~Copyright 1985 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/85/0705-0129S02.20
Zinc-Binding Proteins (Ligands) in Brains of Severely Zinc-Deficient Rats MAr,IUCHAIR EBADI 1'* A N D J A M E S C. W A L L W O R K t :
Department o1:Pharmacology, 1The University of Nebraska College ot:/Vledicine, Omaha, Nebraska 68105; and ~Agricultural Research, United S t a t e s Department of Agriculture, Human Nutrition Research Center, Grand Forks, North Dakota 58202 Received J u n e 14, 1984; Accepted November 8, 1984
ABSTRACT Previous studies from this laboratory reported the presence of a metallothionein-like protein in brain with an apparent estimated molecular weight of 13,000-15,000 daltons. The synthesis of this protein, which incorporates large quantity of cysteine, is stimulated following administration of zinc and copper and is blocked by actinomycin D. In this study, we report that the synthesis of this metallothionein-like protein is considerably lower in brains of severely zinc-deficient rats in comparison with pair-fed or ad libitum fed groups. Furthermore, incubation of partially purified metallothionein-like protein with 65Zn and chromatography on DEAE A-25 Sephadex produced similar elution patterns in the three experimental groups. However, the extent of binding of 6SZn to the rnetallothionein-like protein from the zincdeficient rats was significantly (p < 0.05) lower than the control groups. On the other hand, the total concentration of zinc in brains of zinc deficient rats did not varv fronl control groups. Since the synthesis of this metallothionein-like protein is reduced by zinc deficiency and is stimulated following administration of zinc, we postulate that the free pool of zinc may regulate the synthesis of its binding protein in the brain. *Author to whom all correspondence and reprint requests should be addressed. tPresent Address: Agricultural Research Service, Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street. Boston, MA 02111.
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Index Entries: Zinc deficiency, and brain metallothioneinqike protein; zinc-binding proteins, and Zn deficiency in brain; proteins, in Zn-deficient brains; metallothionein-like protein; in Zn-deficient brains. INTRODUCTION Metallothioneins are low molecular weight proteins that are unusual in their high cysteine content (25-30%) and their high affinity for zinc (Zn2*-), cadmium (Cd2+), and copper (Cu2+). These elements are known to induce metallothioneins in the liver and the kidney (1). Although metallothioneins, soluble cadmium, and zinc-containing proteins have been well identified in liver or kidney of horses, rats, rabbits, chickens, calves, and humans (for review, see ref. 2), zinc-binding proteins (ligands) have not until recently been characterized in brain. Itoh and Ebadi (3) and Itoh et al. (4) detected in brain at least three separate zinc-binding proteins with apparent estimated molecular weights of 13,000-15,000, 25,000, and 210,000 daltons, respectively. The low molecular weight zinc-binding protein resembles in some but not all aspects to a hepatic metallothionein for the following reasons. The synthesis of this protein is stimulated following intracerebroventricular (5), but not intraperitoneal, administration of zinc sulfate (4). Copper, which exists iri large concentration in brain, stimulates the synthesis of the low molecular weight protein, but does not bind to it. Cadmium, which is undetectable in brain, does not stimulate the synthesis of this protein and is a potent inhibitor of zinc-binding (6). The zinc-induced stimulation of the low molecular weight protein is blocked by actinomycin D (7). Furthermore, the zinc-induced protein incorporates 35S-cysteine 24-fold higher than the native unstimulated protein (7). In addition, when the partially purified low molecular weight zinc-binding protein is incubated with zinc-65 and then subjected to anion exchange chromatography (DEAE-A25 Sephadex), the protein peak binding 65Zn and the one incorporating 35S-cysteine depict identical elution profiles (7). Chromatofocusing of zinc-binding proteins eluted from Sephadex G-75 depicts four separate peaks focusing at pHs of 6.8, 6.2, 5.3, and 4.8, respectively (8). By using a nondissociating discontinuous acrylamide gel system, and by applying the low molecular weight protein isolated on anion exchange chromatography, a single protein peak was isolated (6,8). The ability of this purified protein to bind zindmg protein is approximately 100-fold higher than the crude low molecular weight protein isolated on Sephadex G-75. The aforementioned data (4-8), when examined collectively, suggest strongly that a metallothionein-like protein exists in the brain. Since, unlike liver, brain does not participate in detoxification mech-
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anism(s), we hypothesized that the metallothionein-like protein isolated in the brain may be involved in maintaining zinc homeostasis. In order to test the hypothesis, we studied the integrity of this zinc-binding protein in the brains of zinc-deficient rats and in rats in whom the zinc concentration in the brain was altered by intracerebroventricular administration of various doses of zinc. The result of this study which has appeared as an abstract (9) forms the basis of this report.
MATERIALS AND METHODS Chemicals Zinc sulfate and zinc acetate were purchased from Fisher Scientific Company (Fair Lawn, NJ)f. Carrier free 6~Zn (1-10 Ci/g Zn) was purchased from New England Nuclear (Boston, MA). Sephadex G50-80 and Sephadex G75-120 were purchased from Sigma Chemical Company (St. Louis, MO). DEAE A-25 was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). All other chemicals were of the purest grade available.
Animals Zinc-deficient, pair-fed, and ad lib#urn-fed rats were developed according to techniques described by Wallwork and Sandstead (10). The weanling (CRL:[LER]BR) male Long-Evans rats were from the Charles River Breeding Laboratory (Wilmington, MA). The rats were fed a 20% sprayed egg white, biotin-enriched diet with a zinc content of less than 1 mg/kg (It). The diet was modified by the omission of chlortetracyctine hydrochloride and the addition of inositol, 1.0 g/kg of diet (Teklad Mills, Madison, WI). The rats were housed individually in Plexiglass cages in a laminar flow rack located in a humidity- (40-50%) and temperature(25~ controlled room with alternating 12-h periods of light and dark. Food intake and weight changes were measured between 0S:00 and 09:00 h. Wasted food was collected on papers and weighed at the same time. All rats were allowed to adapt to the environment for 5 d. The diet was fed ad libitum, and the drinking water contained 25 ppm zinc (as zinc acetate). To minimize trace metal contamination, we used silicone stoppers and stainless-steel drinking tubes in the water bottles (RodhelmReiss, Inc., Ronsell Rubber Products Division, Belle Mead, NJ). After the adaptation, rats (30-d-old) were randomly assigned to three groups on day 0 of the study. One group (zinc-deficient, 15 animals) was fed the zinc-deficient diet ad libitum and deionized water (Super Q System, Millipore Corporation, Bedford, MA). The two remaining groups were given access to drinking water containing 25 ppm zinc (as zinc acetate). Each rat in the second group (pair-fed, 15 animals) was individually J
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matched by weight with a zinc-deficient rat and pair-fed an amount of diet equaled to the dietary intake of the zinc-deficient rat on the previous day. The third group (ad libitum-fed, 15 animals) was fed the diet ad libitum. During the 30-d dietary regimen, the zinc-deficient rats displayed typical signs of zinc deficiency such as anorexia, cyclic feeding, growth retardation, alopecia, dermatitis surrounding the eyes and on the paws, and hunched posture. After 30 d of zinc deprivation, the animals were flown to Omaha, Nebraska where they were processed the same day for determination of zinobinding proteins. In order to determine the effects of stress of flight on the integrity of zinc-binding proteins, another group of animals shipped from North Dakota was laboratory adapted prior to experimentation. No detectable difference in zinc-binding proteins was noted whether the animal was laboratory adapted or not.
Preparation and Detection of Zinc-Binding Proteins Zinc-deficient, ad-Iibitum, and pair-fed rats were killed by decapitation, and the brains were immediately removed and homogenized in 5 vol of 0.05M Tris-acetic acid (pH 7.5). The homogenates were centrifuged at 750g for 15 min. Then, the supernatants were recentrifuged at 43,000g for 30 min. The pellets were discarded and the supernatants were recentrifuged at 105,000g for 60 rain in a Beckman Model L3-50 ultracentrifuge (Palo Alto, CA). Soluble fractions were concentrated in an Amicon Ultra-filtration Cell using ultrafiltration membranes (YM2, 43 mm Lot AS 02034A) purchased from Amicon Corporation (Danvers, MA). In all cases, 5-10 mL of concentrated supernatant proteins (8-10 mg/mL) were applied to 2.5 x 90 cm Sephadex G-50 or G-75 columns, and the low molecular weight metallothionein-like protein was eluted at 4~ with O.05M Tris-acetic acid buffer (pH 7.4) as discussed by Ebadi et
al. (7).
Stimulation of/Vletallothionein-Like Protein by Zinc Male Sprague-Dawley rats (180-220 g body wt) were purchased from Sasco (Omaha, NE). The animals were housed in stainless steel cages in air-conditioned quarters, laboratory-adapted prior to experiments, and fed a diet of Purina Chow. The zinc content of this diet was 70 ppm which remained constant throughout these experiments. A total of 84 rats were maintained on a 12-h light-dark cycle. Alzet Model 2001 miniosmotic pumps were purchased from Alza (Palo Alto, CA) and sup gically implanted for chronic intracerebroventricular administration of zinc sulfate (test) and 0.9% saline (control) as described by Ebadi et al. (5). The pumps were able to deliver 0.02, 0.05, or 0.22 Ixmol Zn/jxL/h for 48 h in three separate groups of experimental animals consisting of 21 rats. After 48 h, the rats were decapitated and a pool of three brains were
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homogenized in 5 vol of 0.05M Tris acetic acid (pH 7.5) and assayed for metallothionein-like protein as described (7).
DETERMINATION OF ZINC The concentration of zinc in various fractions from the filtration columns were determined with a Perkin-Elmer Atomic Absorption Spectrophotometer, model 306, with ZnSO4. 7H20 as standard. The total concentrations of Zn 2+ were determined according to the procedure of Smeyers-Verbeke et al. (12) as elaborated bv Wuyts et al. (13). In addition, zinc in the whole brain was determined in the high speed supernatant and the resulting pellets.
6~Zinc-Affinity Studies In order to study the binding affinity of zinc for metallothionein-like protein in brains of zinc-deficient rats, and the control groups, the low molecular weight zinc-binding protein (13,000-15,000 daltons) was partially purified on Sephadex G-50 and metallothionein-like protein was isolated, concentrated, and incubated for 2 h at room temperature with 1 ~Ci 65Zn2+. After 2 h at 37~C, the incubation of 6~Zn with metallothionein-like protein continued in the cold room for an additional 16-18 h. The protein-zinc complex was further purified by applying it to a column containing anion exchange medium (DEAE A-25 Sephadex). The metallothionein-like protein was eluted with a linear gradient using Trisacetate buffer (0.05-0.200 rru'vl, pH 7.5) according to the technique published by Karin and Herschman (14).
Determination of Proteins Proteins were determined according to the method of either Bradford (15) or Lowry et al. (16) using bovine serum albumin as standard.
Statistics Significance was determined by univariate and multivariate analyses of variance, followed by Scheffe contrast for the differences in means (17).
RESULTS The intracerebroventricular administration of zinc sulfate (0.02-0.22 p~mol ZnhxL/h for 48 h) increased significantly the synthesis of a metallothionein-like protein from control value of 1-6.5 ~g/mg protein
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(Fig. 1). The synthesis of this metallothionein-like protein in zincdeficient rats was significantly lower than either in the ad libitum group or in the pair-fed group (Fig. 2). Incubation of partially purified metallothionein-like protein with 6~Zn and c h r o m a t o g r a p h y on DEAE A-25 Sephadex p r o d u c e d similar elution patterns in the three experimental groups. However, the extent of binding of 65Zn to the metallothionein-like protein from the zincdeficient rats was significantly (p < 0.05) lower than either zincs u p p l e m e n t e d or ad libitum group (Fig. 3). This data suggests that in zinc d e f i d e n c y state less metallothionein-like protein is synthesized, but what is synthesized is chromatographically similar to those produced by pairfed or ad libitum fed groups. 7.22
,umoies
zinc
~J|
hr -
48hrs
6-
_=
5-
c
=- 4 . 0 5 umoles
zinc j u l
hr - 48
hrs
~a
moles
zinc
ul
hr - 48
hrs
2-
1-
~b
7b
8'o
~o
1do
iio
12 o
EiutiOn Number (3 hi! each)
Fig. 1. Stimulation of the synthesis of a metallothionein-tike protein by zinc delivered intracerebroventricularly by a surgically implanted minipump (5). After 48 h of continuous administration of zinc sulfate (0.02, 0.05, or 0.22/~,mol Zn/~Uh). Rats were decapitated and their brains were immediately homogenized in 5 vol of 0.05M Tris acetic acid buffer (pH 7.5). The high speed (105,000g) supernatant (12 mL containing a total of 96 mg protein) was applied to columns of Sephadex G-75, and the metaUothionien was eluted at 4~C with 0.05M Tris acetic acid buffer (pH 7.5) as described by Ebadi et al. (7). Each elution profile represents the combined average values from three brains using seven separate experiments. The values of zinc-stimulated zinc-binding ligands are significantly higher at their peaks than the control values at least at P < 0.05.
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4-, Pair fed (,)
.3" Ad Libitum (o)
.2.
) N
3,
1.5~
1.3
Pair fed (-)
1.1.
E
.9
~=
.7 84
N
,S'
Ad L i b i t u m (e)
Zinc deficient (,) .3 84
.1.
Elution
Fraction ( 3 m l each)
Fig. 2. Gel filtration chromatography of a metallothionein isolated from brains of zinc-deficient ( i ) ad libitum (Q), and pair-fed (.) rats. Rats were decapitated and their brains were immediately homogenized in 5 vol of 0.05M Tris acetic acid buffer (pH 7.5). The high speed (105,000g) supernatant was concentrated (10 mL containing a total of 85 mg protein) and applied to columns of Sephadex G-75. The metallothionein-like protein was eluted at 4~ with 0.05M Tris acetic acid buffer (pH 7.4) as described by Ebadi et al. (7). Each elution profile from zinc deficient, ,~d libitum, or pair-fed groups represents the combined average values from three rats using five separate experiments. The value of zincbinding ligand in zinc deficiency state at its peak is significantly lower than ad libitum or pair-fed group, at least at P < 0.05.
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Although the zinc-deficiency state manifests in lower synthesis of a metallothionein-like protein, the total subcellular amount of zinc determined in the supernatant or pellet of brains of ad libitum-fed, zinc pairfed, or zinc-deficient groups did not vary significantly (Table 1).
DISCUSSION Preliminary evidence from this laboratory indicated that the synthesis of the low molecular weight zinc-binding protein, a metallothioneinlike protein, was stimulated following the intracerebroventricular administration of zinc (5). In this study, using a far more refined technique, a dose-response effect has been obtained by administering zinc from 0.02-0.22 ~xmol/Zn/~L/h for 48 h through surgically implanted miniosmotic pumps. As it is apparent from Fig. i, the synthesis of the metallothionein-like protein is stimulated from 1 to 6.5 ~g Zn/mg proPair fed , - - ~ 1 4 5
3~1o
4
1
BRAIN
I
_/_._
#
A/ /
mM
L
zso E
I
=
1
Elution Fraction (3ml each) Fig. 3. Gel filtration chromatography depicting the binding of 6SZn into a partially purified metailothionein-]Lke protein. This protein was partially purified on Sephadex G50 (7) concentrated, (40 m[. containing a total of 3.2 mg protein) and incubated with 1 i~Ci ~ Z n + for 2 h at 37~ and then at 4~ for an additional 16-18 h. The metaliothionein binding zinc-65 was further purified by applying it to a column containing anion exchange medium (DEAE A-25 Sephadex). The protein was eiuted with a Linear gradient using Tfis acetate buffer (0-200 rnM, pH 7.5) according to a technique published by Karin and Herschman (14). Each elution profile Dom zinc-deficient (11), ad libitum (O), or pair-fed (.) groups represents the combined average values from three rats using five separate experiments. The extent of binding of ~Zn into metallothioneinLike protein (30 mL containing a tota] of 0.6 mg protein) at its peak (145 raM) was significantly lower in zinc-deficient group than in ad Iibitum or in pair-fed groups at P < 0.05.
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TABLE 1 Total Concentration of Zinc in Brains of ZincDeficient Rats and Their Control Groups ~ Dietary zinc status
Ad Iibitum-fed Pair-fed Zinc-deficient
Pellet zinc, Supernatant zinc, Fg/g dry tissue p.g/mg protein 738 -+ 26.4 745 -+ 14.0 662 - 37.5
0.314 -+ 0.01 0.322 -+ 0.04 0.316 -+- 0.01
"Rats were decapitated and their brains were homogenized in 10 rnM Tris-acetate buffer, p H 7.4. The homogenates were centrifuged in a Beckman refrigerated centrifuge (Model J-21C) at 48,000g for 30 rain. The supernatants were decanted and the pellets were h o m o g e n i z e d in 3 vol of triple-glass distilled water. Aliquots of supernatants and the homogenized pellets were placed in nitric acid prewashed, metal-free, and p r e w e i g h e d 10 x 75 mm culture test tubes (Fisher Scientific C o m p a n y , Pittsburg, PA). The test tubes were dried in a drying oven at I07~C for 18 h. The test tubes were then cooled in a metal-free environment and weighed again, and then 100 p.L of concentrated nitric acid was a d d e d to each test tube. After the dried tissues were allowed to dissolve in nitric acid for 30 min, the test tubes were heated cautiously in boiling water in a hood, but foaming and overboiling were prevented. All b l a n k test tubes received identical treatment. After cooling, the test tubes were centrifuged in an [EC Model CV-5000 centrifuge (International Equipment C o m p a n y , N e e d h a m Heights, MA). Zinc was d e t e r m i n e d by an atomic absorption spectrophotometer according to techniques described by Smeyers-Verbeke et al. (I2) as elaborated by Wuvts et al. (13). Each value represents the m e a n • SEM of at least 15 rats.
tein. F u r t h e r m o r e , in zinc-deficiency, the synthesis of this protein in the brain is r e d u c e d (Fig. 2). The a f o r e m e n t i o n e d studies suggest that the synthesis of the metallothionein-like protein isolated from brain is subject to regulation, in that it is stimulated following administration of zinc a n d inhibited in the zinc-deficiency state. The total concentration of zinc in brains of zinc-deficient rats did n o t vary from the control g r o u p s (Table 1). These studies confirm earlier studies by Wallwork et al. (18), w h o reported that zinc deficiency depressed the concentration of zinc in the olfactory lobe, but n o t in t h e other areas of rat brain studied. Studies in other laboratories have s h o w n that the absorption of zinc is e n h a n c e d in zinc-deficient rats (19-22). W h e t h e r or n o t the transport or absorption of zinc into the brain is enh a n c e d in deficiency state remains to be d e t e r m i n e d . The regional distribution of zinc in rat brain is not uniform (23). Furt h e r m o r e , no direct relation seems to exist b e t w e e n the free concentration of zinc a n d the activity of the zinc metalloenzymes. For example, in
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rat brain the zinc concentration is h i g h e s t in the h i p p o c a m p u s , whereas, the activity of carbonic a n h y d r a s e , a zinc m e t a l l o e n z y m e , is highest in the m e d u l l a oblongata (24). Similarly, in rabbit brain the zinc concentration does not parallel the activity of carbonic a n h y d r a s e (25). In conclusion, the synthesis of a metallothionein-like protein in brain seems to be regulated by free concentration of zinc. H o w e v e r , the mechanism of the stability of zinc in the brain of zinc-deficient rats is n o t clear and deserves additional in-depth investigation.
ACKNOWLEDGMENT M e n t i o n of a t r a d e m a r k or proprietary p r o d u c t does not constitute a guarantee or w a r r a n t y of the p r o d u c t by the US D e p a r t m e n t of Agriculture, a n d does not imply its approval to the exclusion of other p r o d u c t s that m a y also be suitable.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
J. H. R. Kagi and M. Nordberg, Metallothionein Birkhauser, Basel (1979). R. W. Chen, E. J. Vasey, and P. D. Whanger, J. Nutr. 107, 805 (1977). M. Itoh and M. Ebadi, Fed. Proc. 41, 8736 (1982). M. [toh, M. Ebadi, and S. Swanson, J. Neurochem. 41, 823 (1983). M. Ebadi, S. Wilt, R. Ramaley, S. Swanson, and C. Mebus, in Chemical and Biological Aspects of Vitamin B6 Catalysis, A. E. Evangepoulous, ed., Liss, New York, 255 (1984). M. Ebadi, Fed.Proc. 43, 3317 (1984). M. Ebadi, R. J. White, and S. Swanson, in Neurobiology of Zinc, C. J. Frederickson and G. A. Howell, eds., Liss, New York, 1984, Part A, pp. 34-57. M. Ebadi, Trans. Soc. Neurosci. 14, 1062 (1984). M. Ebadi, J. C. Wallwork, S. Swanson, and C. Mebus, Trans. Soc. Neurosci. 13, 402, (1983). J. C. Wallwork and H. H. Sandstead, [. Nutr. 113, 47 (1983). J. M. McKenzie, G. F. Fosmire, and H. H. Sandstead, J. Nutr. 105, I466 (1975). J. Smeyers-Verbeke, E. Defrise-Gussenhoven, G. Ebinger, A. Lowenthal, and D. L. Massart, Clin. Chim. Acta 51, 309 (1974). L. Wuyts, J. Smeyers-Verbeke, and D. L. Massart, Clin. Chim. Acta 72, 405 (1976). M. Karin and H. R. Herschman, Eur. J. Biochem. 107, 395 (1980). M. M. Bradford, Anal. Biochem. 72, 248 (1976). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). H. Scheffe, The Analysis of Variance, Wiley, New York, 1959, pp. 90-137. J. C. Wallwork, D. B. Milne, R. L. Sims, and H. H. Sandstead, [. Nutr. 113, 1895 (1983).
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Zinc-Binding Ligands in Zinc-Deficient Rats 19. 20. 21. 22. 23.
139
E. Weigand and M. Kirchgessner, 1. Nutr. 110, 469 (1980). M. ]. Jackson, D. A. Jones, and R. H. T. Edwards, Br. ]. Nz~tr. 46, 15 (1981). K. T. Smith and R. J. Cousins, I. N~tr. 110, 316 (1980). P. R. Flanagan, J. Haist, and L. S. Valberg, ]. N~tr. 113, 962 (1983). M. Ebadi, M. Itoh, J. Bifano, K. Wendt, and A. Earle, Int. ]. Biochem. 13, 1107
(1981).
24. V. Nair and D. Bau, Brain Res. 31, 185 (1971). 25. M. R. Klee and M. Lieflander, Hoptoe-Seyler's Zeitschrift Physiol. Chem. 341, 143 (1965).
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Zinc-Binding Proteins (Ligands) in Brains of Severely Zinc-Deficient Rats MAr,IUCHAIR EBADI 1'* A N D J A M E S C. W A L L W O R K t :
Department o1:Pharmacology, 1The University of Nebraska College ot:/Vledicine, Omaha, Nebraska 68105; and ~Agricultural Research, United S t a t e s Department of Agriculture, Human Nutrition Research Center, Grand Forks, North Dakota 58202 Received J u n e 14, 1984; Accepted November 8, 1984
ABSTRACT Previous studies from this laboratory reported the presence of a metallothionein-like protein in brain with an apparent estimated molecular weight of 13,000-15,000 daltons. The synthesis of this protein, which incorporates large quantity of cysteine, is stimulated following administration of zinc and copper and is blocked by actinomycin D. In this study, we report that the synthesis of this metallothionein-like protein is considerably lower in brains of severely zinc-deficient rats in comparison with pair-fed or ad libitum fed groups. Furthermore, incubation of partially purified metallothionein-like protein with 65Zn and chromatography on DEAE A-25 Sephadex produced similar elution patterns in the three experimental groups. However, the extent of binding of 6SZn to the rnetallothionein-like protein from the zincdeficient rats was significantly (p < 0.05) lower than the control groups. On the other hand, the total concentration of zinc in brains of zinc deficient rats did not varv fronl control groups. Since the synthesis of this metallothionein-like protein is reduced by zinc deficiency and is stimulated following administration of zinc, we postulate that the free pool of zinc may regulate the synthesis of its binding protein in the brain. *Author to whom all correspondence and reprint requests should be addressed. tPresent Address: Agricultural Research Service, Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street. Boston, MA 02111.
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Index Entries: Zinc deficiency, and brain metallothioneinqike protein; zinc-binding proteins, and Zn deficiency in brain; proteins, in Zn-deficient brains; metallothionein-like protein; in Zn-deficient brains. INTRODUCTION Metallothioneins are low molecular weight proteins that are unusual in their high cysteine content (25-30%) and their high affinity for zinc (Zn2*-), cadmium (Cd2+), and copper (Cu2+). These elements are known to induce metallothioneins in the liver and the kidney (1). Although metallothioneins, soluble cadmium, and zinc-containing proteins have been well identified in liver or kidney of horses, rats, rabbits, chickens, calves, and humans (for review, see ref. 2), zinc-binding proteins (ligands) have not until recently been characterized in brain. Itoh and Ebadi (3) and Itoh et al. (4) detected in brain at least three separate zinc-binding proteins with apparent estimated molecular weights of 13,000-15,000, 25,000, and 210,000 daltons, respectively. The low molecular weight zinc-binding protein resembles in some but not all aspects to a hepatic metallothionein for the following reasons. The synthesis of this protein is stimulated following intracerebroventricular (5), but not intraperitoneal, administration of zinc sulfate (4). Copper, which exists iri large concentration in brain, stimulates the synthesis of the low molecular weight protein, but does not bind to it. Cadmium, which is undetectable in brain, does not stimulate the synthesis of this protein and is a potent inhibitor of zinc-binding (6). The zinc-induced stimulation of the low molecular weight protein is blocked by actinomycin D (7). Furthermore, the zinc-induced protein incorporates 35S-cysteine 24-fold higher than the native unstimulated protein (7). In addition, when the partially purified low molecular weight zinc-binding protein is incubated with zinc-65 and then subjected to anion exchange chromatography (DEAE-A25 Sephadex), the protein peak binding 65Zn and the one incorporating 35S-cysteine depict identical elution profiles (7). Chromatofocusing of zinc-binding proteins eluted from Sephadex G-75 depicts four separate peaks focusing at pHs of 6.8, 6.2, 5.3, and 4.8, respectively (8). By using a nondissociating discontinuous acrylamide gel system, and by applying the low molecular weight protein isolated on anion exchange chromatography, a single protein peak was isolated (6,8). The ability of this purified protein to bind zindmg protein is approximately 100-fold higher than the crude low molecular weight protein isolated on Sephadex G-75. The aforementioned data (4-8), when examined collectively, suggest strongly that a metallothionein-like protein exists in the brain. Since, unlike liver, brain does not participate in detoxification mech-
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anism(s), we hypothesized that the metallothionein-like protein isolated in the brain may be involved in maintaining zinc homeostasis. In order to test the hypothesis, we studied the integrity of this zinc-binding protein in the brains of zinc-deficient rats and in rats in whom the zinc concentration in the brain was altered by intracerebroventricular administration of various doses of zinc. The result of this study which has appeared as an abstract (9) forms the basis of this report.
MATERIALS AND METHODS Chemicals Zinc sulfate and zinc acetate were purchased from Fisher Scientific Company (Fair Lawn, NJ)f. Carrier free 6~Zn (1-10 Ci/g Zn) was purchased from New England Nuclear (Boston, MA). Sephadex G50-80 and Sephadex G75-120 were purchased from Sigma Chemical Company (St. Louis, MO). DEAE A-25 was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). All other chemicals were of the purest grade available.
Animals Zinc-deficient, pair-fed, and ad lib#urn-fed rats were developed according to techniques described by Wallwork and Sandstead (10). The weanling (CRL:[LER]BR) male Long-Evans rats were from the Charles River Breeding Laboratory (Wilmington, MA). The rats were fed a 20% sprayed egg white, biotin-enriched diet with a zinc content of less than 1 mg/kg (It). The diet was modified by the omission of chlortetracyctine hydrochloride and the addition of inositol, 1.0 g/kg of diet (Teklad Mills, Madison, WI). The rats were housed individually in Plexiglass cages in a laminar flow rack located in a humidity- (40-50%) and temperature(25~ controlled room with alternating 12-h periods of light and dark. Food intake and weight changes were measured between 0S:00 and 09:00 h. Wasted food was collected on papers and weighed at the same time. All rats were allowed to adapt to the environment for 5 d. The diet was fed ad libitum, and the drinking water contained 25 ppm zinc (as zinc acetate). To minimize trace metal contamination, we used silicone stoppers and stainless-steel drinking tubes in the water bottles (RodhelmReiss, Inc., Ronsell Rubber Products Division, Belle Mead, NJ). After the adaptation, rats (30-d-old) were randomly assigned to three groups on day 0 of the study. One group (zinc-deficient, 15 animals) was fed the zinc-deficient diet ad libitum and deionized water (Super Q System, Millipore Corporation, Bedford, MA). The two remaining groups were given access to drinking water containing 25 ppm zinc (as zinc acetate). Each rat in the second group (pair-fed, 15 animals) was individually J
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matched by weight with a zinc-deficient rat and pair-fed an amount of diet equaled to the dietary intake of the zinc-deficient rat on the previous day. The third group (ad libitum-fed, 15 animals) was fed the diet ad libitum. During the 30-d dietary regimen, the zinc-deficient rats displayed typical signs of zinc deficiency such as anorexia, cyclic feeding, growth retardation, alopecia, dermatitis surrounding the eyes and on the paws, and hunched posture. After 30 d of zinc deprivation, the animals were flown to Omaha, Nebraska where they were processed the same day for determination of zinobinding proteins. In order to determine the effects of stress of flight on the integrity of zinc-binding proteins, another group of animals shipped from North Dakota was laboratory adapted prior to experimentation. No detectable difference in zinc-binding proteins was noted whether the animal was laboratory adapted or not.
Preparation and Detection of Zinc-Binding Proteins Zinc-deficient, ad-Iibitum, and pair-fed rats were killed by decapitation, and the brains were immediately removed and homogenized in 5 vol of 0.05M Tris-acetic acid (pH 7.5). The homogenates were centrifuged at 750g for 15 min. Then, the supernatants were recentrifuged at 43,000g for 30 min. The pellets were discarded and the supernatants were recentrifuged at 105,000g for 60 rain in a Beckman Model L3-50 ultracentrifuge (Palo Alto, CA). Soluble fractions were concentrated in an Amicon Ultra-filtration Cell using ultrafiltration membranes (YM2, 43 mm Lot AS 02034A) purchased from Amicon Corporation (Danvers, MA). In all cases, 5-10 mL of concentrated supernatant proteins (8-10 mg/mL) were applied to 2.5 x 90 cm Sephadex G-50 or G-75 columns, and the low molecular weight metallothionein-like protein was eluted at 4~ with O.05M Tris-acetic acid buffer (pH 7.4) as discussed by Ebadi et
al. (7).
Stimulation of/Vletallothionein-Like Protein by Zinc Male Sprague-Dawley rats (180-220 g body wt) were purchased from Sasco (Omaha, NE). The animals were housed in stainless steel cages in air-conditioned quarters, laboratory-adapted prior to experiments, and fed a diet of Purina Chow. The zinc content of this diet was 70 ppm which remained constant throughout these experiments. A total of 84 rats were maintained on a 12-h light-dark cycle. Alzet Model 2001 miniosmotic pumps were purchased from Alza (Palo Alto, CA) and sup gically implanted for chronic intracerebroventricular administration of zinc sulfate (test) and 0.9% saline (control) as described by Ebadi et al. (5). The pumps were able to deliver 0.02, 0.05, or 0.22 Ixmol Zn/jxL/h for 48 h in three separate groups of experimental animals consisting of 21 rats. After 48 h, the rats were decapitated and a pool of three brains were
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homogenized in 5 vol of 0.05M Tris acetic acid (pH 7.5) and assayed for metallothionein-like protein as described (7).
DETERMINATION OF ZINC The concentration of zinc in various fractions from the filtration columns were determined with a Perkin-Elmer Atomic Absorption Spectrophotometer, model 306, with ZnSO4. 7H20 as standard. The total concentrations of Zn 2+ were determined according to the procedure of Smeyers-Verbeke et al. (12) as elaborated bv Wuyts et al. (13). In addition, zinc in the whole brain was determined in the high speed supernatant and the resulting pellets.
6~Zinc-Affinity Studies In order to study the binding affinity of zinc for metallothionein-like protein in brains of zinc-deficient rats, and the control groups, the low molecular weight zinc-binding protein (13,000-15,000 daltons) was partially purified on Sephadex G-50 and metallothionein-like protein was isolated, concentrated, and incubated for 2 h at room temperature with 1 ~Ci 65Zn2+. After 2 h at 37~C, the incubation of 6~Zn with metallothionein-like protein continued in the cold room for an additional 16-18 h. The protein-zinc complex was further purified by applying it to a column containing anion exchange medium (DEAE A-25 Sephadex). The metallothionein-like protein was eluted with a linear gradient using Trisacetate buffer (0.05-0.200 rru'vl, pH 7.5) according to the technique published by Karin and Herschman (14).
Determination of Proteins Proteins were determined according to the method of either Bradford (15) or Lowry et al. (16) using bovine serum albumin as standard.
Statistics Significance was determined by univariate and multivariate analyses of variance, followed by Scheffe contrast for the differences in means (17).
RESULTS The intracerebroventricular administration of zinc sulfate (0.02-0.22 p~mol ZnhxL/h for 48 h) increased significantly the synthesis of a metallothionein-like protein from control value of 1-6.5 ~g/mg protein
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(Fig. 1). The synthesis of this metallothionein-like protein in zincdeficient rats was significantly lower than either in the ad libitum group or in the pair-fed group (Fig. 2). Incubation of partially purified metallothionein-like protein with 6~Zn and c h r o m a t o g r a p h y on DEAE A-25 Sephadex p r o d u c e d similar elution patterns in the three experimental groups. However, the extent of binding of 65Zn to the metallothionein-like protein from the zincdeficient rats was significantly (p < 0.05) lower than either zincs u p p l e m e n t e d or ad libitum group (Fig. 3). This data suggests that in zinc d e f i d e n c y state less metallothionein-like protein is synthesized, but what is synthesized is chromatographically similar to those produced by pairfed or ad libitum fed groups. 7.22
,umoies
zinc
~J|
hr -
48hrs
6-
_=
5-
c
=- 4 . 0 5 umoles
zinc j u l
hr - 48
hrs
~a
moles
zinc
ul
hr - 48
hrs
2-
1-
~b
7b
8'o
~o
1do
iio
12 o
EiutiOn Number (3 hi! each)
Fig. 1. Stimulation of the synthesis of a metallothionein-tike protein by zinc delivered intracerebroventricularly by a surgically implanted minipump (5). After 48 h of continuous administration of zinc sulfate (0.02, 0.05, or 0.22/~,mol Zn/~Uh). Rats were decapitated and their brains were immediately homogenized in 5 vol of 0.05M Tris acetic acid buffer (pH 7.5). The high speed (105,000g) supernatant (12 mL containing a total of 96 mg protein) was applied to columns of Sephadex G-75, and the metaUothionien was eluted at 4~C with 0.05M Tris acetic acid buffer (pH 7.5) as described by Ebadi et al. (7). Each elution profile represents the combined average values from three brains using seven separate experiments. The values of zinc-stimulated zinc-binding ligands are significantly higher at their peaks than the control values at least at P < 0.05.
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4-, Pair fed (,)
.3" Ad Libitum (o)
.2.
) N
3,
1.5~
1.3
Pair fed (-)
1.1.
E
.9
~=
.7 84
N
,S'
Ad L i b i t u m (e)
Zinc deficient (,) .3 84
.1.
Elution
Fraction ( 3 m l each)
Fig. 2. Gel filtration chromatography of a metallothionein isolated from brains of zinc-deficient ( i ) ad libitum (Q), and pair-fed (.) rats. Rats were decapitated and their brains were immediately homogenized in 5 vol of 0.05M Tris acetic acid buffer (pH 7.5). The high speed (105,000g) supernatant was concentrated (10 mL containing a total of 85 mg protein) and applied to columns of Sephadex G-75. The metallothionein-like protein was eluted at 4~ with 0.05M Tris acetic acid buffer (pH 7.4) as described by Ebadi et al. (7). Each elution profile from zinc deficient, ,~d libitum, or pair-fed groups represents the combined average values from three rats using five separate experiments. The value of zincbinding ligand in zinc deficiency state at its peak is significantly lower than ad libitum or pair-fed group, at least at P < 0.05.
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Although the zinc-deficiency state manifests in lower synthesis of a metallothionein-like protein, the total subcellular amount of zinc determined in the supernatant or pellet of brains of ad libitum-fed, zinc pairfed, or zinc-deficient groups did not vary significantly (Table 1).
DISCUSSION Preliminary evidence from this laboratory indicated that the synthesis of the low molecular weight zinc-binding protein, a metallothioneinlike protein, was stimulated following the intracerebroventricular administration of zinc (5). In this study, using a far more refined technique, a dose-response effect has been obtained by administering zinc from 0.02-0.22 ~xmol/Zn/~L/h for 48 h through surgically implanted miniosmotic pumps. As it is apparent from Fig. i, the synthesis of the metallothionein-like protein is stimulated from 1 to 6.5 ~g Zn/mg proPair fed , - - ~ 1 4 5
3~1o
4
1
BRAIN
I
_/_._
#
A/ /
mM
L
zso E
I
=
1
Elution Fraction (3ml each) Fig. 3. Gel filtration chromatography depicting the binding of 6SZn into a partially purified metailothionein-]Lke protein. This protein was partially purified on Sephadex G50 (7) concentrated, (40 m[. containing a total of 3.2 mg protein) and incubated with 1 i~Ci ~ Z n + for 2 h at 37~ and then at 4~ for an additional 16-18 h. The metaliothionein binding zinc-65 was further purified by applying it to a column containing anion exchange medium (DEAE A-25 Sephadex). The protein was eiuted with a Linear gradient using Tfis acetate buffer (0-200 rnM, pH 7.5) according to a technique published by Karin and Herschman (14). Each elution profile Dom zinc-deficient (11), ad libitum (O), or pair-fed (.) groups represents the combined average values from three rats using five separate experiments. The extent of binding of ~Zn into metallothioneinLike protein (30 mL containing a tota] of 0.6 mg protein) at its peak (145 raM) was significantly lower in zinc-deficient group than in ad Iibitum or in pair-fed groups at P < 0.05.
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TABLE 1 Total Concentration of Zinc in Brains of ZincDeficient Rats and Their Control Groups ~ Dietary zinc status
Ad Iibitum-fed Pair-fed Zinc-deficient
Pellet zinc, Supernatant zinc, Fg/g dry tissue p.g/mg protein 738 -+ 26.4 745 -+ 14.0 662 - 37.5
0.314 -+ 0.01 0.322 -+ 0.04 0.316 -+- 0.01
"Rats were decapitated and their brains were homogenized in 10 rnM Tris-acetate buffer, p H 7.4. The homogenates were centrifuged in a Beckman refrigerated centrifuge (Model J-21C) at 48,000g for 30 rain. The supernatants were decanted and the pellets were h o m o g e n i z e d in 3 vol of triple-glass distilled water. Aliquots of supernatants and the homogenized pellets were placed in nitric acid prewashed, metal-free, and p r e w e i g h e d 10 x 75 mm culture test tubes (Fisher Scientific C o m p a n y , Pittsburg, PA). The test tubes were dried in a drying oven at I07~C for 18 h. The test tubes were then cooled in a metal-free environment and weighed again, and then 100 p.L of concentrated nitric acid was a d d e d to each test tube. After the dried tissues were allowed to dissolve in nitric acid for 30 min, the test tubes were heated cautiously in boiling water in a hood, but foaming and overboiling were prevented. All b l a n k test tubes received identical treatment. After cooling, the test tubes were centrifuged in an [EC Model CV-5000 centrifuge (International Equipment C o m p a n y , N e e d h a m Heights, MA). Zinc was d e t e r m i n e d by an atomic absorption spectrophotometer according to techniques described by Smeyers-Verbeke et al. (I2) as elaborated by Wuvts et al. (13). Each value represents the m e a n • SEM of at least 15 rats.
tein. F u r t h e r m o r e , in zinc-deficiency, the synthesis of this protein in the brain is r e d u c e d (Fig. 2). The a f o r e m e n t i o n e d studies suggest that the synthesis of the metallothionein-like protein isolated from brain is subject to regulation, in that it is stimulated following administration of zinc a n d inhibited in the zinc-deficiency state. The total concentration of zinc in brains of zinc-deficient rats did n o t vary from the control g r o u p s (Table 1). These studies confirm earlier studies by Wallwork et al. (18), w h o reported that zinc deficiency depressed the concentration of zinc in the olfactory lobe, but n o t in t h e other areas of rat brain studied. Studies in other laboratories have s h o w n that the absorption of zinc is e n h a n c e d in zinc-deficient rats (19-22). W h e t h e r or n o t the transport or absorption of zinc into the brain is enh a n c e d in deficiency state remains to be d e t e r m i n e d . The regional distribution of zinc in rat brain is not uniform (23). Furt h e r m o r e , no direct relation seems to exist b e t w e e n the free concentration of zinc a n d the activity of the zinc metalloenzymes. For example, in
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rat brain the zinc concentration is h i g h e s t in the h i p p o c a m p u s , whereas, the activity of carbonic a n h y d r a s e , a zinc m e t a l l o e n z y m e , is highest in the m e d u l l a oblongata (24). Similarly, in rabbit brain the zinc concentration does not parallel the activity of carbonic a n h y d r a s e (25). In conclusion, the synthesis of a metallothionein-like protein in brain seems to be regulated by free concentration of zinc. H o w e v e r , the mechanism of the stability of zinc in the brain of zinc-deficient rats is n o t clear and deserves additional in-depth investigation.
ACKNOWLEDGMENT M e n t i o n of a t r a d e m a r k or proprietary p r o d u c t does not constitute a guarantee or w a r r a n t y of the p r o d u c t by the US D e p a r t m e n t of Agriculture, a n d does not imply its approval to the exclusion of other p r o d u c t s that m a y also be suitable.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
J. H. R. Kagi and M. Nordberg, Metallothionein Birkhauser, Basel (1979). R. W. Chen, E. J. Vasey, and P. D. Whanger, J. Nutr. 107, 805 (1977). M. Itoh and M. Ebadi, Fed. Proc. 41, 8736 (1982). M. [toh, M. Ebadi, and S. Swanson, J. Neurochem. 41, 823 (1983). M. Ebadi, S. Wilt, R. Ramaley, S. Swanson, and C. Mebus, in Chemical and Biological Aspects of Vitamin B6 Catalysis, A. E. Evangepoulous, ed., Liss, New York, 255 (1984). M. Ebadi, Fed.Proc. 43, 3317 (1984). M. Ebadi, R. J. White, and S. Swanson, in Neurobiology of Zinc, C. J. Frederickson and G. A. Howell, eds., Liss, New York, 1984, Part A, pp. 34-57. M. Ebadi, Trans. Soc. Neurosci. 14, 1062 (1984). M. Ebadi, J. C. Wallwork, S. Swanson, and C. Mebus, Trans. Soc. Neurosci. 13, 402, (1983). J. C. Wallwork and H. H. Sandstead, [. Nutr. 113, 47 (1983). J. M. McKenzie, G. F. Fosmire, and H. H. Sandstead, J. Nutr. 105, I466 (1975). J. Smeyers-Verbeke, E. Defrise-Gussenhoven, G. Ebinger, A. Lowenthal, and D. L. Massart, Clin. Chim. Acta 51, 309 (1974). L. Wuyts, J. Smeyers-Verbeke, and D. L. Massart, Clin. Chim. Acta 72, 405 (1976). M. Karin and H. R. Herschman, Eur. J. Biochem. 107, 395 (1980). M. M. Bradford, Anal. Biochem. 72, 248 (1976). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). H. Scheffe, The Analysis of Variance, Wiley, New York, 1959, pp. 90-137. J. C. Wallwork, D. B. Milne, R. L. Sims, and H. H. Sandstead, [. Nutr. 113, 1895 (1983).
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Zinc-Binding Ligands in Zinc-Deficient Rats 19. 20. 21. 22. 23.
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E. Weigand and M. Kirchgessner, 1. Nutr. 110, 469 (1980). M. ]. Jackson, D. A. Jones, and R. H. T. Edwards, Br. ]. Nz~tr. 46, 15 (1981). K. T. Smith and R. J. Cousins, I. N~tr. 110, 316 (1980). P. R. Flanagan, J. Haist, and L. S. Valberg, ]. N~tr. 113, 962 (1983). M. Ebadi, M. Itoh, J. Bifano, K. Wendt, and A. Earle, Int. ]. Biochem. 13, 1107
(1981).
24. V. Nair and D. Bau, Brain Res. 31, 185 (1971). 25. M. R. Klee and M. Lieflander, Hoptoe-Seyler's Zeitschrift Physiol. Chem. 341, 143 (1965).
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