discussed in the following paper (13), formation of Cu-chelatin in yeast is dependent upon exposure of the cells to copper and requires de nouo protein synthesis as demonstrated by the strong inhibitory effect of cycloheximide. It is uncertain whether the induction in yeast is also under transcriptional control as in rat liver. Our results on the inducible nature of Cu-chelatin conflict with the conclusion of Bloomer and Sourkes (2) that the protein is constitutive. It might be pointed out, however, that they did not use either cycloheximide or actinomycin D in their experiments to test the inducibility of the protein. Bremner and Davies (3) reported that injection of cycloheximide simultaneously with CuS04 into rats led to a 90% reduction in labeling of the rat liver copperbinding protein. The results presented in this report are in agreement with the observation of Bremner and Davies. The ability of metal ions to regulate the synthesis of metalloproteins is known. The ferritin content of mammalian tissues is regulated by iron through a translational mechansim (14,151. One postulated mechanism suggests that binding of iron to nascent apoferritin polypeptides on the free polysomes relieves an inhibition of apoferritin synthesis (16). As discussed in the first paper in this series (17), cadmium administration to rats results in the accumulation of metallothionein. Squibb and Cousins (18) reported that the induction was blocked by pretreatment of the animals with either cycloheximide or actinomycin D. Davies et al. (19) and Webb (20) also found that cycloheximide inhibited the metal-induced formation of thionein. In support of a transcriptional mechanism, Weser et al. (21, 22) reported that cadmium and zinc stimulated the incorporation of [6-14C]orotate into nuclear RNA. The content of Cu-chelatin formed in rat liver after a single subcutaneous injection of CuClz is depleted 50% in less than 40 h.
ET
AL.
In contrast, Shaikh and Lucis (23) found that 2 weeks after a single subcutaneous injection of CdCl, into rats, hepatic Cd, Znthionein was present at a concentration unchanged from that seen 1 day after the injection. The half-time for Cd, Zn-thionein turnover in the rat presumably exceeds 100 days (24). The significance of the widely different turnover times for Cu-chelatin and the thionein remains to be investigated. While no biological role for Cu-chelatin has been defined, these studies suggest that the protein can function to sequester free copper ions which are potentially cytotoxic. The rapid induction response to high levels of the metal would permit the protein to minimize the disruption of cellular functions by free copper ions. Finally, another significant feature of the copper-dependent induction of Cu-chelatin is apparent from the data in Table I. The marked enhancement in the incorporation of [3H]lysine into newly synthesized protein in livers of Cu-treated animals must result, to a large extent, from the synthesis of Cu-chelatin. This system therefore represents one of the few instances in which the rate of synthesis of a single protein is altered to such an extent in response to an extraneous stress as to make it the predominant single product of hepatic protein synthesis under these circumstances. REFERENCES 1. WINGE, D. R., PREMAKUMAR, R., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem. Biophys. 170, 253-266. 2. BLOOMER, L. C., AND SOURKES, T. L. (1973) Biochem. Med. 8, 78-91. 3. BREMNER, I., AND DAVIES, N. T. (1974) B&hem. Sot. Trans. 2, 425-427. 4. PREMAKUMAR, R., WINGE, D. R., ANDRAJAGOPALAN, K. V. (1974) Fed. Proc. 33, 1385. 5. JOVIN, T., CHRAMBACH, A., AND NAUGHTON, M. A. (1964) Anal. Biochem. 9, 351-369. 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. SCHNEIDER, W. C. (1957) in Methods in Enzymoiogy (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 680, Academic Press, New York. 8. WANG, T. Y. (1967) in Methods in Enzymology
INDUCTION
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