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The accumulation of copper by rat liver slices
ARCRIVES OF BIOCHEMISTRYAND BIOPHYSICS83, 538-547 (1959)
The Accumulation of Copper by Rat Liver Slices’ Paul Saltman, Ted Alex and Bruce McCornack From the Department of Biochemistry Southern
and Nutrition, School of Medicine, California, Los Angeles, California
University
of
Received January 23, 1959 INTRODUCTION
The role of copper as an essential trace element in the metabolism of animals, plants, and bacteria has long been recognized. Several excellent reviews concerning the nutritional aspects of this ion are available (1, 2), as well as its mechanism of participation in various enzymic reactions (3). Copper is intimately involved in human pathological conditions (4, 5). The most interesting example of the disruption of normal copper metabolism is seen in Wilson’s disease where the lack of ceruloplasmin, the normal serum protein carrier of this ion, permits the accumulation of tremendous amounts of copper in the central nervous system as well as in the liver (6). It has been demonstrated (7) in some cases of iron storage disease that there is a concomitant increased deposition of copper above normal levels. We felt that it would be of interest to investigate the normal mechanisms by which copper can be metabolized by liver tissues with the hope that we could gain insight into some of the biochemical lesions associated with pathological conditions. It will be shown in this paper that copper is accumulated by rat liver slices by a mechanism which is not directly dependent upon energy production by the cell. It appears that the metabolism involves the sorption of the copper by some copper-binding entity within the cell. The kinetics of this reaction have been studied in some detail, as well as other chemical and physical properties of the system. MATERIALS
AND METHODS
Male rats 2OO-300g. were sacrificed by decapitation. The livers were immediately removed and placed in chilled Krebs-Ringer solution (K-R), and slices were prepared with the conventional Stadie-Riggs microtome. The slices were pooled and blotted with filter paper, and aliquots of lOO-200 mg. (wet weight) were placed in chilled 2O-ml. beakers containing the reaction mixture. 1 This research was supported in part by a contract with the U. S. Atomic Energy Commission and by the Smith, Kline and French Foundation. The facilities of the Allen Hancock Foundation were generously provided. 538
ACCUMULATION OF COPPER
539
Because of the low solubility of copper hydroxide at physiological pH’s, it, was necessary to supply the ion as a citrate complex. Copper citrate was prepared by mixing equimolar concentrations of copper nitrate and citric acid, adjusting the pH to 7.0, and then diluting to the desired concentration with K-R. Radioactive Cue4 was obtained from the Oak Ridge National Laboratories as copper nitrate in 0.2 N HNO, . The citrate complex of the Cue’ was prepared as described above. Suitable dilutions of the radioactive copper citrate were prepared in K-R. The total volume of the reaction mixture was 5.0 ml. of K-R, adjusted to pH 7.0, in which was dissolved copper citrate, the Cu”4, and any other compound under investigation. The slices were incubated in a Dubnoff metabolic shaking incubator. Temperature, unless otherwise stated, was 37°C. The rate of shaking was 80 cycles/ min. After the desired period of incubation the slices were removed, washed three times with chilled K-R, and digested wet with 2.0 ml. of H2SOd-HN03 (1:l of the concentrated acids). Near the end of the digestion period, several drops of H102 were added to complete the digestion. Total copper was determined calorimetrically by a modification of the dithizone method (8). The colored complex was extracted into carbon tetrachloride, and the optical density was determined at 540 rnp. Total copper values were corrected to 1.0 g. wet weight of tissue. Radioactivity was determined on the total digest in a scintillation well counter, with a pulse height analyzer set at the 0.5-mev. peak. Activity was corrected to 1.0 g. of wet weight,. Each experiment
was run in triplicate; the data are expressedas the average of three values. Experiments were designed to study the efflux of accumulated copper. Slices were permitted to take up Cue4 for a suitable period of time. The slices were washed and transferred to either copper-free or coppel-citrate-containing K-R solution. At, various intervals after transfer, the slices were washed again and digested, and their activity measured. In order to be certain that the preparation of the liver slices did not destroy the integrity of the cells, two types of experiments were carried out. Through the cooperation of Dr. John Webb and Mr. Phillip Hollander of our Department of Pharmacology, we were able to measure the electric potential of single cells from intact lobes as well as slices of liver. These techniques have been described in detail (9). The resting potentials for both lobes and slices were in the same range, 9-13 mv. All of the electrical properties of both preparations appeared the same. When surface-active agents were added, the resting potential immediately fell to 0 mv. Microscopic examination of fixed tissue slices by Drs. Butt and Bernick, Department of Pathology, indicated that 90% or better of the cells were intact. The primary site of copper binding as revealed by staining with rubianic acid (10) was within the intact cells. Examination by high-power phase microscopy of slices incubated with surface-active agents confirmed the electric potential data. Rupture of the cell membranes was clearly seen. RESULTS
The Kinetics of Copper Accumulation Slices were incubated in K-R solution containing 5 pg. copper/ml. (cupric citrate with a specific activity of 8.1 X lo3 counts/min./pg. copper). At the times indicated in Fig. 1, the slices were removed for the determination of both total, as well as radioactive copper. A significant observation in this experiment is that the rate of uptake of radioactive copper closely parallels the rate of accumulation of total copper. It is apparent, therefore,
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FIG. 1. Accumulation of copper by rat liver slices as a function of time. Slices were incubated in 5.0 ml. of Krebs-Ringer buffer, pH 7.0, containing 5rg. Cu/ml. as cupric citrate (specific activity 8.1 X 108 counts/min./pg. Cu). At intervals slices were washed and their radioactivity measured. The slices were then digested in H&WHNOI and total copper concentration determined by the dithizone method.
that ion-exchange phenomena are playing a relatively minor role in the accumulation process. Further, this experiment indicates that it is experimentally valid to follow uptake by means of radioactivity measurements alone and thus avoid the tedious total copper analyses. The time course of copper uptake by liver slices was similar to the result obtained in our previous studies (11) on iron accumulation. We therefore applied the kinetic formulations developed for iron metabolism to our copper studies. The rate of uptake of the ion is dependent upon the number and accessibility of binding sites. The total number of sites should be proportional to the total amount of copper sorbed after an infinite time. Thus the rate can be expressed as: @Q/d0 = W&.9- Q>
(1)
where Q = the amount of copper at t, Q8 = the amount of copper after infinite time, and k = the rate constant. Integration of Eq. (1) results in the expression: ln [(Qs - Q>/(Q8- Q&l = -kt
(2)
where Q0 = the amount of copper initially present. Figure 2 presents the data from an uptake experiment carried out at 37” and 0”. It is clear that not only is the rate enhanced at the higher temperature, but also that the capacity of the copper-binding entity has increased. If these data are plotted in semilogarithmic fashion as suggested by Eq. (2), the two straight lines seen in Fig. 3 are obtained. The slopes equal -k/2.3. The insert in Fig. 3 shows the values for the rate constants ob-
ACCUMULATION
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FIG. 2. The uptake
of copper by rat liver slices at 0’ and 37”. Two series of slices were incubated at 0” and 37’, respectively, in 5.0 ml. of Krebs-Ringer buffer, pH 7.0, containing 5 pg. Cu/ml. as cupric citrate. At intervals, slices were removed, washed, and digested in H2S04-HN03 and total copper determined by the dithizone method.
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FIG. 3. Determination of rate constants for the uptake of copper by rat liver slices. The data data from from Fig. 2 are shown shown plotted plotted as as log log (Q. (Q. - Q)/(Qs Q)/(Qs - Qo) Qo) vs. vs. t. The slopes of the straight lines are equal to -k/2.3. The insert indicates the values for k so obtained. tained. The apparent activation tion is 1300 cal./mole/deg.
Eflux
energy
obtained
from the Arrhenius
equa-
and Exchange of Copper in Liver Slices
It seemed of interest to study the kinetics of the efllux ously accumulated by liver slices both into a copper-free
of copper previand copper-con-
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ALEX
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MCCORNACK
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Minutes FIG. 4. The rate of efflux of radiocopper from rat liver slices. Slices were incubated in 5.0 ml. Krebs-Ringer buffer containing 5 pg. &/ml. as copper citrate (specific activity, 6 X lo4 counts/min./pg. Cu). At 15 and 45 min. a series of slices was removed to 5.0 ml. of Krebs-Ringer buffer either with (+) or without (0) 5 pg. &/ml. as nonradioactive cupric citrate. Other slices were permitted to continue to accumulate Cu6’ (a). The slices were washed and radioactivity measured.
medium. To do this, slices previously incubated with CUDSfor 15 or 45 min. were permitted to lose or exchange the CUDSinto K-R with or without nonradioactive copper present. A control series was allowed to accumulate CUDSover the entire period to follow the time course of uptake for total copper. Figure 4 presents the data from such an exneriment. Since the efllux of CUDSinto a copper-containing medium is measurably enhanced, it seems likely that some ion-exchange phenomena are operative in copper metabolism. However, the role of the exchange seems rather slight in comparison with similar situations observed with other ions in other tissues (12). It can be seen from these data that the presence of nonradioactive copper approximately doubles the amount of CUDSfrom the slice which is available for release. It should also be noted that a significant percentage of the total CUDSaccumulated is so firmly bound as to be unavaiiable either for efllux or exchange. taining
The Uptake of Copper us. External Concentration of Copper Liver slices were incubated in the presence of various external copper concentrations for 20 min. The activity of the CUDSin the medium was in direct proportion to the total copper concentration in the solution. Figure 5 presents the data from such an experiment. The shape of the uptake vs. concentration curve appears to be sigmoid. These data are not susceptible to analysis by the method of Lineweaver and Burk, as were the results of similar studies on iron metabolism (13).
ACCUMULATION
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Buffer
FIG. 5. Effect of external copper concentration on the initial rate of uptake of copper by rat liver slices. Slices were incubated for 20 min. in 5.0 ml. of Krebs-Ringer buffer containing various concentrations of radioactive cupric citrate. The activity in the buffer was directly proportional to the copper concentration (specific activity, 110 counts/min./pg. Cu). The slices were washed and radioactivity measured.
The E$ect of pH on Accumulation Copper can be accumulated over a range of pH from 5.0 to 7.3. The K-R solution was adjusted to the indicated pH prior to the incubation, and the pH was checked at the end of the 20-min. incubation period to insure pH regulation. Above pH 7.3, the copper precipitated, thus limiting the pH range which could be studied. The E$ect of Pretreatment at Various Temperatures Much of the evidence for the mechanism of copper accumulation points to the participation of binding sites on or within the cell. Experiments were designed to modify the properties of these sites by pretreatment at elevated temperatures. Liver slices were placed in 5 ml. of pH 7 buffer solution without added copper, and were incubated for 20 min. at the various temperatures indicated. They were then transferred to Cuc4 solution and permitted to accumulate copper at 37” for 30 min. The results are presented in Fig. 6. It can be seen that the pretreatment at elevated temperatures enhances the rate of accumulation. In many experiments the total amount of copper accumulated in 30 min. by the slices pretreated at 60” exceeded the total accumulated by the 37” slices at their equilibrium level. These results point to irreversible changes in the total number and/or capacity
of the binding
sites and their accessibility.
The Effect of Various Chemical Agents on Copper Accumulation Several metabolic inhibitors were tested for their effect on copper accumulation. Cells were preincubated for 15 min. without copper at 37” in
the buffer solution
containing
the inhibitor,
washed, and transferred
to
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SALTMAN,
b, 4000 F
ALEX
AND
MCCORNACK
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8 7 2 2000 .p. $ s 8
Preincubafion
Tempera&e
I _ 100
PC/
FIG. 6. Effect of pretreatment at various temperatures on copper accumulation. Slices were preincubated for 20 min. at various temperatures in 5.0 ml. of KrebsRinger buffer, pH 7.0, in the absence of copper. The slices were then transferred to 5.0 ml. of Krebs-Ringer containing 5 pg. Cu/ml. as cupric citrate (specific activity, 520 counts/min./pg. Cu). After incubation for 30 min. at 37”, the slices were washed and radioactivity measured.
fresh buffer solution containing both inhibitor and CUDS.The slices were allowed to accumulate the ion for 20 min., and the activity of the slices was measured. Table I presents the results of this experiment. Both dinitrophenol and sulfhydryl inhibitors manifest little effect on the copper uptake. The possibility that sulfhydryl groups are intimately concerned with this process is further negated by the results with glutathione and TABLE Effect of Various
Inhibitors
I
on the Uptake
of Copper by Rat Liver
Slices
Liver slices were preincubated for 15 min. at 37” in 5.0 ml. of Krebs-Ringer buffer, pH 7.0, containing the inhibitor, but no copper. The slices were washed and transferred to fresh Krebs-Ringer containing both the inhibitor and 5 fig. Cu/ml. as cupric citrate (specific activity, 7.0 X lo4 counts/min./ml.). After incubating 20 min., the slices were washed three times and radioactivity was determined. A control containing no inhibitor was carried through the same procedure. Condition
Concentration
Per cent activity of control
M 2,4-Dinitrophenol 2,4-Dinitrophenol Iodoacetate p-Chloromercuribenzoate p-Chloromercurisulfonate Cysteine Glutathione
lo-” 5 x lo-4 10-a 10-d 10-4 10-a 10-s
106 97 99 108 103 98 114
ACCUMULATION
OF
COPPER
545
cysteine. Surface-active agents such as Cutscum and Rocca13 had no effect. This would point to a rather indirect participation of the membrane as a barrier to permeability. Several di- and trivalent ions were tested including Zn++, Ni++, Mg++, Co++, and Fe+++. No significant effect on CUDSaccumulation by these ions was observed. DISCUSSION
The physical and chemical properties of the system or systems operative in the uptake of copper are remarkably similar to those observed for the accumulation of iron (11, 13, 14) and of zinc.4 All evidence points to a mechanism for the accumulation of copper which is not linked directly to the energy-generating metabolism of the cells. Brown and Justus (15) have recently demonstrated that iron transport into and through everted intestinal sacs is a passive process. Maynard (16) has shown that such passive mechanisms are responsible for the metabolism of manganese both in vitro and in vivo. Some insight into the nature of the process can be gained from a consideration of the results of the kinetic studies. The experimental data satisfy the theoretical equation. This indicates that some copper binding sites, capable of being saturated, exist within the cell. The rate at which the copper accumulates is dependent upon the rate constant, k, and is a function of the number and availability of copper-binding sites. Although pretreatment at high temperatures does increase the total number of sites available, there is little change in k. It appears that the cell membrane does not act as a permeability barrier to the entrance of copper. The fact that surface-active agents did not affect the rate or amount of copper accumulation indicates that, unlike yeast cells (17), the binding sites are not dependent upon the intact cell membranes. The energy of activation of the over-all uptake process as measured by the increase of Ic with temperature is approximately 1300 cal./mole/deg. This points to the participation of some physical process as the rate-limiting reaction. But there is still the possibility that other enzymic reactions are involved. We have not been able, as yet, to gain insight into any of these non-rate-limiting steps. We have considered that copper chelates are the only forms in which the ion could be presented to the cells. However, this is the same state in which most, if not all, heavy-metal ions are found under physiological conditions. Chelates of copper with organic and amino acids, sugars, as well as proteins, are to be expected. The amount of each copper chelate will * Isooctylphenoxypolyethoxyethanol. 3 High molecular alkyl-dimethyl benzyl ammonium chloride. 4 Saltman, P., and Boroughs, H., unpublished experiments.
546
SALTMAN, ALEX AND MCCORNACK
depend upon both the affinity for the ion and the concentration of the binding agent. Recent reports of a number of copper-binding proteins in the blood and liver give support to the concept that some system, analogous to ferritin for iron metabolism, is operative in the uptake of copper by the liver. One of the most exciting findings concerning copper-binding proteins of liver has been presented by Uzman et al. (ES), who found an abnormal copperbinding protein present in cases of hepatolenticular disease (Wilson’s disease). This protein was not found in normal tissues but was present in very high concentration in diseased liver. Further, it was demonstrated that liver homogenates from patients with Wilson’s disease had the ability to take up and store very large amounts of copper compared with normals. Copper that is bound by rat liver slices can be mobilized as indicated in the efflux experiment. But a major fraction of this accumulated copper seems to be unavailable for transport. In this respect the copper system behaves quite similarly to that for iron. The possibility that nucleic acids or their derivatives play an important role in copper accumulation should also be considered. Frieden and Alles (19) have demonstrated that copper is bound with great avidity to such compounds as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The sigmoid curve observed when uptake is measured against copper concentration could be interpreted as a “self-denaturation.” At low concentrations, only the primary action of copper accumulation is observed, However, at higher concentrations the secondary effect of the copper itself, participating in denaturation phenomena, leads to an enhanced uptake by making accessible previously unavailable sites. The data from the concentration experiments do not permit the determination of the afhnity of the copper for the sites. Much evidence seems to be accumulating for the importance of passive, rather than active, transport processes in the accumulation of trace metal ions, These systems depend upon the sorption of ions onto binding sites on or in the cell. The sites have a rather high degree of specificity with respect to the chemical nature of the ion being sorbed. In the two pathological conditions involving copper and iron,6 where abnormally high deposition of the ion takes place in the tissue, abnormal metal-binding proteins are synthesized and seem to be directly responsible for this accumulation. SUMMARY
1. The accumulation of copper by rat liver slices has been studied with respect to the effect of various chemical and physical conditions. 6 Unpublished
results from this laboratory.
ACCUMULATION OF COPPER
547
2. This accumulation is not directly coupled to metabolic energy. 3. The kinetics of uptake are of the first order and indicate that specific sites on or in the cell bind the copper. These sites can be saturated. A rate equation is derived to account for these observed results. 4. The copper-binding sites are affected by temperature but not by sulfhydryl inhibitors or activators, dinitrophenol, or surface-active agents. 5. The relationship of the mechanism of accumulation to some pathological conditions is discussed. REFERENCES 1. UNDERWOOD, E. J., “Trace 2.
3. 4.
5. 6. 7. 8. 9.
10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
Elements in Human and Animal Nutrition.” Academic Press, New York, 1956. MCELROY, W. D., AND GLASS, B., “Copper Metabolism.” Johns Hopkins Press, Baltimore, 1950. MAHLER, H. R., “Currents in Biochemical Research” (D. E. Green, ed.), p. 251. Interscience Publ. Co., New York, 1956. BUTT, E. M., NUSBAU?M, R. E., GILMOUR, T. C., AND DIDIO, S. L., Am. J. Clin. Pathol. 30, 479 (1958). SCHROEDER, H. A., Advances in Internal Med. 8,259 (1956). BEARN, A. G., Am. J. Med. 22,747 (1957). BUTT, E. M., NUSBAUM, R. W., GILMOUR, T. Cl., AND DIDIO, S. L., Am. J. Clin. Pathol. 24, 385 (1954). Determination of Trace Metals,” 2nd ed. p. 326. SANDELL, E. B., “Calorimetric Interscience Publ. Co., New York, 1959. HOLLANDER, P., AND WEBB, J. L., Circulation Research 2,604 (1955). Technic and Practical Histochemistry,” p. 266. LILLIE, R. D., “Histopathologic The Blakiston Co., New York, 1954. SALTMAN, P., FRISCH, H. L., FISKIN, R. D., AND ALEX, T., J. BioZ. Chem. 221, 777 (1956). KREBS, H. A., EGGLESTON, L. V., AND TERNER, C., Biochem. J. 48, 530 (1951). SALTMAN, P., FISKIN, R. D., AND BELLINGER, S. B., J. BioZ. Chem. 220,741 (1956). SALTMAN, P., FISKIN, R. D., BELLINFER, S. B., AND ALEX, T., J. BioZ. Chem. 220, 751 (1956). BROWN, E. B., AND JUSTUS, B. W., Am. J. Physiol. 194,319 (1958). MAYNARD, L. S., Ann. N. Y. Acad. Sci. 42,227 (1958). ROTHSTEIN, A., Symposia Sot. Exptl. BioZ. No. 8, 165 (1954). UZMAN, L. I,., IBER, R. L., AND CHALMERS, T. C., Am. J. CZin. Sci. 231,511 (1956). FRIEDEN, E., AND ALLES, J., J. BioZ. Chem. 230,797 (1958).
The Accumulation of Copper by Rat Liver Slices’ Paul Saltman, Ted Alex and Bruce McCornack From the Department of Biochemistry Southern
and Nutrition, School of Medicine, California, Los Angeles, California
University
of
Received January 23, 1959 INTRODUCTION
The role of copper as an essential trace element in the metabolism of animals, plants, and bacteria has long been recognized. Several excellent reviews concerning the nutritional aspects of this ion are available (1, 2), as well as its mechanism of participation in various enzymic reactions (3). Copper is intimately involved in human pathological conditions (4, 5). The most interesting example of the disruption of normal copper metabolism is seen in Wilson’s disease where the lack of ceruloplasmin, the normal serum protein carrier of this ion, permits the accumulation of tremendous amounts of copper in the central nervous system as well as in the liver (6). It has been demonstrated (7) in some cases of iron storage disease that there is a concomitant increased deposition of copper above normal levels. We felt that it would be of interest to investigate the normal mechanisms by which copper can be metabolized by liver tissues with the hope that we could gain insight into some of the biochemical lesions associated with pathological conditions. It will be shown in this paper that copper is accumulated by rat liver slices by a mechanism which is not directly dependent upon energy production by the cell. It appears that the metabolism involves the sorption of the copper by some copper-binding entity within the cell. The kinetics of this reaction have been studied in some detail, as well as other chemical and physical properties of the system. MATERIALS
AND METHODS
Male rats 2OO-300g. were sacrificed by decapitation. The livers were immediately removed and placed in chilled Krebs-Ringer solution (K-R), and slices were prepared with the conventional Stadie-Riggs microtome. The slices were pooled and blotted with filter paper, and aliquots of lOO-200 mg. (wet weight) were placed in chilled 2O-ml. beakers containing the reaction mixture. 1 This research was supported in part by a contract with the U. S. Atomic Energy Commission and by the Smith, Kline and French Foundation. The facilities of the Allen Hancock Foundation were generously provided. 538
ACCUMULATION OF COPPER
539
Because of the low solubility of copper hydroxide at physiological pH’s, it, was necessary to supply the ion as a citrate complex. Copper citrate was prepared by mixing equimolar concentrations of copper nitrate and citric acid, adjusting the pH to 7.0, and then diluting to the desired concentration with K-R. Radioactive Cue4 was obtained from the Oak Ridge National Laboratories as copper nitrate in 0.2 N HNO, . The citrate complex of the Cue’ was prepared as described above. Suitable dilutions of the radioactive copper citrate were prepared in K-R. The total volume of the reaction mixture was 5.0 ml. of K-R, adjusted to pH 7.0, in which was dissolved copper citrate, the Cu”4, and any other compound under investigation. The slices were incubated in a Dubnoff metabolic shaking incubator. Temperature, unless otherwise stated, was 37°C. The rate of shaking was 80 cycles/ min. After the desired period of incubation the slices were removed, washed three times with chilled K-R, and digested wet with 2.0 ml. of H2SOd-HN03 (1:l of the concentrated acids). Near the end of the digestion period, several drops of H102 were added to complete the digestion. Total copper was determined calorimetrically by a modification of the dithizone method (8). The colored complex was extracted into carbon tetrachloride, and the optical density was determined at 540 rnp. Total copper values were corrected to 1.0 g. wet weight of tissue. Radioactivity was determined on the total digest in a scintillation well counter, with a pulse height analyzer set at the 0.5-mev. peak. Activity was corrected to 1.0 g. of wet weight,. Each experiment
was run in triplicate; the data are expressedas the average of three values. Experiments were designed to study the efflux of accumulated copper. Slices were permitted to take up Cue4 for a suitable period of time. The slices were washed and transferred to either copper-free or coppel-citrate-containing K-R solution. At, various intervals after transfer, the slices were washed again and digested, and their activity measured. In order to be certain that the preparation of the liver slices did not destroy the integrity of the cells, two types of experiments were carried out. Through the cooperation of Dr. John Webb and Mr. Phillip Hollander of our Department of Pharmacology, we were able to measure the electric potential of single cells from intact lobes as well as slices of liver. These techniques have been described in detail (9). The resting potentials for both lobes and slices were in the same range, 9-13 mv. All of the electrical properties of both preparations appeared the same. When surface-active agents were added, the resting potential immediately fell to 0 mv. Microscopic examination of fixed tissue slices by Drs. Butt and Bernick, Department of Pathology, indicated that 90% or better of the cells were intact. The primary site of copper binding as revealed by staining with rubianic acid (10) was within the intact cells. Examination by high-power phase microscopy of slices incubated with surface-active agents confirmed the electric potential data. Rupture of the cell membranes was clearly seen. RESULTS
The Kinetics of Copper Accumulation Slices were incubated in K-R solution containing 5 pg. copper/ml. (cupric citrate with a specific activity of 8.1 X lo3 counts/min./pg. copper). At the times indicated in Fig. 1, the slices were removed for the determination of both total, as well as radioactive copper. A significant observation in this experiment is that the rate of uptake of radioactive copper closely parallels the rate of accumulation of total copper. It is apparent, therefore,
540
SALTMAN,
I
ALEX
20
AND
Minutes
MCCORNACK
40
FIG. 1. Accumulation of copper by rat liver slices as a function of time. Slices were incubated in 5.0 ml. of Krebs-Ringer buffer, pH 7.0, containing 5rg. Cu/ml. as cupric citrate (specific activity 8.1 X 108 counts/min./pg. Cu). At intervals slices were washed and their radioactivity measured. The slices were then digested in H&WHNOI and total copper concentration determined by the dithizone method.
that ion-exchange phenomena are playing a relatively minor role in the accumulation process. Further, this experiment indicates that it is experimentally valid to follow uptake by means of radioactivity measurements alone and thus avoid the tedious total copper analyses. The time course of copper uptake by liver slices was similar to the result obtained in our previous studies (11) on iron accumulation. We therefore applied the kinetic formulations developed for iron metabolism to our copper studies. The rate of uptake of the ion is dependent upon the number and accessibility of binding sites. The total number of sites should be proportional to the total amount of copper sorbed after an infinite time. Thus the rate can be expressed as: @Q/d0 = W&.9- Q>
(1)
where Q = the amount of copper at t, Q8 = the amount of copper after infinite time, and k = the rate constant. Integration of Eq. (1) results in the expression: ln [(Qs - Q>/(Q8- Q&l = -kt
(2)
where Q0 = the amount of copper initially present. Figure 2 presents the data from an uptake experiment carried out at 37” and 0”. It is clear that not only is the rate enhanced at the higher temperature, but also that the capacity of the copper-binding entity has increased. If these data are plotted in semilogarithmic fashion as suggested by Eq. (2), the two straight lines seen in Fig. 3 are obtained. The slopes equal -k/2.3. The insert in Fig. 3 shows the values for the rate constants ob-
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FIG. 2. The uptake
of copper by rat liver slices at 0’ and 37”. Two series of slices were incubated at 0” and 37’, respectively, in 5.0 ml. of Krebs-Ringer buffer, pH 7.0, containing 5 pg. Cu/ml. as cupric citrate. At intervals, slices were removed, washed, and digested in H2S04-HN03 and total copper determined by the dithizone method.
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FIG. 3. Determination of rate constants for the uptake of copper by rat liver slices. The data data from from Fig. 2 are shown shown plotted plotted as as log log (Q. (Q. - Q)/(Qs Q)/(Qs - Qo) Qo) vs. vs. t. The slopes of the straight lines are equal to -k/2.3. The insert indicates the values for k so obtained. tained. The apparent activation tion is 1300 cal./mole/deg.
Eflux
energy
obtained
from the Arrhenius
equa-
and Exchange of Copper in Liver Slices
It seemed of interest to study the kinetics of the efllux ously accumulated by liver slices both into a copper-free
of copper previand copper-con-
542
SALTMAN,
I
ALEX
I
20
AND
I
MCCORNACK
I
40
I
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60
Minutes FIG. 4. The rate of efflux of radiocopper from rat liver slices. Slices were incubated in 5.0 ml. Krebs-Ringer buffer containing 5 pg. &/ml. as copper citrate (specific activity, 6 X lo4 counts/min./pg. Cu). At 15 and 45 min. a series of slices was removed to 5.0 ml. of Krebs-Ringer buffer either with (+) or without (0) 5 pg. &/ml. as nonradioactive cupric citrate. Other slices were permitted to continue to accumulate Cu6’ (a). The slices were washed and radioactivity measured.
medium. To do this, slices previously incubated with CUDSfor 15 or 45 min. were permitted to lose or exchange the CUDSinto K-R with or without nonradioactive copper present. A control series was allowed to accumulate CUDSover the entire period to follow the time course of uptake for total copper. Figure 4 presents the data from such an exneriment. Since the efllux of CUDSinto a copper-containing medium is measurably enhanced, it seems likely that some ion-exchange phenomena are operative in copper metabolism. However, the role of the exchange seems rather slight in comparison with similar situations observed with other ions in other tissues (12). It can be seen from these data that the presence of nonradioactive copper approximately doubles the amount of CUDSfrom the slice which is available for release. It should also be noted that a significant percentage of the total CUDSaccumulated is so firmly bound as to be unavaiiable either for efllux or exchange. taining
The Uptake of Copper us. External Concentration of Copper Liver slices were incubated in the presence of various external copper concentrations for 20 min. The activity of the CUDSin the medium was in direct proportion to the total copper concentration in the solution. Figure 5 presents the data from such an experiment. The shape of the uptake vs. concentration curve appears to be sigmoid. These data are not susceptible to analysis by the method of Lineweaver and Burk, as were the results of similar studies on iron metabolism (13).
ACCUMULATION
pg. Cu /ml
OF
COPPER
543
Buffer
FIG. 5. Effect of external copper concentration on the initial rate of uptake of copper by rat liver slices. Slices were incubated for 20 min. in 5.0 ml. of Krebs-Ringer buffer containing various concentrations of radioactive cupric citrate. The activity in the buffer was directly proportional to the copper concentration (specific activity, 110 counts/min./pg. Cu). The slices were washed and radioactivity measured.
The E$ect of pH on Accumulation Copper can be accumulated over a range of pH from 5.0 to 7.3. The K-R solution was adjusted to the indicated pH prior to the incubation, and the pH was checked at the end of the 20-min. incubation period to insure pH regulation. Above pH 7.3, the copper precipitated, thus limiting the pH range which could be studied. The E$ect of Pretreatment at Various Temperatures Much of the evidence for the mechanism of copper accumulation points to the participation of binding sites on or within the cell. Experiments were designed to modify the properties of these sites by pretreatment at elevated temperatures. Liver slices were placed in 5 ml. of pH 7 buffer solution without added copper, and were incubated for 20 min. at the various temperatures indicated. They were then transferred to Cuc4 solution and permitted to accumulate copper at 37” for 30 min. The results are presented in Fig. 6. It can be seen that the pretreatment at elevated temperatures enhances the rate of accumulation. In many experiments the total amount of copper accumulated in 30 min. by the slices pretreated at 60” exceeded the total accumulated by the 37” slices at their equilibrium level. These results point to irreversible changes in the total number and/or capacity
of the binding
sites and their accessibility.
The Effect of Various Chemical Agents on Copper Accumulation Several metabolic inhibitors were tested for their effect on copper accumulation. Cells were preincubated for 15 min. without copper at 37” in
the buffer solution
containing
the inhibitor,
washed, and transferred
to
544
SALTMAN,
b, 4000 F
ALEX
AND
MCCORNACK
I
I
I
I
I 20
I
I 60
I
I
8 7 2 2000 .p. $ s 8
Preincubafion
Tempera&e
I _ 100
PC/
FIG. 6. Effect of pretreatment at various temperatures on copper accumulation. Slices were preincubated for 20 min. at various temperatures in 5.0 ml. of KrebsRinger buffer, pH 7.0, in the absence of copper. The slices were then transferred to 5.0 ml. of Krebs-Ringer containing 5 pg. Cu/ml. as cupric citrate (specific activity, 520 counts/min./pg. Cu). After incubation for 30 min. at 37”, the slices were washed and radioactivity measured.
fresh buffer solution containing both inhibitor and CUDS.The slices were allowed to accumulate the ion for 20 min., and the activity of the slices was measured. Table I presents the results of this experiment. Both dinitrophenol and sulfhydryl inhibitors manifest little effect on the copper uptake. The possibility that sulfhydryl groups are intimately concerned with this process is further negated by the results with glutathione and TABLE Effect of Various
Inhibitors
I
on the Uptake
of Copper by Rat Liver
Slices
Liver slices were preincubated for 15 min. at 37” in 5.0 ml. of Krebs-Ringer buffer, pH 7.0, containing the inhibitor, but no copper. The slices were washed and transferred to fresh Krebs-Ringer containing both the inhibitor and 5 fig. Cu/ml. as cupric citrate (specific activity, 7.0 X lo4 counts/min./ml.). After incubating 20 min., the slices were washed three times and radioactivity was determined. A control containing no inhibitor was carried through the same procedure. Condition
Concentration
Per cent activity of control
M 2,4-Dinitrophenol 2,4-Dinitrophenol Iodoacetate p-Chloromercuribenzoate p-Chloromercurisulfonate Cysteine Glutathione
lo-” 5 x lo-4 10-a 10-d 10-4 10-a 10-s
106 97 99 108 103 98 114
ACCUMULATION
OF
COPPER
545
cysteine. Surface-active agents such as Cutscum and Rocca13 had no effect. This would point to a rather indirect participation of the membrane as a barrier to permeability. Several di- and trivalent ions were tested including Zn++, Ni++, Mg++, Co++, and Fe+++. No significant effect on CUDSaccumulation by these ions was observed. DISCUSSION
The physical and chemical properties of the system or systems operative in the uptake of copper are remarkably similar to those observed for the accumulation of iron (11, 13, 14) and of zinc.4 All evidence points to a mechanism for the accumulation of copper which is not linked directly to the energy-generating metabolism of the cells. Brown and Justus (15) have recently demonstrated that iron transport into and through everted intestinal sacs is a passive process. Maynard (16) has shown that such passive mechanisms are responsible for the metabolism of manganese both in vitro and in vivo. Some insight into the nature of the process can be gained from a consideration of the results of the kinetic studies. The experimental data satisfy the theoretical equation. This indicates that some copper binding sites, capable of being saturated, exist within the cell. The rate at which the copper accumulates is dependent upon the rate constant, k, and is a function of the number and availability of copper-binding sites. Although pretreatment at high temperatures does increase the total number of sites available, there is little change in k. It appears that the cell membrane does not act as a permeability barrier to the entrance of copper. The fact that surface-active agents did not affect the rate or amount of copper accumulation indicates that, unlike yeast cells (17), the binding sites are not dependent upon the intact cell membranes. The energy of activation of the over-all uptake process as measured by the increase of Ic with temperature is approximately 1300 cal./mole/deg. This points to the participation of some physical process as the rate-limiting reaction. But there is still the possibility that other enzymic reactions are involved. We have not been able, as yet, to gain insight into any of these non-rate-limiting steps. We have considered that copper chelates are the only forms in which the ion could be presented to the cells. However, this is the same state in which most, if not all, heavy-metal ions are found under physiological conditions. Chelates of copper with organic and amino acids, sugars, as well as proteins, are to be expected. The amount of each copper chelate will * Isooctylphenoxypolyethoxyethanol. 3 High molecular alkyl-dimethyl benzyl ammonium chloride. 4 Saltman, P., and Boroughs, H., unpublished experiments.
546
SALTMAN, ALEX AND MCCORNACK
depend upon both the affinity for the ion and the concentration of the binding agent. Recent reports of a number of copper-binding proteins in the blood and liver give support to the concept that some system, analogous to ferritin for iron metabolism, is operative in the uptake of copper by the liver. One of the most exciting findings concerning copper-binding proteins of liver has been presented by Uzman et al. (ES), who found an abnormal copperbinding protein present in cases of hepatolenticular disease (Wilson’s disease). This protein was not found in normal tissues but was present in very high concentration in diseased liver. Further, it was demonstrated that liver homogenates from patients with Wilson’s disease had the ability to take up and store very large amounts of copper compared with normals. Copper that is bound by rat liver slices can be mobilized as indicated in the efflux experiment. But a major fraction of this accumulated copper seems to be unavailable for transport. In this respect the copper system behaves quite similarly to that for iron. The possibility that nucleic acids or their derivatives play an important role in copper accumulation should also be considered. Frieden and Alles (19) have demonstrated that copper is bound with great avidity to such compounds as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The sigmoid curve observed when uptake is measured against copper concentration could be interpreted as a “self-denaturation.” At low concentrations, only the primary action of copper accumulation is observed, However, at higher concentrations the secondary effect of the copper itself, participating in denaturation phenomena, leads to an enhanced uptake by making accessible previously unavailable sites. The data from the concentration experiments do not permit the determination of the afhnity of the copper for the sites. Much evidence seems to be accumulating for the importance of passive, rather than active, transport processes in the accumulation of trace metal ions, These systems depend upon the sorption of ions onto binding sites on or in the cell. The sites have a rather high degree of specificity with respect to the chemical nature of the ion being sorbed. In the two pathological conditions involving copper and iron,6 where abnormally high deposition of the ion takes place in the tissue, abnormal metal-binding proteins are synthesized and seem to be directly responsible for this accumulation. SUMMARY
1. The accumulation of copper by rat liver slices has been studied with respect to the effect of various chemical and physical conditions. 6 Unpublished
results from this laboratory.
ACCUMULATION OF COPPER
547
2. This accumulation is not directly coupled to metabolic energy. 3. The kinetics of uptake are of the first order and indicate that specific sites on or in the cell bind the copper. These sites can be saturated. A rate equation is derived to account for these observed results. 4. The copper-binding sites are affected by temperature but not by sulfhydryl inhibitors or activators, dinitrophenol, or surface-active agents. 5. The relationship of the mechanism of accumulation to some pathological conditions is discussed. REFERENCES 1. UNDERWOOD, E. J., “Trace 2.
3. 4.
5. 6. 7. 8. 9.
10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
Elements in Human and Animal Nutrition.” Academic Press, New York, 1956. MCELROY, W. D., AND GLASS, B., “Copper Metabolism.” Johns Hopkins Press, Baltimore, 1950. MAHLER, H. R., “Currents in Biochemical Research” (D. E. Green, ed.), p. 251. Interscience Publ. Co., New York, 1956. BUTT, E. M., NUSBAU?M, R. E., GILMOUR, T. C., AND DIDIO, S. L., Am. J. Clin. Pathol. 30, 479 (1958). SCHROEDER, H. A., Advances in Internal Med. 8,259 (1956). BEARN, A. G., Am. J. Med. 22,747 (1957). BUTT, E. M., NUSBAUM, R. W., GILMOUR, T. Cl., AND DIDIO, S. L., Am. J. Clin. Pathol. 24, 385 (1954). Determination of Trace Metals,” 2nd ed. p. 326. SANDELL, E. B., “Calorimetric Interscience Publ. Co., New York, 1959. HOLLANDER, P., AND WEBB, J. L., Circulation Research 2,604 (1955). Technic and Practical Histochemistry,” p. 266. LILLIE, R. D., “Histopathologic The Blakiston Co., New York, 1954. SALTMAN, P., FRISCH, H. L., FISKIN, R. D., AND ALEX, T., J. BioZ. Chem. 221, 777 (1956). KREBS, H. A., EGGLESTON, L. V., AND TERNER, C., Biochem. J. 48, 530 (1951). SALTMAN, P., FISKIN, R. D., AND BELLINGER, S. B., J. BioZ. Chem. 220,741 (1956). SALTMAN, P., FISKIN, R. D., BELLINFER, S. B., AND ALEX, T., J. BioZ. Chem. 220, 751 (1956). BROWN, E. B., AND JUSTUS, B. W., Am. J. Physiol. 194,319 (1958). MAYNARD, L. S., Ann. N. Y. Acad. Sci. 42,227 (1958). ROTHSTEIN, A., Symposia Sot. Exptl. BioZ. No. 8, 165 (1954). UZMAN, L. I,., IBER, R. L., AND CHALMERS, T. C., Am. J. CZin. Sci. 231,511 (1956). FRIEDEN, E., AND ALLES, J., J. BioZ. Chem. 230,797 (1958).