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Isolation of three copper metallothionein isoforms from the blue crab (Callinectes sapidus)
Aquatic Toxicology. 20 ( 1991) 25-34 Elsevier
e
Duke University
tine Laboramy. Beaufort. NC 4U.S.
(Received 23 November 1990; revision received 7 Mar& I
In the blue crab Callinectes sapidus. a direct correlation exists between levels o ein (CuMT) and Cu(I)-hemocyanin during the molt cycle (Engel and B makes these organisms unique model systems to study the involvement o tion and metal detoxification. To further clarify the role of MT in metal-regu determined how many different MT isoforms are present in blue crabs and how spond to elevated dietary levels of Cu. Anion-exchange HPLC showed two Znisoforms, eluting at 40 and 87 mM Tris, in hepatopancreas from control crabs. CuMT-II, and CuMT-III), eluting at 42, 55 -md 93 mM Tris. were present in treated animals. CuMT-III was induced to a much greater extent than CuMTof the metal-free MTs by reverse-phase HPLC and amino acid analysis show Cu-treated animals had a unique amino acid composition indicating the presence of t I MTs. Functional implications of these findings are discussed.
Key words: Metallothionein; Crustacea; Amino acid; Crab; HPLC, Copper
INTRODUCTION
Metallothioneins are a class of cysteine-rich, low molecula proteins originally isolated from the renal cortex of the horse 1957). Although metallothioneins appear to be involved in metal homeostasis and detoxification, the biological functions of the proteins are not fully underst (Hamer, 1986). Ubiquitous throughout phylogeny, MT has been shown to exi three classes (Fowler et al., 1987). Class I MTs are found in animals and consist of 58-63 amino acids, 18 or 20 of which are cysteines. Class II primarily in yeast and are likewise rich in cystei homology when compared to Class I proteins
Correspondence to: D. Schlenk, Duke University Marine Laboratory, Beaufort, NC 285 16, U.S.A. 0166-445X/91/$3.50 @ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
organisms (Grill et al., 1985). With
es and Goering, I9
;
auser et al., 1983). n shown to be induo at the level of transcription by heavy metals (i.e. copy of other agents (i.e. glucocorticoid hordeprivation, and elevated oxygen tension) Jahroudi et al., 1990; Sadhu and Gedamu, 1988;
r (Cu) induces three isoforms in the American levance of the occurthe involvement of and Brouwer, 1987; during hemocyanin different from the form that donates Cu(I), via glutathione, to protein during hemocyanin synthesis (Brouwer et al., 1989; 1991). These suggested the possibility that MT isoforms in marine crustacea may t functions and are differentially regulated. Consequently, the se of this study was to examine the effects of dietary Cu administration on MT
Blue crabs C. sapidus were obtained from Hoopers Crab Farm (Marshallberg, NC.). Animals were kept in tanks with flow-through seawater at ambient temperature (approximately 22°C). Crabs were exposed to CuSO4 in 2-day intervals by feeding them fish (approximately IO-20 g pieces) injected with approximately 5 mg of CUSOJ. After 5 dosages, the hepatopancreas of the crabs was excised and frozen in liquid nitrogen. Frozen tissues were thawed, homogenized in buffer (10 mM Tris-HCl pH 8.2, 2 mM /%mercaptoethanol) and centrifuged at 20 000 x g for 30 min to remove cellular debris. Following a second centrifugation at 100000 x g, the resulting supernatant was applied to a Sephadex G-75 column (2.5 x 50 cm). The column was eluted with 10 mM Tris-HCl, pH 8.2 with 2 mM /I-mercaptoethanol at a flow rate of 30 h. All buffers were cold and saturated with nitrogen, and fractions were collected in a nitrogen atmosphere. Metal content was measured using a Perkin Elmer 5000 Atomic Absorption Spectrophotometer. Metal containing fractions corresponding to 10000 Daltons (Ve/Vo = 2.5) were pooled, concentrated using an Amicon YM-2 membrane and injected on to a Beckman Gold HPLC system containing a Spherogel
0
20
40
60
60
Fractions Fig. 1. Sephadex G-75 profile of the cytosolic fraction from control crab hepatopancreas. U tions represent pooled material for ion-exchange chromatography.
TSK DEAE-3SW anion-exchange column (21.5 mm i.d. x 15 cm). isoforms were eluted at room temperature using a linear gradient from 10 HCl (pH 7.6) 2 mM j%mercaptoethanol to 200 mM Tris-HCl (pH 7 captoethanol over 60 min at a flow rate of 6 ml/min. Reverse-phase HPLC of CuMT gave unsatisfactory results, presumably because of Cu-induced disulfide bridge formation. To prevent oxidation of sullhydryl groups, CuMT isoforms were demetallized by acidification to pH 0.5. HCl was removed by extensive dialysis against water. ApoMT samples were reduced with dithiothreitol in 6 M guanidine HCl and alkylated with iodoacetic acid (Brouwer et al., 1984), lyophilized and chromatographed via reversed-phase HPLC utilizing a Synchropak RP-4 column (Synchrom, Inc.) with a gradient of O-245 acetonitrile in 0.1% trifluoroacetic acid. After acid hydrolysis of the apoprotein the amino acids were converted to their phenylthiocarbamyl derivatives and analyzed by reversed-phase HPLC on a ClgPTH Ultrasphere column (4.6 mm i.d. x 25 cm) (Heinrikson and Meredith, 1984). RESULTS
Significant increases in Cu content in the metallothionein region of the Sephadex elution profile were observed in Cu-treated animals (Figs. 1 and 2). The in hepatopancreas from control crabs contained 55% and 25% of the total Zn and Cu, respectively. The predominance of Zn over Cu in the MT fractions indicates that
0
20
40
60
80
100
Fractions Fig. 2. Sephadex G-75 profile of the cytosolic fraction from Cu-treated crab hepatopancreas. Underlined fractions represent pooled material for ion-exchange chromatography.
were in the premolt stage (Engel and Brouwer, 1987). In Cu-exand 61 ydof the total Zn and Cu, respectively, was located in the Two major Cu- and Zn-containing peaks were resolved from HPLC ion-exchange chromatography of the cytosolic fraction from control crab hepatopancreas (Fig. 3), while in Cu-treated crabs three peaks were observed (Fig. 4). In control animals, the first of these proteins eluted at a Tris concentration of 40 mM. This form appeared to be associated primarily with Zn and to a lesser degree Cu. A broad second peak of smaller intensity also associated with Cu and Zn eluted at approximately 87 mM Tris. Three MT isoforms eluted at 42 mM (CuMT-I), 55 mM (CuMT-II), and 93 mM (CuMT-III) Tris in samples from Cu-treated crabs (Fig. 4). CuMT-I and CuMT-II contained twice the amount of Cu as that of the two ZnCuMTs of control crabs, but lacked Zn. CuMT-III contained nearly three times more copper than CuMT-I or CuMT-II. Carboxymethylated MTs from Cu-exposed crabs were further purified by reversed-phase HPLC. Retention times for CuMT-I and CuMT-II were very similar, eluting at 22.6 and 23.0 min, respectively. CuMT-III eluted as a single peak at 23.7 min. Attempts to further purify MT from control animals were unsuccessful due to the small amounts of these proteins present.
0.8
A
LL
0.6
a ii
s
0.4
ZE
0.2
0.0
a
0
10
Fig. 3. HPLC ion~x~~a~ge protie of pooled fraction obtained after of cytosot from the hepatopancreas of contro1 crabs- Underarm fractious represent reverse-phase chromatography.
1.21 1.01
-
copper
--+-
Zinc
0.8 s 0.6 8
0.4
20
Time (min) Fig. 4. HPLC ion-exchange profile of pooled fractions obtained after !kphadex G-75 chro of cytosol from the hepatopancreas of Cu-treated crabs. Underlined fractions rep-t for reverse-phase chromatography.
-III were composed of a proximately 28
aromatic amino acid residues, e amino acid compositions of Cu was distinct in t at it possessed more glycine, aspartic ne, but less glutamic acid than either Cu
at metallothionein plays an important role in molt cycle of the blue crab C. sapidus(Engel cant changes occur in the metals bound to first is at the beginning of premolt, where metals ominantly Cu to Zn, presumably correlated with rend an increased rate of Zn-carbonic anhydrase synthed occurs 90 min after ecdysis, where there T, correlated with the degradation of hemocyanin. The e papershell stages, where the MT once again becomes Cu protein, and this change is correlated with hemocyanin synthesis. Resies was limited to size-fractionation chromatography. ether the observed changes in MT represent changes in one or more T isoforms, and whether the MT isoform involved in sequestration (detoxification) of Cu released upon hemocyanin breakdown is the same as the form ay provide Cu, via glutathione (see below), for hemocyanin biosynthesis. To understanding of these fascinating processes studies are required at the T structure/function and MT gene regulation. To address the quesTABLE I Amino acid compositions of Cu-metallothioneins from the hepatopancreas of the blue crab. MT-I
ASK Tbr Ser Glx pro
2.0 4.9 4.4 6.2 5.9
GlY Ala Val
7.4 2.3 1.1
CYS LYS Arg
16.6 7.7 0.9
MT-II
MT-III
2.3 4.6 4.3 6.1 5.8 6.4 2.2 1.2 16.9 8.4 1.2
3.8 4.6 7.4 2.9 4.3 8.4 3.6 0.4 17.0 7.2 -
The values are displayed as the number of residues per molecule, assuming that each MT contains 58 amino acids (Brouwer et al., 1989). Each value represents the average of two CuMT preparations.
tion of whether isoforms are coo ately crabs, we have begun to characterize the structure an MT proteins. er treatment with
topancreas from the American lobster high Cu levels were present (Brouwer et al., 19 levels of serine and aspartic acid in were also observed in the lobster C II were not res a steeper Tris-Chloride gradient. Therefore, C cal to CuMT-II in our previous study which is with Cu possessed three CuMT isoforms that differed in amino from rat liver ZnMT (Bremner and Young, 1976) and Cd (Winge et al., 1981), suggesting that Cu- and Cd-inducible distinct proteins. In Cu-treated crabs, CuMT-III was significantly greater t or CuMT-II, indicating differential regulation of pression of MT genes has been examined in hum Gedamu, 1988; Jahroudi et al., 1990; Olsson et al., 1990). In humans, t least 4 to 5 different MT-I and 1 MT-II isoforms (Kagi a tional genes have been characterized with 5 expressing ing MT-II (Hamer, 1986). The differential pattern of induction, which also specific (Schmidt and Hamer, 1986; Jahroudi et al., 19 MTs carry out distinct functions in the processes of cation, and that cells may modulate their metal toire of expressed MT genes (Schmidt and Hamer, 1986). How differential regulation of MT isoforms may affect cellular metal m is still unclear. The preferential expression of CuMT-III foIlowing Cu e crab hepatopancreas was also observed in studies with lobster, suggesting that CuMT-III may be involved primarily in the detoxification of Cu (Brouwer et al., 1989). Recently, it has been shown that the three CuMT isoforms from lobster interact with the tripeptide, glutathione (Brouwer et al., 1991). Whereas the third (CuMT-III) forms a stable 1:l CuMT-glutathione complex, the first two forms (CuMT-I/II) make a transient complex with glutathione, with subsequent re Cu as a Cu-glutathione species. The latter can restore the oxygen-binding ca of Cu-free hemocyanin, suggesting that the CuMT-I/II isoforms may be in via glutathione, as a donor of Cu(1) for the activation of Cu-requiring e proteins. Such observations strongly suggest th distinct physiological roles: the first two forms (C
ses, whe~as the thin fog
(C r. This functional distinction
more rapid than that
may be directly correwhich eiute at 40 and ing at 42 and 93 mM animals. Low levels of the control proteins prohibited further pusition analysis. We will clarify the nature of the conc nucleotide probes prepared from MT mRNAs from will also be used to further our understanding of studying MT isoform
arch was fade by grants from the National Institute of Environmental Sciences ESOX’l31 and ~~~~4.
~~~~et, I. and B.W. Young, 1976. Isolation of (copper, zinc)-thioneins from the livers of copper-injected rats. Biochem. J. 157,517-520. Bro~wer, rouwer-Hoexum and D-W. Engel, 1984. Cadmium a~um~ation by the blue crab (Cnlinvolvement of hemocyanin and characterization of cad~um-binding proteins. Mar. ~~~~~~S Environ. Res. l4,71-88. Brouwer, M., P. Whaling and D.W. Engel, 1986. Copper-metallothioneins in the American lobster HomaI&W ~~r~c~~; ~tentiai role as copper(l) donors tu a~hem~yanin. Environ. Health Perspect. 65,93-Brouwer, M., D.R. Winge and W.R. Gray, 1989. Structural and functional diversity of copper-metallo~~~~e~ns from the American lobster yogurts ~rneric~~. J. Inorg. Biochem. 35,289-303. Brouwer, M., T. Brouwer-H~xum and R. Cashon, 1991. Crustaceans as models for metal metabolism: III. Interaction of lobster and mammalian metallothioneins with glutathione. Mar. Environ. Res., in Pm. Cain, R. and B.L. GriIiiths, 1984. A compa~son of isometauothionein synthesis in rat liver after partial hepat~tomy and parenteral zinc injection. B&hem. J. 217,85-92. Engel, D.W. and M. Brouwer, 1987. Metal regulation and molting in the blue crab, Callinectes sapidus: me~lot~onein function in metal metabolism. Biol. Bull. 173,239-25 I. Engel, D.W. and M. Brouwer, 1991. Short-term metallothionein and copper changes in blue crabs at ecdysis. Biol. Bull., 180,4%7-452. Fowler, B.A., C.E. Hildebrand, Y. Kojima and M. Webb, 1987. Nomenclature of Metallotbioneins. In: Me~~lo~onei~ II, edited by J.H.R. Kagi and J. Kojima, Birder Verlag, B~l~Boston~ pp. 19-22.
Grill, E., EL. W~~na~k~ peptides of higher plants Hamer, D., I986. Metallothioneins. H~n~k~n, R.L. and SC. zenith, chromatogra~by: p~o~~rn~ d Jahroudi, N., R. Foster, J. Pricetial regulation of the human rneta~~otb~on~~ genes. J. BioL Cbem. 265 Kagi, J.H.R. and 1. Koj II, edited by J.H.R. K Klauser. S., J.H.R. Ka and isolated samples of MTs by reverse phase 71-80. Margoshes, M. and B.L. Vallee, 1957. A cadmium protein from the equine See. 79,4813-4814_ Glsson, P., S.J. Hyllner, M. ~fa~!~ab, T. Ande~~ and L. emu, 1 ein gene expression in primary cultures of rainbow trout hepat chim. Biophys. Acta IO49,78-82. Richards, R.I., A. Heguy and M. K&n, 1984. St~ctu~~ and ~~~t~o thionein la gene: differential indM~tion by metd ions and ~~~~~0 Sadhu, C. and L. Gedamu, 1988. Regulation of human rnetal~ot~o~~~ 2679-2684. Schmidt, C.J. and D-H. Namer, 1986. Ceh s~fi~ity and an e&et of pas on buman expression. Proc. Natl. Acad. Sci. USA 83,3346-3350. Suzuki, K-T. and M. Yamamura, 1980. Changes of metal contents and isometahothio tissues after cadmium loading. B&hem. Pharmacol. 29,24O-2412. Waalkes, M.P. and P.L. Goering, 1990. Me~llot~onei~ and other ~d~~-b~~ng ~0~~ velopments. Winge, D.R. and K.A. Miklossy, 1982a. Differences in the polymorphic forms of metaJJotbionein. Arch. Bioehem. Biophys. 214,80--88. Winge, D.R. and K.A. Miklossy, 1982b. Domain nature of metahothionein. J. Biol. Chem. 257.374&3746. Winge, D.R., B.L. Geller and J. Garvey, 1981. Isolation of copper thionein from rat Biver.Arch. Biophys. 208,160-166.
e
Duke University
tine Laboramy. Beaufort. NC 4U.S.
(Received 23 November 1990; revision received 7 Mar& I
In the blue crab Callinectes sapidus. a direct correlation exists between levels o ein (CuMT) and Cu(I)-hemocyanin during the molt cycle (Engel and B makes these organisms unique model systems to study the involvement o tion and metal detoxification. To further clarify the role of MT in metal-regu determined how many different MT isoforms are present in blue crabs and how spond to elevated dietary levels of Cu. Anion-exchange HPLC showed two Znisoforms, eluting at 40 and 87 mM Tris, in hepatopancreas from control crabs. CuMT-II, and CuMT-III), eluting at 42, 55 -md 93 mM Tris. were present in treated animals. CuMT-III was induced to a much greater extent than CuMTof the metal-free MTs by reverse-phase HPLC and amino acid analysis show Cu-treated animals had a unique amino acid composition indicating the presence of t I MTs. Functional implications of these findings are discussed.
Key words: Metallothionein; Crustacea; Amino acid; Crab; HPLC, Copper
INTRODUCTION
Metallothioneins are a class of cysteine-rich, low molecula proteins originally isolated from the renal cortex of the horse 1957). Although metallothioneins appear to be involved in metal homeostasis and detoxification, the biological functions of the proteins are not fully underst (Hamer, 1986). Ubiquitous throughout phylogeny, MT has been shown to exi three classes (Fowler et al., 1987). Class I MTs are found in animals and consist of 58-63 amino acids, 18 or 20 of which are cysteines. Class II primarily in yeast and are likewise rich in cystei homology when compared to Class I proteins
Correspondence to: D. Schlenk, Duke University Marine Laboratory, Beaufort, NC 285 16, U.S.A. 0166-445X/91/$3.50 @ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
organisms (Grill et al., 1985). With
es and Goering, I9
;
auser et al., 1983). n shown to be induo at the level of transcription by heavy metals (i.e. copy of other agents (i.e. glucocorticoid hordeprivation, and elevated oxygen tension) Jahroudi et al., 1990; Sadhu and Gedamu, 1988;
r (Cu) induces three isoforms in the American levance of the occurthe involvement of and Brouwer, 1987; during hemocyanin different from the form that donates Cu(I), via glutathione, to protein during hemocyanin synthesis (Brouwer et al., 1989; 1991). These suggested the possibility that MT isoforms in marine crustacea may t functions and are differentially regulated. Consequently, the se of this study was to examine the effects of dietary Cu administration on MT
Blue crabs C. sapidus were obtained from Hoopers Crab Farm (Marshallberg, NC.). Animals were kept in tanks with flow-through seawater at ambient temperature (approximately 22°C). Crabs were exposed to CuSO4 in 2-day intervals by feeding them fish (approximately IO-20 g pieces) injected with approximately 5 mg of CUSOJ. After 5 dosages, the hepatopancreas of the crabs was excised and frozen in liquid nitrogen. Frozen tissues were thawed, homogenized in buffer (10 mM Tris-HCl pH 8.2, 2 mM /%mercaptoethanol) and centrifuged at 20 000 x g for 30 min to remove cellular debris. Following a second centrifugation at 100000 x g, the resulting supernatant was applied to a Sephadex G-75 column (2.5 x 50 cm). The column was eluted with 10 mM Tris-HCl, pH 8.2 with 2 mM /I-mercaptoethanol at a flow rate of 30 h. All buffers were cold and saturated with nitrogen, and fractions were collected in a nitrogen atmosphere. Metal content was measured using a Perkin Elmer 5000 Atomic Absorption Spectrophotometer. Metal containing fractions corresponding to 10000 Daltons (Ve/Vo = 2.5) were pooled, concentrated using an Amicon YM-2 membrane and injected on to a Beckman Gold HPLC system containing a Spherogel
0
20
40
60
60
Fractions Fig. 1. Sephadex G-75 profile of the cytosolic fraction from control crab hepatopancreas. U tions represent pooled material for ion-exchange chromatography.
TSK DEAE-3SW anion-exchange column (21.5 mm i.d. x 15 cm). isoforms were eluted at room temperature using a linear gradient from 10 HCl (pH 7.6) 2 mM j%mercaptoethanol to 200 mM Tris-HCl (pH 7 captoethanol over 60 min at a flow rate of 6 ml/min. Reverse-phase HPLC of CuMT gave unsatisfactory results, presumably because of Cu-induced disulfide bridge formation. To prevent oxidation of sullhydryl groups, CuMT isoforms were demetallized by acidification to pH 0.5. HCl was removed by extensive dialysis against water. ApoMT samples were reduced with dithiothreitol in 6 M guanidine HCl and alkylated with iodoacetic acid (Brouwer et al., 1984), lyophilized and chromatographed via reversed-phase HPLC utilizing a Synchropak RP-4 column (Synchrom, Inc.) with a gradient of O-245 acetonitrile in 0.1% trifluoroacetic acid. After acid hydrolysis of the apoprotein the amino acids were converted to their phenylthiocarbamyl derivatives and analyzed by reversed-phase HPLC on a ClgPTH Ultrasphere column (4.6 mm i.d. x 25 cm) (Heinrikson and Meredith, 1984). RESULTS
Significant increases in Cu content in the metallothionein region of the Sephadex elution profile were observed in Cu-treated animals (Figs. 1 and 2). The in hepatopancreas from control crabs contained 55% and 25% of the total Zn and Cu, respectively. The predominance of Zn over Cu in the MT fractions indicates that
0
20
40
60
80
100
Fractions Fig. 2. Sephadex G-75 profile of the cytosolic fraction from Cu-treated crab hepatopancreas. Underlined fractions represent pooled material for ion-exchange chromatography.
were in the premolt stage (Engel and Brouwer, 1987). In Cu-exand 61 ydof the total Zn and Cu, respectively, was located in the Two major Cu- and Zn-containing peaks were resolved from HPLC ion-exchange chromatography of the cytosolic fraction from control crab hepatopancreas (Fig. 3), while in Cu-treated crabs three peaks were observed (Fig. 4). In control animals, the first of these proteins eluted at a Tris concentration of 40 mM. This form appeared to be associated primarily with Zn and to a lesser degree Cu. A broad second peak of smaller intensity also associated with Cu and Zn eluted at approximately 87 mM Tris. Three MT isoforms eluted at 42 mM (CuMT-I), 55 mM (CuMT-II), and 93 mM (CuMT-III) Tris in samples from Cu-treated crabs (Fig. 4). CuMT-I and CuMT-II contained twice the amount of Cu as that of the two ZnCuMTs of control crabs, but lacked Zn. CuMT-III contained nearly three times more copper than CuMT-I or CuMT-II. Carboxymethylated MTs from Cu-exposed crabs were further purified by reversed-phase HPLC. Retention times for CuMT-I and CuMT-II were very similar, eluting at 22.6 and 23.0 min, respectively. CuMT-III eluted as a single peak at 23.7 min. Attempts to further purify MT from control animals were unsuccessful due to the small amounts of these proteins present.
0.8
A
LL
0.6
a ii
s
0.4
ZE
0.2
0.0
a
0
10
Fig. 3. HPLC ion~x~~a~ge protie of pooled fraction obtained after of cytosot from the hepatopancreas of contro1 crabs- Underarm fractious represent reverse-phase chromatography.
1.21 1.01
-
copper
--+-
Zinc
0.8 s 0.6 8
0.4
20
Time (min) Fig. 4. HPLC ion-exchange profile of pooled fractions obtained after !kphadex G-75 chro of cytosol from the hepatopancreas of Cu-treated crabs. Underlined fractions rep-t for reverse-phase chromatography.
-III were composed of a proximately 28
aromatic amino acid residues, e amino acid compositions of Cu was distinct in t at it possessed more glycine, aspartic ne, but less glutamic acid than either Cu
at metallothionein plays an important role in molt cycle of the blue crab C. sapidus(Engel cant changes occur in the metals bound to first is at the beginning of premolt, where metals ominantly Cu to Zn, presumably correlated with rend an increased rate of Zn-carbonic anhydrase synthed occurs 90 min after ecdysis, where there T, correlated with the degradation of hemocyanin. The e papershell stages, where the MT once again becomes Cu protein, and this change is correlated with hemocyanin synthesis. Resies was limited to size-fractionation chromatography. ether the observed changes in MT represent changes in one or more T isoforms, and whether the MT isoform involved in sequestration (detoxification) of Cu released upon hemocyanin breakdown is the same as the form ay provide Cu, via glutathione (see below), for hemocyanin biosynthesis. To understanding of these fascinating processes studies are required at the T structure/function and MT gene regulation. To address the quesTABLE I Amino acid compositions of Cu-metallothioneins from the hepatopancreas of the blue crab. MT-I
ASK Tbr Ser Glx pro
2.0 4.9 4.4 6.2 5.9
GlY Ala Val
7.4 2.3 1.1
CYS LYS Arg
16.6 7.7 0.9
MT-II
MT-III
2.3 4.6 4.3 6.1 5.8 6.4 2.2 1.2 16.9 8.4 1.2
3.8 4.6 7.4 2.9 4.3 8.4 3.6 0.4 17.0 7.2 -
The values are displayed as the number of residues per molecule, assuming that each MT contains 58 amino acids (Brouwer et al., 1989). Each value represents the average of two CuMT preparations.
tion of whether isoforms are coo ately crabs, we have begun to characterize the structure an MT proteins. er treatment with
topancreas from the American lobster high Cu levels were present (Brouwer et al., 19 levels of serine and aspartic acid in were also observed in the lobster C II were not res a steeper Tris-Chloride gradient. Therefore, C cal to CuMT-II in our previous study which is with Cu possessed three CuMT isoforms that differed in amino from rat liver ZnMT (Bremner and Young, 1976) and Cd (Winge et al., 1981), suggesting that Cu- and Cd-inducible distinct proteins. In Cu-treated crabs, CuMT-III was significantly greater t or CuMT-II, indicating differential regulation of pression of MT genes has been examined in hum Gedamu, 1988; Jahroudi et al., 1990; Olsson et al., 1990). In humans, t least 4 to 5 different MT-I and 1 MT-II isoforms (Kagi a tional genes have been characterized with 5 expressing ing MT-II (Hamer, 1986). The differential pattern of induction, which also specific (Schmidt and Hamer, 1986; Jahroudi et al., 19 MTs carry out distinct functions in the processes of cation, and that cells may modulate their metal toire of expressed MT genes (Schmidt and Hamer, 1986). How differential regulation of MT isoforms may affect cellular metal m is still unclear. The preferential expression of CuMT-III foIlowing Cu e crab hepatopancreas was also observed in studies with lobster, suggesting that CuMT-III may be involved primarily in the detoxification of Cu (Brouwer et al., 1989). Recently, it has been shown that the three CuMT isoforms from lobster interact with the tripeptide, glutathione (Brouwer et al., 1991). Whereas the third (CuMT-III) forms a stable 1:l CuMT-glutathione complex, the first two forms (CuMT-I/II) make a transient complex with glutathione, with subsequent re Cu as a Cu-glutathione species. The latter can restore the oxygen-binding ca of Cu-free hemocyanin, suggesting that the CuMT-I/II isoforms may be in via glutathione, as a donor of Cu(1) for the activation of Cu-requiring e proteins. Such observations strongly suggest th distinct physiological roles: the first two forms (C
ses, whe~as the thin fog
(C r. This functional distinction
more rapid than that
may be directly correwhich eiute at 40 and ing at 42 and 93 mM animals. Low levels of the control proteins prohibited further pusition analysis. We will clarify the nature of the conc nucleotide probes prepared from MT mRNAs from will also be used to further our understanding of studying MT isoform
arch was fade by grants from the National Institute of Environmental Sciences ESOX’l31 and ~~~~4.
~~~~et, I. and B.W. Young, 1976. Isolation of (copper, zinc)-thioneins from the livers of copper-injected rats. Biochem. J. 157,517-520. Bro~wer, rouwer-Hoexum and D-W. Engel, 1984. Cadmium a~um~ation by the blue crab (Cnlinvolvement of hemocyanin and characterization of cad~um-binding proteins. Mar. ~~~~~~S Environ. Res. l4,71-88. Brouwer, M., P. Whaling and D.W. Engel, 1986. Copper-metallothioneins in the American lobster HomaI&W ~~r~c~~; ~tentiai role as copper(l) donors tu a~hem~yanin. Environ. Health Perspect. 65,93-Brouwer, M., D.R. Winge and W.R. Gray, 1989. Structural and functional diversity of copper-metallo~~~~e~ns from the American lobster yogurts ~rneric~~. J. Inorg. Biochem. 35,289-303. Brouwer, M., T. Brouwer-H~xum and R. Cashon, 1991. Crustaceans as models for metal metabolism: III. Interaction of lobster and mammalian metallothioneins with glutathione. Mar. Environ. Res., in Pm. Cain, R. and B.L. GriIiiths, 1984. A compa~son of isometauothionein synthesis in rat liver after partial hepat~tomy and parenteral zinc injection. B&hem. J. 217,85-92. Engel, D.W. and M. Brouwer, 1987. Metal regulation and molting in the blue crab, Callinectes sapidus: me~lot~onein function in metal metabolism. Biol. Bull. 173,239-25 I. Engel, D.W. and M. Brouwer, 1991. Short-term metallothionein and copper changes in blue crabs at ecdysis. Biol. Bull., 180,4%7-452. Fowler, B.A., C.E. Hildebrand, Y. Kojima and M. Webb, 1987. Nomenclature of Metallotbioneins. In: Me~~lo~onei~ II, edited by J.H.R. Kagi and J. Kojima, Birder Verlag, B~l~Boston~ pp. 19-22.
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