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Influence of parenteral zinc and actinomycin D on tissue zinc uptake and the synthesis of a zinc-binding protein
BIOINORGANIC
CHEMISTR Y 4,2 1%224( 1975)
Influence of Parenteral Zinc and Actinomycin on Tissue Zinc Uptake and the Synthesis of a Zinc -Binding Protein * t MARK
P. RICHARDS
Department
and ROBERT
of Nutrition,
New Brunswick,
Cook
215
D
J. COUSINS
College.
Rutgers
University,
New Jersey 08903 ABSTRACT
Parenterally administered zinc markedly increased the incorporation of I 4C-cystine and 6 5 Zn into a low mo1ecula.r weight zinc binding protein (ZnBP) isolated from liver cytoplasm of rats fed an adequate amount of zinc. This zinc load significantly increased the zinc content in the liver. The increase in hepatic ainc content was inhibited by actinomycin D indicating that DNA-dependent RNA synthesis is required for zinc uptake into liver. Antinomycin D also produced a concomitant decrease in ZnBP synthesis indicating that this protein may be involved in the uptake mechanism in cells. Zinc repletion also stimulated the synthesis of hepatic ZnBP in zinc deficient rats. This stimulation was also prevented by prior administration of actinomycin D. A similar effect was observed in the intestinal mucosal cells. The data collectively indicate that the control of the synthesis of ZnBP which occurs at the transcriptional level of protein synthesis is responsive to zinc status and thus may have a function in zinc metabolism.
INTRODUCTION A number of studies have shown zinc metabolism is controlled by a homeostatic mechanism (1.21, i.e., zinc absorption appears to be related in some way to body stores of zinc. Concomitant to these studies, some investigators have postulated a role for a low molecular weight protein in the absorption process [ 3-5 1, however, the exact role of this protein(s), if any, has yet to be elucidated_ Specifically, a low molecular weight protein has been demonstrated by Van Campen in the intestinal mucosa which is capable of binding the cations Zinc, copper and cadmium [4] _ In addition, this protein has properties similar to
*Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers University-The State University of New Jersey, New Brunswick, New Jersey 08903. z t Supported in part by the Nutrition Foundation, innc. Future Leader Grant No. 458. 0 American Elsevier Publishing Company, Inc., 1975.
216
MARK
P_ RICHARDS
AND ROBERT
J. COUSINS
a metalloprotein of unknown function previously isolated “metallothionein”, from kidney and liver [ 6,7 1_ This report describes experiments designed to investigate the influence of zinc on the synthesis of a low molecular weight zinc binding protein (ZnBP) in liver and intestinal mucosa and the possible role of ZnBP in cellular zinc uptake.
MATERIAL Animals
AND METHODS
and Diets
A total of one hundred and twenty-three male rats of the Sprague-Dawiey strain (Charles River) were maintained on Purina Rat Chow, ad libitum, until they weighed 150 g, at which time they were used for experiments_ In those experiments, where zinc depleted rats were used, the animals were maintained as above until they weighed 300 g and were then fed a biotinenriched zinc deficient diet [81_ ad lib&urn, for two weeks.
Isotopes Nuclear Zinc-65 as 65ZnC1a (4.5 mCi/mg) was obtained from New E&and and was diluted to 20 pCi/mt with 09% NaCl prior to injection_ DL-cystine 3-r4C obtained from Schwarz-Mann with a specific activity of 30 mCi;mmole was diluted to 3_0 yCi/ml with 0.9% NaCl prior to injection.
Synthesis
of ZnBP
The rats were injected, intraperitoneally, with 5 PCi of 65Zn, 5 PCi of r4C-cystine and with either 2.0 mg of Zn*+ as ZnS04 in 0.9% NaCl or 0.9% NaCl. in some experiments actinomycin D (Sigma Chemical Company) in 50% propylene glycol was administered subcutaneously (0.8 mg/kg body weight) four hours before zinc was administered [9 I_ Three rats received each of the three treatment combinations. The rats were then fasted for twelve hours and killed by decapitation_ The livers were excised, weighed, washed in cold 0.9% NaC.1 and immediately homogenized in a glass-teflon homogenizer with an equal volume of O-25 M sucrose solution buffered at pH 8.6 with 10 mM Tris-HCl. in the experiments with zinc depleted rats mucosal cells were obtained from a 30 cm segment of the duodenal end of the small intestine that was perfused with 10 ml of ice cold 0.9% NaCl. The segment was split open and the cells harvested with a glass microscope slide using the method of Crane and Mandelstam [ 10 ] _ Mucosal homogenates were prepared as described above. Each homogenate was centrifuged at 42,000 xg and 4OC for 30 min. The supematant solution was removed and recentifuged at 42,000 g for 2 h_ Five milliliters of each final supernatant
ZINC-BINDING
PROTEIN
217
was applied to a 2.6 X 50 cm column of Sephadex G-75. The column was eluted with 10 mM Tris-HCl buffer (ph 8.6) containing 0.02% sodium azide. Ahquots (2 ml) of each fraction were dissolved in 15 ml of a toluene-based scintillation cocktail containing 7.0 g PPO and 200 ml of Bio-Solv BBS-3 (Beckman Instruments, Inc.) per liter. The 6 ’ Zn and r 4 C content was measured in separate channels of a Beckman LS-233 liquid scintillation spectrometer equipped with an automatic external standardization system. 6 ‘Zn and r4C were counted with channel discriminator settings of O-150 and 300-1000, respectively, at a gain setting of 650. Data are presented as disintegrations per min (DPM) calculated from standard curves. Each experimental treatment was repeated three times with one rat per replicate and the data presented in each chromatographic profile are representative of data obtained. Time Course of Liver and Serum Zinc A total of ninety-six rats divided into two groups of 64 and 32 rats were injected intraperitoneally with either 2 mg of Zn*+ as ZnSO4 in 0.9% NaCl (zinc loaded) or with 0.9% NaCl, respectively. One-half of the zinc loaded rats had received actinomycin D in 50% propylene glycol (0.8 mg/kg body weight) four hours prior to zinc administration. At 0, 4, 8, 12, 16, 20, 24 and 48 hours post-zinc injection, four animals from each of the three injection groups (zinc loaded; zinc loaded plus actinomycin D; 0.9% NaCl control) were killed by decapitation_ The livers were excised, weighed, dried in crucibles overnight at 10S°C and ashed at 550°C for 24 h. The ash was dissolved in 6N HCl and dihrted to an appropriate volume. The zinc content was determined by atomic absorption spectrophotometry (AAS). Serum zinc was determined by AAS using serum diluted with deionized water.
RESULTS The photon spectra exhibited by 65 Zn and r4 C are shown in Fig. 1 _ The isotope pair is clearly separable by liquid scintillation counting_ The crossover of 14C photons into the ” Zn counting window was 11% or less in quenched samples. The chromatographic profile of the soluble proteins from control (0.9% NaCl-injected) rat liver cytoplasm is shown in the top portion of Fig. 2. The ’ 4 C radioactivity separated into 2 major peaks: a high molecular weight fraction which was the first peak eluted from the column, and a second peak corresponding to the elution of small peptides and amino acids. The majority of the 65 Zn eluted from the column was associated with the high molecular weight protein fractions with a smaller amount found to be associated with the low molecular weight fractions_ In contrast, the middle profile (Fig. 2) shows the elution of i4C and 65 Zn from liver cytosol of zinc loaded rats, i.e., those rats injected with 2 mg Zn*’ (ip.) The 14C content of the first and last peaks was nearly identical to that found in the control (non zinc-loaded) rats. However, it
218
MARK
P_ RICHARDS
AidD ROBERT
J. COUSINS
FIG_ l_ Photon spectra of 6 s2n and ’ 4C obtained with a liquid scintillationspectrometer by counting 2% windows with the amplifiergainset for counting 3 H_ Aqueous solutionsof * * Zo Cl= and”C-cystine were dissolvedin toluenecountingsolution containing20% BBS-3 solubilizer. is evident that there was a substantial increase in I 4 C-cystine incorporation into a second peak which corresponds to a protein of 1O-14,000 daltons and which has been designated as zinc binding protein (ZnBP). The incorporation of “C-cystihe into this second peak (ZrrBP) suggests that the synthesis of hepatic ZnBP was increased in response to the zinc load. Furthermore, the increase in ZnBP synthesis is associated with a concomitment increase in the total amount of 65 Zn bound by the liver. The lower profile (Fig. 2) was obtained from animals which were treated with actinomycin D (0.8 mg/kg subcutaneously) 4 h prior to administration of the zinc load. The effect on ZnBP synthesis is clear as into ZnBP fractions_ there was a virtual absence of * 4 C-cystine incorporation Not only was ZnBP synthesis in response to a zinc load reduced, but total liver 65Zn accumulation was also reduced. As expected, the actinomycin D administration also decreased the incorporation of r 4C-cystine into the other fractions_ These data appear to indicate that when protein synthesis is blocked at the transcriptional level, the liver fails to accumulate the additional zinc (65 Zn) which is concomitant with a virtual absence of ZnBP synthesis. The influence of zinc loading on the uptake of Zn*+ into rat liver is shown in the top half of Fig_ 3. Over a 48 h period the control rats exhibited a small but nonsignificant fluctuation in liver zinc content. In contrast, those rats injected with 2 mg of Zn** showed a significant (P < 0.05) elevation in liver zinc from twelve hours post Zn’+ mjection _ that remained significantly elevated above the control animals even at 48 h. This indicates that the liver is able to rapidly accumulate zinc, but that zinc transport from the liver to extra-hepatic sites
ZINC-BINDKNG
PROTEIN
219
UVER CoNNa
FRLCTION
NUMBER
FIG. 2. Sephsdex G-75 chromatography of rat liver cytosol. The 6 s Zn and ’ 4 C content was mtxsuzed by liquid sdntillation counting. The radioactivity plotted is that measured in a 2 ml aliquot of each 5 ml fraction_ Top profile: Rats were injected with ‘9C-cystine and 65Zn 12 h before sacrike. Middle profile: Rats were injected with * 4 C-cystine and 6 * Zn and2 mg Zna* (zinc [Faded) I2 h before sacrifice. Bottom profile: Rats were injected with actinomycin D (0.8 mg/kg-B)
4 h prior to injection with ’ 4C-cystine and 45Zn and 2 mg
Zn”+ (zinc loaded) and were sacrificed 12h after the latter.
ZINC
DeFlclENT
FIG-I Sephadex G-75 chromatography of rat liver cytosol. The 65Zn and 14C content was measured by liquid scintillation counting. The radioactivity plotted is that measured in a 2 ml atiquot of each 5 ml fraction_ Top proffie: Zinc depleted rats injected with “C-qsfine, and asZn 12h before scrifice. Middle profile: Zinc depleted ;ats injected with z ’ C-cystine, 65Zn and 2 mg of ZN*+ (zinc loaded) 12 h before sacrif’ice. Bottom profile: Zinc depleted rats injected with “C-cystine, ’ 'Zn and 2 mg of Zn” (zinc loaded) 12 h before sacrifice and actinomycin D I6 h before sacrifice.
1 LIVER ZINC
I
--ACT.D*Zn ----~Acr.D*zn T
--
CONTROL
d0 FIG. 3. The effect of parenteral zinc and actinomycin D on liver and serum zinc in rats fed a zinc adequate diet. Each point represents the mean * SE&f of four rats. - - - -, Control nts: Injected
occurs at a slower rate perhaps due to a certain amount of storage. When actinomycin D was administered 4 h prior to the zinc load, liver zinc was not significantly (P > 0.05) increased compared to the control group for the first 24 h. However, liver zinc in the actinomycin D treated group did increase significantly (P < 0.001) above the control group by 48h indicating that when the influence of actinomycin D had diminished (between 24-36 h) then zinc accumulated in the liver in response to the zinc load. Thus it appears that hepatic protein synthesis is necessary for zinc uptake into the liver. The bottom portion of Fig. 3 shows the influence of the treatments on serum zinc level. it is clear that the zinc loaded groups exhibited a significant (P< 0.05) increase in serum zinc compared to the control group which did not show a significant fluctuation during the 48 h comparison period. Initially, both zinc loaded groups showed a similar increase in serum zinc. However, by 12 h post-injection, the non-actinomycin D treated animals showed a significant (P < 0.001) drop in serum zinc as opposed to the actinomycin D treated group which continued to show a significant (P < 0.001) elevation of serum zinc over the other groups for an additional eight hour period (to twenty hours post-injection)_ Twenty-four hours after the zinc load, the serum zinc level in the actinomycin D group was not significantly different from the other groups. These data suggest that hepatic protein synthesis is necessary for the uptake of
222
MARK P. RICHARDS AND ROBERT
J. COUSINS
zinc from blood into liver since the influence of the drug must diminish prior to a reduction in serum zinc and a concomitant increase in liver zinc. The influence of a zinc load to liver ZnBP biosynthesis was investigated in zinc depleted rats (Fig_ 4). The incorporation of ’ 4 Ccystine and6’ Zn into the ZnBP region of the profile was evident in the zinc depleted animal (Top profile), The zinc load (2 mg Zn*‘) markedly increased the incorporation of ’ 4C-cystine and 6s Zn into ZnBP and also increased 6 5 Zn incorporation into the high molecular weight fractions_ When actinomycin D was administered prior to the zinc load, the incorporation of t4C-cystine and 6s Zn into ZnBP was virtually abolished_ These data suggest that zinc could induce the formation of ZnBP to sequester plasma zinc and that ZnBP is directly invoived in the uptake of zinc into the liver cells. This possibility is likely since actinomycin D was shown to block both ZnBP synthesis and Zn uptake into liver_ The results of the chromatography of the soluble mucosal proteins (Fig. 5) show results similar to those obtained with liver_ The zinc load markedly increased the incorporation of both r4C and 65 Zn into the ZnBP. Actinomycin D treated rats did not respond with a similar increase indicating that ZnBP synthesis is controlled via a similar mechanism in both liver and mucosal cells.
DISCUSSION The results presented in this report provide evidence that zinc is able to influence the synthesis of a low molecular weight zinc binding protein. Previous investigations have shown that radio-zinc will associate with a protein similar in molecular weight to ZnBP [3,4,5] _ However, the present study demonstrates that the synthesis of the binding protein is influenced by zinc status. The regulation of the synthesis of a specific binding protein could be an integral part of the mechanism responsible for zinc homeostasis It appears from the results on zinc uptake in response to the zinc load that DNA-dependent RNA synthesis is required for the uptake of zinc into liver cells. Moreover, the only main change in the soluble hepatic proteins in the zinc loaded rats compared to control rats was an increase in the synthesis of ZnBP. This suggests that ZnBP, formed in response to zinc, is involved in the process of zinc uptake by the cell_ ZnBP is undoubtedly a metallothionein-like protein [ 6,7 ] ; since relatively large amounts of r4 C-cystine are incorporated, thus a high binding affinity for zinc would be anticipated. Therefore, it is easy to envision how such a protein could function as a sequestering agent within the cytoplasm to facilitate cellular zinc uptake_ It is of considerable interest that COX [ 111 has demonstrated that mRNA and protein synthesis are required for zinc uptake into HeLa cells in culture_ Evans and coworkers have clearly demonstrated that a zinc load imposed on zinc depleted rats markedly decreased zinc absorption and concomitantly elevated mucosal cell zinc 12 1. Our experiments have shown that with similar zinc depleted rats the zinc load increased ZnBP synthesis_ Therefore, it does not appear likely that ZnBP functions in the absorption process per se. The amount
ZINC-BINDING
PROTEIN
223
MUCOSA I-
Do-
-2h
0
O-
10.000
30
i I_
::
0i IO
!
0 20
I&300
-Zn +zn
+Act.D
FRACTION
0 NUMBER
>
FIG. 5. Sephadex G-75 chromarography of rat intestinal mucosal cytosol. The 652n and I *C con:ent was measured by liquid scintillation countin g. The radioactivity plotted is that measure; in a 2 ml aliquot of each 5 ml fraction. Top profile: Zinc depleted rats injected with ‘* C-cystine and ssZn 12 h before sacrifice. Middle profile: Zinc depleted rats injected with 14C-cystine, 65Zn and 2 mg of Zn’+ (zinc loaded) 12 h before sacrifice. Bottom profile: Zinc depleted rats injected with t 4 Ccystine, 6 s Zn and 2 mg of Zn*+ (zinc loaded) 12 h before sacrifice and actinomycin D 16 h before sacrifice.
224
MARK P_ RICHARDS AND ROBERT
J. COUSINS
of zinc transferred
to plasma albumin at the serosal surface is probably the rate in zinc absorption [ 12]_ Since the mucosal cell is a short-lived cell, it is possible that zinc imprints new mucosal cells with the ability to synthesize ZnBP. Thus ZnBP would subsequently function to sequester dietary zinc, prevent its transfer to plasma albumin and permit excretion of the sequestered zinc via the desquamation of mucosa epithelium. This hypothesis is 24 h is required for zinc to initiate events that tenable since approximately depress zinc absorption in zinc depleted rats [ 21, which is sufficient time for the synthesis of new protein. Experiments are now in progress to characterize the hepatic and mucosal ZnBP and to evaluate their function in zinc metabolism. determining
factor
REFERENCES 1. W_ 3. hiiller, Amer. J_ Clin Nutr. 22, 1323 (1969). 2_ G. W. Evans, C. I. Grace, and C. H&n,Proc- Sot- Exp. Biol Med. 143.723 (1973). 3. B. C. Starcher,J. Nutr. 97,321 (1969). 4. D. R_ Van Campen and T. J. Kowalski, Proc. Sot. Exp. Biol. Med. 136,294 (1971). 5 F_ A. Suso and H. %I_Edwards. Jr.,Proc_ tic_ Exp. Biol. Med. 137,306 (1971)_ 6. J. H. R. Kagi and B. L. ValIee,J. Biol. Chem. 237.3460 (1960). 7. R H. 0. Buhkr and J. H. R. I(@. FEBS Letters 39,229 (1974). 8_ R. W. Luecke, M_ E_ Ohm, and B. V. Baltzer, J. Nufr_ 94,344 (1968). V. K. S. Sqilibb and R. J. Cousins, Environ. Physiol Biochem. 4,24 (1974). 10. R_ K. Crane and P. hiandelstam, Biochem. Biophys. AC&Z45.460 (1960). 11_ R. P. Cos,Science 165,196 (1969). 12. G. IV. Evans, C. I. Grace, and C. Hahn, Bioinorganic Chem_ 3,115 (1974). Received Mzy 22. 1974; revised August 13. 1974.
CHEMISTR Y 4,2 1%224( 1975)
Influence of Parenteral Zinc and Actinomycin on Tissue Zinc Uptake and the Synthesis of a Zinc -Binding Protein * t MARK
P. RICHARDS
Department
and ROBERT
of Nutrition,
New Brunswick,
Cook
215
D
J. COUSINS
College.
Rutgers
University,
New Jersey 08903 ABSTRACT
Parenterally administered zinc markedly increased the incorporation of I 4C-cystine and 6 5 Zn into a low mo1ecula.r weight zinc binding protein (ZnBP) isolated from liver cytoplasm of rats fed an adequate amount of zinc. This zinc load significantly increased the zinc content in the liver. The increase in hepatic ainc content was inhibited by actinomycin D indicating that DNA-dependent RNA synthesis is required for zinc uptake into liver. Antinomycin D also produced a concomitant decrease in ZnBP synthesis indicating that this protein may be involved in the uptake mechanism in cells. Zinc repletion also stimulated the synthesis of hepatic ZnBP in zinc deficient rats. This stimulation was also prevented by prior administration of actinomycin D. A similar effect was observed in the intestinal mucosal cells. The data collectively indicate that the control of the synthesis of ZnBP which occurs at the transcriptional level of protein synthesis is responsive to zinc status and thus may have a function in zinc metabolism.
INTRODUCTION A number of studies have shown zinc metabolism is controlled by a homeostatic mechanism (1.21, i.e., zinc absorption appears to be related in some way to body stores of zinc. Concomitant to these studies, some investigators have postulated a role for a low molecular weight protein in the absorption process [ 3-5 1, however, the exact role of this protein(s), if any, has yet to be elucidated_ Specifically, a low molecular weight protein has been demonstrated by Van Campen in the intestinal mucosa which is capable of binding the cations Zinc, copper and cadmium [4] _ In addition, this protein has properties similar to
*Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers University-The State University of New Jersey, New Brunswick, New Jersey 08903. z t Supported in part by the Nutrition Foundation, innc. Future Leader Grant No. 458. 0 American Elsevier Publishing Company, Inc., 1975.
216
MARK
P_ RICHARDS
AND ROBERT
J. COUSINS
a metalloprotein of unknown function previously isolated “metallothionein”, from kidney and liver [ 6,7 1_ This report describes experiments designed to investigate the influence of zinc on the synthesis of a low molecular weight zinc binding protein (ZnBP) in liver and intestinal mucosa and the possible role of ZnBP in cellular zinc uptake.
MATERIAL Animals
AND METHODS
and Diets
A total of one hundred and twenty-three male rats of the Sprague-Dawiey strain (Charles River) were maintained on Purina Rat Chow, ad libitum, until they weighed 150 g, at which time they were used for experiments_ In those experiments, where zinc depleted rats were used, the animals were maintained as above until they weighed 300 g and were then fed a biotinenriched zinc deficient diet [81_ ad lib&urn, for two weeks.
Isotopes Nuclear Zinc-65 as 65ZnC1a (4.5 mCi/mg) was obtained from New E&and and was diluted to 20 pCi/mt with 09% NaCl prior to injection_ DL-cystine 3-r4C obtained from Schwarz-Mann with a specific activity of 30 mCi;mmole was diluted to 3_0 yCi/ml with 0.9% NaCl prior to injection.
Synthesis
of ZnBP
The rats were injected, intraperitoneally, with 5 PCi of 65Zn, 5 PCi of r4C-cystine and with either 2.0 mg of Zn*+ as ZnS04 in 0.9% NaCl or 0.9% NaCl. in some experiments actinomycin D (Sigma Chemical Company) in 50% propylene glycol was administered subcutaneously (0.8 mg/kg body weight) four hours before zinc was administered [9 I_ Three rats received each of the three treatment combinations. The rats were then fasted for twelve hours and killed by decapitation_ The livers were excised, weighed, washed in cold 0.9% NaC.1 and immediately homogenized in a glass-teflon homogenizer with an equal volume of O-25 M sucrose solution buffered at pH 8.6 with 10 mM Tris-HCl. in the experiments with zinc depleted rats mucosal cells were obtained from a 30 cm segment of the duodenal end of the small intestine that was perfused with 10 ml of ice cold 0.9% NaCl. The segment was split open and the cells harvested with a glass microscope slide using the method of Crane and Mandelstam [ 10 ] _ Mucosal homogenates were prepared as described above. Each homogenate was centrifuged at 42,000 xg and 4OC for 30 min. The supematant solution was removed and recentifuged at 42,000 g for 2 h_ Five milliliters of each final supernatant
ZINC-BINDING
PROTEIN
217
was applied to a 2.6 X 50 cm column of Sephadex G-75. The column was eluted with 10 mM Tris-HCl buffer (ph 8.6) containing 0.02% sodium azide. Ahquots (2 ml) of each fraction were dissolved in 15 ml of a toluene-based scintillation cocktail containing 7.0 g PPO and 200 ml of Bio-Solv BBS-3 (Beckman Instruments, Inc.) per liter. The 6 ’ Zn and r 4 C content was measured in separate channels of a Beckman LS-233 liquid scintillation spectrometer equipped with an automatic external standardization system. 6 ‘Zn and r4C were counted with channel discriminator settings of O-150 and 300-1000, respectively, at a gain setting of 650. Data are presented as disintegrations per min (DPM) calculated from standard curves. Each experimental treatment was repeated three times with one rat per replicate and the data presented in each chromatographic profile are representative of data obtained. Time Course of Liver and Serum Zinc A total of ninety-six rats divided into two groups of 64 and 32 rats were injected intraperitoneally with either 2 mg of Zn*+ as ZnSO4 in 0.9% NaCl (zinc loaded) or with 0.9% NaCl, respectively. One-half of the zinc loaded rats had received actinomycin D in 50% propylene glycol (0.8 mg/kg body weight) four hours prior to zinc administration. At 0, 4, 8, 12, 16, 20, 24 and 48 hours post-zinc injection, four animals from each of the three injection groups (zinc loaded; zinc loaded plus actinomycin D; 0.9% NaCl control) were killed by decapitation_ The livers were excised, weighed, dried in crucibles overnight at 10S°C and ashed at 550°C for 24 h. The ash was dissolved in 6N HCl and dihrted to an appropriate volume. The zinc content was determined by atomic absorption spectrophotometry (AAS). Serum zinc was determined by AAS using serum diluted with deionized water.
RESULTS The photon spectra exhibited by 65 Zn and r4 C are shown in Fig. 1 _ The isotope pair is clearly separable by liquid scintillation counting_ The crossover of 14C photons into the ” Zn counting window was 11% or less in quenched samples. The chromatographic profile of the soluble proteins from control (0.9% NaCl-injected) rat liver cytoplasm is shown in the top portion of Fig. 2. The ’ 4 C radioactivity separated into 2 major peaks: a high molecular weight fraction which was the first peak eluted from the column, and a second peak corresponding to the elution of small peptides and amino acids. The majority of the 65 Zn eluted from the column was associated with the high molecular weight protein fractions with a smaller amount found to be associated with the low molecular weight fractions_ In contrast, the middle profile (Fig. 2) shows the elution of i4C and 65 Zn from liver cytosol of zinc loaded rats, i.e., those rats injected with 2 mg Zn*’ (ip.) The 14C content of the first and last peaks was nearly identical to that found in the control (non zinc-loaded) rats. However, it
218
MARK
P_ RICHARDS
AidD ROBERT
J. COUSINS
FIG_ l_ Photon spectra of 6 s2n and ’ 4C obtained with a liquid scintillationspectrometer by counting 2% windows with the amplifiergainset for counting 3 H_ Aqueous solutionsof * * Zo Cl= and”C-cystine were dissolvedin toluenecountingsolution containing20% BBS-3 solubilizer. is evident that there was a substantial increase in I 4 C-cystine incorporation into a second peak which corresponds to a protein of 1O-14,000 daltons and which has been designated as zinc binding protein (ZnBP). The incorporation of “C-cystihe into this second peak (ZrrBP) suggests that the synthesis of hepatic ZnBP was increased in response to the zinc load. Furthermore, the increase in ZnBP synthesis is associated with a concomitment increase in the total amount of 65 Zn bound by the liver. The lower profile (Fig. 2) was obtained from animals which were treated with actinomycin D (0.8 mg/kg subcutaneously) 4 h prior to administration of the zinc load. The effect on ZnBP synthesis is clear as into ZnBP fractions_ there was a virtual absence of * 4 C-cystine incorporation Not only was ZnBP synthesis in response to a zinc load reduced, but total liver 65Zn accumulation was also reduced. As expected, the actinomycin D administration also decreased the incorporation of r 4C-cystine into the other fractions_ These data appear to indicate that when protein synthesis is blocked at the transcriptional level, the liver fails to accumulate the additional zinc (65 Zn) which is concomitant with a virtual absence of ZnBP synthesis. The influence of zinc loading on the uptake of Zn*+ into rat liver is shown in the top half of Fig_ 3. Over a 48 h period the control rats exhibited a small but nonsignificant fluctuation in liver zinc content. In contrast, those rats injected with 2 mg of Zn** showed a significant (P < 0.05) elevation in liver zinc from twelve hours post Zn’+ mjection _ that remained significantly elevated above the control animals even at 48 h. This indicates that the liver is able to rapidly accumulate zinc, but that zinc transport from the liver to extra-hepatic sites
ZINC-BINDKNG
PROTEIN
219
UVER CoNNa
FRLCTION
NUMBER
FIG. 2. Sephsdex G-75 chromatography of rat liver cytosol. The 6 s Zn and ’ 4 C content was mtxsuzed by liquid sdntillation counting. The radioactivity plotted is that measured in a 2 ml aliquot of each 5 ml fraction_ Top profile: Rats were injected with ‘9C-cystine and 65Zn 12 h before sacrike. Middle profile: Rats were injected with * 4 C-cystine and 6 * Zn and2 mg Zna* (zinc [Faded) I2 h before sacrifice. Bottom profile: Rats were injected with actinomycin D (0.8 mg/kg-B)
4 h prior to injection with ’ 4C-cystine and 45Zn and 2 mg
Zn”+ (zinc loaded) and were sacrificed 12h after the latter.
ZINC
DeFlclENT
FIG-I Sephadex G-75 chromatography of rat liver cytosol. The 65Zn and 14C content was measured by liquid scintillation counting. The radioactivity plotted is that measured in a 2 ml atiquot of each 5 ml fraction_ Top proffie: Zinc depleted rats injected with “C-qsfine, and asZn 12h before scrifice. Middle profile: Zinc depleted ;ats injected with z ’ C-cystine, 65Zn and 2 mg of ZN*+ (zinc loaded) 12 h before sacrif’ice. Bottom profile: Zinc depleted rats injected with “C-cystine, ’ 'Zn and 2 mg of Zn” (zinc loaded) 12 h before sacrifice and actinomycin D I6 h before sacrifice.
1 LIVER ZINC
I
--ACT.D*Zn ----~Acr.D*zn T
--
CONTROL
d0 FIG. 3. The effect of parenteral zinc and actinomycin D on liver and serum zinc in rats fed a zinc adequate diet. Each point represents the mean * SE&f of four rats. - - - -, Control nts: Injected
occurs at a slower rate perhaps due to a certain amount of storage. When actinomycin D was administered 4 h prior to the zinc load, liver zinc was not significantly (P > 0.05) increased compared to the control group for the first 24 h. However, liver zinc in the actinomycin D treated group did increase significantly (P < 0.001) above the control group by 48h indicating that when the influence of actinomycin D had diminished (between 24-36 h) then zinc accumulated in the liver in response to the zinc load. Thus it appears that hepatic protein synthesis is necessary for zinc uptake into the liver. The bottom portion of Fig. 3 shows the influence of the treatments on serum zinc level. it is clear that the zinc loaded groups exhibited a significant (P< 0.05) increase in serum zinc compared to the control group which did not show a significant fluctuation during the 48 h comparison period. Initially, both zinc loaded groups showed a similar increase in serum zinc. However, by 12 h post-injection, the non-actinomycin D treated animals showed a significant (P < 0.001) drop in serum zinc as opposed to the actinomycin D treated group which continued to show a significant (P < 0.001) elevation of serum zinc over the other groups for an additional eight hour period (to twenty hours post-injection)_ Twenty-four hours after the zinc load, the serum zinc level in the actinomycin D group was not significantly different from the other groups. These data suggest that hepatic protein synthesis is necessary for the uptake of
222
MARK P. RICHARDS AND ROBERT
J. COUSINS
zinc from blood into liver since the influence of the drug must diminish prior to a reduction in serum zinc and a concomitant increase in liver zinc. The influence of a zinc load to liver ZnBP biosynthesis was investigated in zinc depleted rats (Fig_ 4). The incorporation of ’ 4 Ccystine and6’ Zn into the ZnBP region of the profile was evident in the zinc depleted animal (Top profile), The zinc load (2 mg Zn*‘) markedly increased the incorporation of ’ 4C-cystine and 6s Zn into ZnBP and also increased 6 5 Zn incorporation into the high molecular weight fractions_ When actinomycin D was administered prior to the zinc load, the incorporation of t4C-cystine and 6s Zn into ZnBP was virtually abolished_ These data suggest that zinc could induce the formation of ZnBP to sequester plasma zinc and that ZnBP is directly invoived in the uptake of zinc into the liver cells. This possibility is likely since actinomycin D was shown to block both ZnBP synthesis and Zn uptake into liver_ The results of the chromatography of the soluble mucosal proteins (Fig. 5) show results similar to those obtained with liver_ The zinc load markedly increased the incorporation of both r4C and 65 Zn into the ZnBP. Actinomycin D treated rats did not respond with a similar increase indicating that ZnBP synthesis is controlled via a similar mechanism in both liver and mucosal cells.
DISCUSSION The results presented in this report provide evidence that zinc is able to influence the synthesis of a low molecular weight zinc binding protein. Previous investigations have shown that radio-zinc will associate with a protein similar in molecular weight to ZnBP [3,4,5] _ However, the present study demonstrates that the synthesis of the binding protein is influenced by zinc status. The regulation of the synthesis of a specific binding protein could be an integral part of the mechanism responsible for zinc homeostasis It appears from the results on zinc uptake in response to the zinc load that DNA-dependent RNA synthesis is required for the uptake of zinc into liver cells. Moreover, the only main change in the soluble hepatic proteins in the zinc loaded rats compared to control rats was an increase in the synthesis of ZnBP. This suggests that ZnBP, formed in response to zinc, is involved in the process of zinc uptake by the cell_ ZnBP is undoubtedly a metallothionein-like protein [ 6,7 ] ; since relatively large amounts of r4 C-cystine are incorporated, thus a high binding affinity for zinc would be anticipated. Therefore, it is easy to envision how such a protein could function as a sequestering agent within the cytoplasm to facilitate cellular zinc uptake_ It is of considerable interest that COX [ 111 has demonstrated that mRNA and protein synthesis are required for zinc uptake into HeLa cells in culture_ Evans and coworkers have clearly demonstrated that a zinc load imposed on zinc depleted rats markedly decreased zinc absorption and concomitantly elevated mucosal cell zinc 12 1. Our experiments have shown that with similar zinc depleted rats the zinc load increased ZnBP synthesis_ Therefore, it does not appear likely that ZnBP functions in the absorption process per se. The amount
ZINC-BINDING
PROTEIN
223
MUCOSA I-
Do-
-2h
0
O-
10.000
30
i I_
::
0i IO
!
0 20
I&300
-Zn +zn
+Act.D
FRACTION
0 NUMBER
>
FIG. 5. Sephadex G-75 chromarography of rat intestinal mucosal cytosol. The 652n and I *C con:ent was measured by liquid scintillation countin g. The radioactivity plotted is that measure; in a 2 ml aliquot of each 5 ml fraction. Top profile: Zinc depleted rats injected with ‘* C-cystine and ssZn 12 h before sacrifice. Middle profile: Zinc depleted rats injected with 14C-cystine, 65Zn and 2 mg of Zn’+ (zinc loaded) 12 h before sacrifice. Bottom profile: Zinc depleted rats injected with t 4 Ccystine, 6 s Zn and 2 mg of Zn*+ (zinc loaded) 12 h before sacrifice and actinomycin D 16 h before sacrifice.
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MARK P_ RICHARDS AND ROBERT
J. COUSINS
of zinc transferred
to plasma albumin at the serosal surface is probably the rate in zinc absorption [ 12]_ Since the mucosal cell is a short-lived cell, it is possible that zinc imprints new mucosal cells with the ability to synthesize ZnBP. Thus ZnBP would subsequently function to sequester dietary zinc, prevent its transfer to plasma albumin and permit excretion of the sequestered zinc via the desquamation of mucosa epithelium. This hypothesis is 24 h is required for zinc to initiate events that tenable since approximately depress zinc absorption in zinc depleted rats [ 21, which is sufficient time for the synthesis of new protein. Experiments are now in progress to characterize the hepatic and mucosal ZnBP and to evaluate their function in zinc metabolism. determining
factor
REFERENCES 1. W_ 3. hiiller, Amer. J_ Clin Nutr. 22, 1323 (1969). 2_ G. W. Evans, C. I. Grace, and C. H&n,Proc- Sot- Exp. Biol Med. 143.723 (1973). 3. B. C. Starcher,J. Nutr. 97,321 (1969). 4. D. R_ Van Campen and T. J. Kowalski, Proc. Sot. Exp. Biol. Med. 136,294 (1971). 5 F_ A. Suso and H. %I_Edwards. Jr.,Proc_ tic_ Exp. Biol. Med. 137,306 (1971)_ 6. J. H. R. Kagi and B. L. ValIee,J. Biol. Chem. 237.3460 (1960). 7. R H. 0. Buhkr and J. H. R. I(@. FEBS Letters 39,229 (1974). 8_ R. W. Luecke, M_ E_ Ohm, and B. V. Baltzer, J. Nufr_ 94,344 (1968). V. K. S. Sqilibb and R. J. Cousins, Environ. Physiol Biochem. 4,24 (1974). 10. R_ K. Crane and P. hiandelstam, Biochem. Biophys. AC&Z45.460 (1960). 11_ R. P. Cos,Science 165,196 (1969). 12. G. IV. Evans, C. I. Grace, and C. Hahn, Bioinorganic Chem_ 3,115 (1974). Received Mzy 22. 1974; revised August 13. 1974.