metallothionein and the subcellular localization of mercury and cadmium in the california sea lion

Comp. Biochem. Physiol., 1977, Vol. 57C~ pp. 45 to 53. Perglamon Press. Printed in Great Britain

M E T A L L O T H I O N E I N A N D THE SUBCELLULAR LOCALIZATION OF M E R C U R Y A N D C A D M I U M

IN THE CALIFORNIA SEA LION

S. S. LEE*, B. R. MATE?, K. T. YON DER TRI~:NCK +, R, A. RIMERMAN AND D. R. BUHLER

Department of Agricultural Chemistry and Environmental Health Sciences Center, Oregon State University, Corvallis. OR 97331, U.S.A.

(Received 14 September 1976)

Abstract--1. Cadmium is primarily localized in the soluble fl'actions of kidney (60-65'!o) and liver (43-58~o) of the California sea lion.

2. In contrast, most of the mercury is concentrated in the nuclear fractions of kidney (35M7{'o) and liver (59-66~%) of this species.

3. Two mercury (cadmium)-binding proteins with properties similar to metallothionein from other species have been isolated from sea lion kidney and liver and partially characterized.

INTRODUCTION

Substantial amounts of mercury (Hg) have been found in livers and kidneys of the California sea lion (Zalo- phus cali fornianus californianus) in our laboratory (Buhler et al., 1975) and in these tissues from other marine mammals (Gaskin et al., 1972; Freeman & Horne, 1973; Gaskin et al., 1973; Heppleston & French, 1973; Koeman et al., 1973; Anas, 1974; Gas- kin et al., 1974). Mercury levels as high as 700/~g/g (Anas, 1974) and 765/~g/g (Koeman et al., 1973) thus have been reported in the livers of healthy marine mammals. Most of the liver Hg, however, is in the inorganic form (Freeman & H o r n e . 1973; Buhler et al., 1975).

Jakubowski et al. (1970) observed that approxi- mately 5(~75% of inorganic Hg present in liver and kidney of the rat was bound to a low molecular weight protein, subsequently identified as metallothionein. Similarly, Ellis & Fang (197l) found a substantial amount of Hg bound to a protein corresponding to metallothionein in the vital organs of rats exposed to either organic or inorganic Hg. Metallothionein isolated from the liver of patients treated with Hg diuretics also contained this metal (Pulido et al., 1966).

Metallothionein, first discovered by Margoshes & Vallee (1957), binds almost all of the cadmium (Cd) in horse renal cortex and has subsequently been detected in tissue from a number of mammals (Mar- goshes & Vallee. 1957: K~igi & Vallee, 1961; Pulido et al., 1966; Jakubowski et al., 1970; Shaikh & Lucis, 1971; Nordberg et al., 1972; Winge & Rajagopalan, 1972; Weser et al., 1973; K~igi et al., 1974). While the physiological function of metallothionein is still unknown, it has been postulated that this low mol-

* Present address: Department of Life Sciences, Virginia State College, Petersburg, VI 23803, U.S.A.

t Department of Oceanography, Oregon State Univer- sity, Marine Sciences Center, Newport, OR 97365, U.S.A.

~.Present address: University of Freiburg, Freiburg, Federal Republic of Germany.

45

ecular weight protein functions as a protective buffer against Hg and other toxic heavy metals (Piscator, 1966). Since metallothionein is inducible by pretreat- ment of laboratory animals with Cd or Hg (Chen et al., 1975a; Piotrowski et al., 1974), this is suggestive evidence supporting the hypothesis. Toxic effects appear when the Cd-binding capacity of the renal metallothionein is exceeded (Piotrowski & Bolanowska, 1970), More recent work (Kimura et al., 1974), however, suggests that the protective role of metallothionein is confined primarily to the kidney.

Metallothionein has also recently been detected in fur and grey seals (Olafson & Thompson, 1974). The present communication reports the subcellular distribution of Hg and Cd and the partial purification of Hg (Cd)-binding proteins similar to metallothionein from the soluble fraction of liver and kidney of Cali- fornia sea lions.

MATERIALS AND METHODS

Materials and chemicals

Healthy California sea lions (Zalophus ealiJornianus californianus) were obtained along the Oregon Coast under permits issued by the Oregon State Game Commission or the National Marine Fisheries Service. Kidneys, livers, and samples of muscle were removed and a portion cooled to 0°C for immediate use. The remainder was then frozen at -2f fC. Reference proteins and biochemicals were obtained from Sigma Chemical Company (St. Louis, MO) and all other chemicals were analytical grade. Sephadex G-75 and G-200 (Pharmacia) and DEAE-cellulose 32 (Whatman) were used for column chromatography. The predominant form of horse renal metallothionein (K~igi et al., 1974) was prepared from horse kidneys by the methods described below for sea lions.

Subcellular fractionation o f sea lion tissue

To prepare subcellular fractions, about 5 g of tissue were first homogenized in 0.25 M sucrose solution containing 1 mM EDTA in an omnimixer for 30sec. Additional homogenization was effected in a Potter Elvehjem hom- ogenizer and these homogenates were subjected to differen-

46 S.S. LEE et al.

tial centrifugation at 1.2 x 104, 1 x 105, and 1.4 x 10 6 0-rain. To determine Hg and Cd concentrations, 0.5 1.0 g of each fraction was digested with 4 ml concentrated HNO3 at 90'C for 1 hr and diluted to 10 ml. The digested sample was cooled to 4°C so that the fat, essentially free of metal, could be separated from the rest of the solution and discarded. Concentrations of Cd were determined in a Perkin Elmer Model 403 atomic absorption spectrometer equipped with model HGA-2000 graph- ite furnace. Total and inorganic Hg concentrations were measured using a Coleman Mercury Analyzer according to the method of Magos (1971) except that 1 N H2SO¢ was used instead of 16 N H2SO 4. Selenium (Se) was determined by the fluorimetric method of Hoffman et al, (1968) except that decahydronaphthalene (J. T. Baker, Analyzed Reagent) was used in place of cyclohexane. Samples were assayed on a Turner Model 111 fluorometer filtered with a 365 nm primary filter (excitation) and a combination of 525 nm and 520 nm secondary filter (fluorescence). Protein was routinely determined by the method of Lowry et al. (1951). Because of a low content of aromatic amino acids and an abundance of sulfhydryl groups, however, this assay may underestimate metallothionein when bovine serum albumin (BSA) was used as a standard. Van Kley & Hale (1976) have recently shown that color yield in the Lowry assay is not solely dependent on the presence of aromatic amino acids in a variety of peptides.

Statistical analysis Correlations between the cadmium and mercury in tis-

sues and that occurring in metallothionein were determined by use of the Statistical Interactive Programming System (SIPS) (Guthrie et al., 1974) in a CDC-3300 com- puter.

Isolation and pur!fication of Hg (Cd)-binding proteins Five grams of sea lion kidney, liver or muscle was

minced, ground under liquid nitrogen into a fine powder, and then dissolved in 10 ml of 0.01 M Tris-Cl buffer, pH 8.6, containing 0.05 M NaC1 plus 0.02°0 sodium azide as a preservative. The homogenate was centrifuged at 1.4 × 106 g-rain.

The supernatant and pellet were separated and the Cd, Hg, and protein content of tissue, supernatant, and pellet were determined before carrying out further steps. The 1.4 x 10 ~ g-min supernatant fraction was applied to either a Sephadex G-75 (2.5 x 5.0 × 100 cm) or Sephadex G-200 column (2.5 x 100 cm) at 4'C. The absorbancy at 250 and 280 nm of the effluent fractions was determined on a Gil- ford Model 2000 spectrophotometer. Cadmium in the frac-

tions was determined by use of the APDC-M1BK extrac- tion procedure (Slavin, 1968) and atomic absorption spec- trophotometry. Total Hg concentrations were determined as described previously. The fractions which exhibited a low absorbancy and high metal content (V~/Vo = 2.1) were pooled and diluted 10 times with 0.01 M Tris-CI buffer, pH 8.6. This solution was passed through a Whatman DE-32 column (2.5 x 42 cm) which was pre-equilibrated with 0.01 M Tri~HCI, pH 8.6, at 4'C. The column was eluted with a gradient consisting of (~1 M NaCI in 0.01 M Tris CI, pH 8.6. Protein, Cd, and Hg contents of the effluent were monitored as described above. Aliquots for each purification step were examined by disc electrophoresis according to the method of Davis (1964).

RESULTS

Subcellular distribution of heavy metals

The subcellular distribution of Cd, Hg, and protein in sea lion liver, kidney, and muscle are shown in Tables 1-3. The highest concentration of Cd was present in the 1.4 x 106 o-min supernatant (soluble) fractions of all three tissues but appreciable Cd also appeared in the nuclear fractions. In contrast, Hg was localized primarily in the nuclear fraction. Although appreciable Hg was found in the soluble fraction from kidney and muscle, a very small proport ion of the Hg appeared in this fraction from liver. The liver and kidney microsomes, however, contained the highest concentrations of both Cd and Hg on a protein basis.

Separation of Cd (Hg)-binding proteins

Typical elution profiles of the 1.4 x 106 o-min supernatant fractions from sea lion liver and kidney on Sephadex G-75 and Sephadex G-200 are shown in Figs 1 and 2. Four Hg (Cd)-binding peaks were detected on Sephadex G-75 and five peaks were found on the Sephadex G-200 column. Neither Cd nor Hg was detectable in the two peaks of lowest molecular weight from sea lion liver or kidney supernatants. Peak I (Ve/Vo = 1), a major Hg-binding component, was eluted at the void volume in both Sephadex G-75 and Sephadex G-200 columns, The MW of the proteins in this fraction was estimated to be 200,000 or larger. Peak lI, which eluted at the void volume on G-75 but separated (Ve/Vo = 1.55) on G-200 contained

Table 1. Subcellular distribution of mercury, cadmium and protein in sea lion liver*

Animal Cd Hg Protein number Fraction /lg/g ~.~; yg/g % mg/g %

19+ Nuclear 0.20 16.7 30.4 59.0 36.6 6.1 Mitochondrial 0.32 26.7 12.0 23.3 39.4 6.6 Microsomal 0.10 8.3 6.0 11.7 0.6 0.1 Soluble 0.58 48.3 1,6 3.0 524 87.3

20+ + Nuclear 0.16 27.2 2%7 65.2 44.7 14.3 Mitochondrial 0.02 2.7 12,3 27.0 17.7 5.7 Microsomal 0.01 2.0 2.2 4.9 0.5 0.2 Soluble 0.41 58.0 1.3 2.9 249 79.9

21§ Nuclear 0.26 38.2 80.9 66.0 52.6 15.3 Mitochondrial 0.12 17.7 34.5 28.2 20.9 6.0 Microsomal 0.01 1.5 4.3 3.5 1.1 0.3 Soluble 0.29 42.7 2.8 2.3 270 78.4

* On a wet weight basis. -t The intact liver contained 51.5/~g/g Hg and 1.2 yg/g Cd. :~ The intact liver contained 45.6/~g/g Hg and 0.60 #g/g Cd. § The intact liver contained 122.5 yg/g Hg and 0.68 #g/g Cd.

Mercury (cadmium)-binding proteins

Table 2. Subcellular distribution of mercury, cadmium and protein in sea lion kidney*

47

Animal Cd Hg Protein number Fraction pg/g % pg/g o/~, mg/g

197 Nuclear 1.8 16.2 4.5 47.0 38.5 25.8 Mitochondrial 2.2 19.1 1.7 18.3 29.0 1,5 Microsomal 0.50 5.5 1.5 16,1 4.4 0.3 Soluble 6.8 60.3 1.8 18.6 281 72.4

20:]: Nuclear 1.2 20.4 0.44 35.1 29.1 14.0 Mitochondrial 0.90 15.4 0.26 20.2 14.1 5.1 Microsomal 0.13 2.2 0.05 4.1 3.2 1.1 Soluble 3.7 62.1 0.51 40.1 222 79.8

21§ Nuclear 0.96 19.4 1.4 40.9 46.6 13.9 Mitochondrial 0.59 11.9 0.75 22.4 26.8 8.0 Microsomal 0.17 3,5 0.20 5.9 5.5 1.6 Soluble 3.2 65.2 1.0 20.8 257 76.5

* On a wet weight basis. t The intact kidney contained 9.6/~g/g Hg and 11.3 #g/g Cd. :~ The intact kidney contained 1.6 pg/g Hg and 6.1 pg/g Cd.

The intact kidney contained 3.2 1~g/g Hg and 3.75 ~tg/g Cd.

little Cd but a large amount of Hg in the liver and less Hg in the kidney. An appreciable amount of Se in ratios essentially equimolar to that of Hg also appeared in that fraction. The MW of the proteins in this peak were estimated at 150,000 or larger. Hemoglobin was a major constituent of peak III which contained very little Cd or Hg. A high concentration of these metals but no Se, however, appeared in peak IV (Ve/V o = 2.0-2.1), a component eluted just after horse heart cytochrome c (MW = 12,500) marker. The elution volume of the metal binding protein in peak IV corresponded to that for metallothionein from a number of other species (Margoshes & Vallee, 1957; K~igi & Vallee, 1961; Pulido et al., 1966; Jakubowski et al., 1970; Shaikh & Lucis, 1971 ; Ellis & Fang, 1971; Nordberg et al., 1972; Winge & Raja- hopalan, 1972; Weser et al., 1973; Kfigi et al., 1974). Peak V, present only in frozen tissue, apparently was a degradation product of the metallothionein Peak IV, since there was a proportional decrease in the metallothionein peak IV when peak V was present.

A typical elution profile of the 1.4 x 106 0-rain supernatant fraction from sea lion muscle on Sephadex G-75 is shown in Fig. 3. Four Hg-binding peaks and one small Cd-binding peak were observed. All of the bound Hg was exclusively present as methylmercury. The void volume peak (V~/V o = 1.0) and the second peak (V,.IV,, = 1.5) contained most of the

Hg. Very little Hg appeared in the hemoglobin peak which was eluted in between these two peaks. A third reddish-brown peak (V~/V o = 1.83) bound both Hg and a small amount of Cd, contained a high concentration of myoglobin. If metallothionein was present in sea lion muscle, it was masked by the high myoglobin content of this fraction. The fourth low MW peak (V,,/V o = 2.57) in muscle contained only a small amount of Hg and exhibited a high A2so and a low A2so.

Distribution of Cd and Hg in tissues and 1.4 x 1060-min supernatant fractions from several animals are. summarized in Tables 3 and 4. Peaks I and II and peak IV (metallothionein), were always the major areas of Hg-binding in sea lion liver and kidney supernatant fractions, while Cd was predomi- nantly localized in peak IV (metallothionein). Sea lion metallothionein thus contained 20-30~ of the Hg and 30-60~o of the Cd in kidney while only 2 - 3 ~ of the Hg and 25-50~o of the Cd in liver appeared in this fraction. Mercury concentrations tended to increase in the kidney nuclear and mitochondrial fractions with increasing concentrations of Hg in the organ but there was no similar trend for Hg in the liver or for Cd in either of those tissues.

A correlation was found between levels of metallothionein in both liver and kidney and the levels of Cd and Hg in those tissues (Fig. 4). The correlation

Table 3. Subcellular distribution of mercury, cadmium, and protein in sea lion muscle*

Animal Cd Hg Protein number Fraction #g/g o/~ ~g/g o/ o mg/g ~o

197 Nuclear 0.040 29.9 0.91 88.3 103 25.8 Mitochondrial 0.004 3.2 0.003 0.3 5.9 1.5 Microsomal 0.004 3.2 0.003 0.3 1.0 0.3 Soluble 0.080 63.8 0.11 11.1 289 72.4

20:~ Nuclear 0.05 32.7 0.36 83.9 98 25.3 Mitochondrial 0.013 8.7 0.003 0.7 0.3 0.1 Microsomal 0.015 9.6 0.006 1.4 0.8 0.2 Soluble 0.080 49.0 0.06 14.0 288 74.4

* On a wet weight basis. t The intact muscle contained 1.03/~g/g Hg and 0.12,ug/g Cd. :~ The intact muscle contained 0.43/~g/g Hg and 0.08/~g/g Cd.

48 S.S. L ~ et al.

2 , 4 I I t I t 12 end

2 . 0 .4 I 0

' i i

1.6 i 8 i

I ' a I.z I = 6 g I I

I I 0 . 8 I L 4

=L I ' , ~ 1 0 .4 I I 2

%~ 5o 7o 90 ,,o ,~o ,~o-

6 0 C i i i i t 6

5 0 0 ~ B 5

-~ 4 0 0 ~ 4

~oo , 3

=~ 200 ~ 2 i l I ]z i i /

I 0 0 I / I

% 2 0 4 0 6 0 8 0 I 0 0 12() 0 F r a c t i o n N u m b e r

Fig. 1. Separation of Hg (Cd)-binding proteins of sea lion No. 19 liver by gel filtration. A. Sephadex G-75 column (2.5 x 100cm). Five grams of tissue were homogenized in 0.01 M Tris-CL pH 8.6, containing 0.05M NaCI and 0.02 NAN3. Proteins in the 1.4 x 106 0-rain supernatant were eluted with 0.01 M Tris-C1, pH 8.6, at a flow rate of 20ml/hr. B. Sephadex G-200 (2.5 × 100 cm), 4.3 g of liver was homogenized as above and the 1.4 × 106 g-rain supernatant was applied on the column. Proteins were eluted with the homogenization buffer at a flow rate of 24.8 ml/hr. Az~o ( . . . . . ); Hg ( ~ - - - t ) ; and Cd (A A).

6 I I ~ I I I 6

5 ,~ 5 - - i i I I E I i I

I I t~ 4 # i l 4

i i i I I 1 ~ I I

t, 3 , ' I ' t l ; I ( ' ' I I 2 , , 2 --

i i I t t - ' ' ] ~ 1

0 .i ._ . " - r _ _ . , , . _ 0 5 0 5 0 7 0 9 0 I10 150 150

6 o 0 , [ , , , 16

5 0 0 B 5 i

-E 4 0 0 4 fir

3oo '/ ' o~

I : ," i 2 0 0 ~t t'~ 2 , , ; \ ,,, I ! I I I

,oe I / " I \ , ,

I / |

0 ' • ~ '0 2 0 4 0 6 0 8 0 I 0 0 17'0

F r a c t i o n N u m b e r

Fig. 2. Separation of Hg (Cd)-binding proteins of sea lion No. 19 kidney by gel filtration. A. Sephadex G-75 column (2.5 × 100 am). The experimental conditions and flow rate were identical to Fig. IA. B. Sephadex G-200 (2.5 × 100 am), 4.3 g of kidney was homogenized in 0.1 M phosphate, pH 7.5, containing 0.02% NaN3 and the 1.4 × 106 o-min supernatant was applied on the column. Proteins were eluted with the homogenization buffer at a flow rate of 15.2 ml/hr. A280 ( . . . . . ); Hg ( O - ~ O ) ; and

Cd (A--A).

coefficients for the Hg, the Cd, or the Cd + Hg bound to metal lothionein versus the total metal content of the liver are respectively, 0.95, 0.94, and 0.86, while the corresponding coefficients for sea lion kidney are 0.95, 0.97, and 0.97. These results indicate that the amount of metal bound to metal lothionein in sea lion liver and kidney increases proport ionally with increasing tissue levels of the two heavy metals. A slightly better correlation was found in the kidney than in the liver.

Characterization o f sea lion metallothionein

Further purification of crude metal lothionein (Peak IV, Fig. 2A) of sea lion kidney on DEAE-cellulose is shown in Fig. 5. About 2(~30% of the Hg and Cd washed through the column before application of the NaC1 gradient (not shown), but two forms of metal lothionein containing most of the metal were resolved by NaC1 gradient elution. Chromatography of 1.4 x 106 0-min supernatant fraction from sea lion liver on Sephadex G-75 and of the resulting peak IV on DEAE-cellulose (not shown) also revealed two forms of metallothionein, wh ich exhibited elution properties similar to those of the kidney protein. The kidney form I and II metallothioneins were purified 445- and 149-fold, respectively with respect to specific activity (Table 6), resulting in about 14 mg of the purified proteins from 100 g of tissue. Each step of the

isolation procedure was moni tored by disc electrophoresis. The two kidney forms eluted from DEAE- cellulose each gave one mtuor band by disc electro-

4 0 (

0

--r 2O(

( ,9

IO(

_ _ . t 0 5 0

I T r ~ 4 1 i

I I ' , ,5

Ill I I ! I I ! ! I I I t t I 0

I i

'L', r i t !

5

o I 0 0 150 2 0 0 2 5 0

F r a c t i o n N u m b e r

O c o tM

Fig. 3. Separation of Hg (Cd)-binding proteins of sea lion No. 19 muscle by gel filtration. Sephadex G-75 column (5 x 82cm), 150g of muscle was homogenized in 0.02M Tris C1, pH 8,6 and the 7.0 x 105 g-rain supernatant was applied on the column. Proteins were eluted with the homogenization buffer at a flow rate of 80ml/hr. A28o

( . . . . . ); Hg (e @); and Cd ( A - - A ) .


Table 4. Distribution of cadmium in sea lion tissues and soluble fractions

49

Animal Tissue number

% Cd distribution Cd in % of tissue Cd in within 1.4 x 106 ,q-min

Tissue tissue 1.4 x 106 0-min supernatant* age ( ~ t g / g ) supernatant I & II III 1V V

~o tissue Cd in metallothionein

Kidney 12 frozen 8 40.5 12 I0 69 9 ,, 20 flesh 10 95.0 18 21 61 0 ,, 19 fresh 16 56.5 8 11 80 0 ,, 17 frozen 20 50.2 7 20 54 18 ,, 18 frozen 27 74.0 15 18 50 18

Liver 4 frozen 0.52 81.5 39 8 28 25 ,, 18 frozen 2.2 41.0 33 33 24 8 ,, 11 frozen 2.55 41.0 25 17 42 17 ,, 19 flesh 3.85 79.0 21 21 58 0 , 20 fresh 5.00 55.3 7 20 72 0

31.4 57.7 45.5 36.6 48.1

43.2 13.7 23.9 46.0 39.9

* Peak numbers following Sephadex G-75 chromatography (Fig. 1A and 2A).

phoresis but some minor contamination was evident. The metal content and extinction coefficients of the

kidney forms ! and II metallothioneins are shown in Table 7. The Ezs0 for the form I protein from sea lion kidney is 1.0 x 104 l/mole cm which is similar to that observed for horse kidney Cd-thionein (Mar- goshes & Vallee, 1957). The Azso/A28 o ratio for the sea lion kidney forms I and II metallothionein were respectively, 10 and 5. A ratio of 18 has been reported (Kiigi & Vallee, 1961) for horse kidney metallothionein. Sea lion kidney form I metallothionein contained 7.2 g-atom of (Hg + Cd) per mole of protein while the form II protein contained 14g-atom of (Hg + Cd) per mole of protein.

The MW of both forms of the sea lion kidney metallothionein were estimated to be 7100 using the molecular weight calibration curve for a Sephadex G-75 column as shown in Fig. 6. Similar results (not shown) were obtained with metallothionein from sea lion liver.

DISCUSSION

Although a majority of Cd occurring in the cell cytosol of sea lion liver or kidney is associated pri-

_ 3[ , , /

- 0 40 80 120 Liver Hg (~g/g)

4 i I 1

4 8 12 Kidney Hg (~g/g)

marily with metallothionein, Hg in liver or kidney occurs mainly in the nuclear and mitochondrial fractions (Tables 1 and 2), perhaps bound to membranes. Factors such as dietary Se and protein have been sug- gested to be important in the prevention of mercury toxicity (Parizek & Ost/tdalovfi, 1967; Henrittson et al., 1969; Ganther et al., 1972). In sea lion liver (R. A. Rimerman & D. R. Buhler, unpublished results) and in grey sea lion liver (Koeman et al., 1973), most of the tissue Hg and Se occurs in the nuclear fraction and a 1:1 Hg:Se molar ratio has been found in the tissues and nuclear fractions of both marine mammals. A similar Hg-Se correlation also has been observed in the livers of nine species of marine mammals (Koeman et al., 1975). Burk et al. (1974) also found equivalent molar amounts of Hg and Se in purified plasma proteins from rats that had received simultaneous administration of HgCI 2 and NaSeO3. These investigators speculated that the Se bound to protein sulfhydryl groups and that Hg then attached to the Se.

Much of the Hg present in the cell cytosol is bound to high molecular weight proteins other than metallothionein. It is unlikely, therefore, that the latter protein is the only cellular constituent responsible for

Liver Cd (/u.g/g)

° /

0 I0 20 Kidney Cd (~g/g)

6[ l i i [

2 ~ I I ° 1 4-

°o 40 80 120 Liver Cd + Hg ( ~ g / g )

1 6 f i i

= 8

z ÷ 4

3 0 I I I

Io 2 0 3 o Kidney Cd -I-Hg (~g/g)

Fig. 4. Correlation curve between the Cd and Hg content of (MT; peak IV) and the metal content of sea lion kidney and liver.

( l i p 5 7 ' I C - - I )

50 S.S. LEE et al.

I I I I I

"~ 0.8 0.4

,,% o 0.6 ~ , ~ 0.3 8 T

"o 0.4 0.2

gg\\ /5'./; ',, 0.2 , ; . ; ] ,: o..

O, : - " l -~4-J O 20 40 60 80 ~ 1 0 0 120 Froction Number

Fig. 5. Purification of metallothionein on DEAE-cellulose column (2.5 x 52 cm). The ion exchanger was preequili- brated with 0.01 M Tris-C1, pH 8.6. Proteins were eluted from the column with a 0-1.0M NaCI linear gradient: 0-0.6 M NaCI gradient ( - - ) ; A 2 5 0 ( . . . . . ); Hg (O I) ;

and Cd ( A - - A ) .

protection of animals against high Hg concentrations. Metallothionein may, however, act as a buffer to sequester Hg and Cd into the cell cytosol in light of the work of Piotrowski & Bolanowska (1970) showing that toxic effects of Hg and Cd are evident when the amount of these heavy metals exceeds the binding capacity of the cytosol protein, metallothionein.

Metallothioneins from liver and kidney of the Cali-

fornia sea lion share several common characteristics of metallothioneins from the other sources as reported by many investigators (Margoshes & Vallee, 1957; K~igi & Vallee, 1961; Pulido e t al., 1966; Jaku- bowski e t al., 1970; Shaikh & Lucius, 1971 ; Nordberg e t al., 1972; Winge & Rajagopalan, 1972; Weser et al., 1973; K~igi e t al., 1974; Olafson & Thompson, 1974): (1) low MW of 6000-12,000; (2) V,. /V o ratio on Sephadex G-75 column of 2.O-2.1; (3) high content of heavy metals on a molar basis; (4) binding of 30-60% of the tissue Cd; (5) higher absorbance at 250 nm than at 280 nm; and (6) a high cysteine content. Preliminary studies show that sea lion kidney form I metallothionein contains at least 10 moles of cysteine per mole of protein. Cd-binding proteins with properties similar to those of metallothionein also have been detected in other aquatic organisms. The apparent MW of these proteins was 9000 and 10,000 for the grey seal (Olafson & Thompson, 1974) and fur seal, respectively (Olafson & Thompson, 1974), 11,000 for the copper rock fish (Olafson & Thompson, 1974) and 10,000-12,000 for the blue-green alga (Mac- Lean e t al., 1972).

Conflicting values, however, have been reported for the MW of metallothionein isolated from various animals. K~igi et al. (1974) and Nordberg et al. (1972) found a value of 6,600 for the molecular weight of horse and rabbit kidney metallothioneins. In contrast, MW of 10,000-12,000 have been reported for rat and mouse metallothionein (Shaikh & Lucis, 1972;

Table 5. Distribution of mercury in sea lion tissues and soluble fractions

Animal Tissue Tissue number age

% Hg distribution within Hg in % of tissue Hg in 1.4 x 106 0-rain tissue 1.4 × 106 0-rain supernatant* % tissue Hg in (/~g/g) supernatant I & II III IV V metallothionein

Kidney 17 frozen 1.11 43.4 23 8 36 33 29.9 20 fresh 1.9 48.5 50.6 8.6 50.8 0 24.6 12 frozen 5.0 52.5 45 15.0 31.0 8.8 21.0

,, 19 fresh 16.0 45.0 36.4 15 47 1.3 21.7 ,, 18 frozen 27 42.5 41.0 22 22 15.0 14.5

Liver 4 frozen 3.7 12.0 47 28 20 5 3.0 ,, 19 fresh 50 4.6 50 6.0 44 0.0 2.0 ,, 20 fresh 65.0 5.9 44 2 54 0 3.2

18 frozen 90 6.9 63 8.5 15.4 13.5 2.0 ,, 11 frozen 97 4.1 28 7.3 55 10.7 3.0

* Peak numbers following Sephadex G-75 chromatography (Fig. 1A and 2A).

Table 6. Purification of sea lion kidney metallothionein*

Total Cd Total Hg Total proteint Specific activity Steps (~g) (#g) (mg) % (#g metal/rag protein)

Tissue homogenate 40 25 880 100 0.074 1.4 x 106 0-min

supernatant 24 15 450 51 0.087 Sephadex G-75

peaks III and IV 10.7 5 16.6 2.0 0.95 DEAE-cellulose

form l metallothionein 6.2 3.3 0.29 0.033 32,9 form II metallothionein 3.4 1.1 0.40 0.045 l 1.0

* Yield from 5 g of tissue. ? Protein determined by the method of Lowry et al. (1951).


Table 7. Comparison of the metal contents and extinction coefficients metallothionein

of sea lion and horse kidney

51

Hg Cd Metallothionein (g-atom/ (g-atom/ E250

Animal form mole protein)* mole protein)* 1/mole/cm x 104

Sea lion 1 2.6 2.6 1.0 II 5.0 8.8 2.9

Horset | 0 3.0{ 0.8§

* Protein was determined by the method of Lowry et al. (1951). t Values are derived from K~igi & Vallee (1961). + Also containing 3.0g atom zinc/mote and 0.1 g atom copper/mole of protein. § Estimated on a basis of the relative ez5 o values for Zn- and Cd-metallothioneins (K~igi & Vallee.

1961).

Winge & Rajagopalan, 1972). These discrepancies appear to be due in part to the method of MW determination used. The longated shape of native metallothionein (axial ratio of prolate ellipsoid is 6) changes its elution behavior on gel chromatography relative to globular protein standards, which makes the molecular weight of metallothionein estimated by this method too high (K~igi et al., 1974). A discrepancy may also result from the method of isolation. Tech- niques using organic solvents have been shown to result in the oxidation and dimerization of metallothionein (K~igi e t al., 1974). Species differences in the amino acid composition (Weser et al., 1973) and MW (Olafson & Thompson, 1974) of metallothionein also are reported along with variations in the metal content of metallothionein isolated from different organs of a given species (K~igi et al., 19741.

The MW of sea lion kidney (Fig, 6) and liver metallothionein found in the present study was 7100. Metallothionein from other species such as the horse (K~igi & Vallee, 1961) is rich in Cd and zinc yet contains little or no Hg. The partial replacement of Cd by Hg in sea lion metallothionein would be expected to slightly increase the MW of metallothionein from

2.2

2.0

o f . 8

> 1,6

1.4

1.2

i I I I [ I

A B

C

E

I I I I I I 3.8 4.0 4.2 4.4 4.6 4.8

Log Moleculer Weight

Fig. 6. Determination of the molecular weight of sea lion kidney metallothionein on a Sephadex G-75 column (2.5 x 100cm). The MW of the proteins used were: (A) 6,600 for horse kidney metallothionein, form I; (B) 7,100 (calculated) for sea lion kidney metallothionein, forms I and II; (C) 12,500 for cytochrome c; (D) 25,000 for chymo-

trypsinogen A; and (E) 45,000 for ovalabumin.

the 6600 value reported for horse (K~igi et al., 1974) or rabbit (Nordberg et al., 1972). Binding of Hg to the protein in place of Cd or Zn also may induce conforrnational changes which influence the retention of the protein on Sephadex columns and hence the calculated MW.

Multiple forms of metallothionein also have been observed. These variants differ by their behaviour on ion exchange chromatography or electrophoresis, which indicates charge differences. Such forms could arise from differences in amino acid composition (K~igi et al., 1974) or in metal substitution. Two forms have been detected in the liver of rabbit (Nordberg et al.. 1972), man (Bfihler & K~igi, 1974), rat (Shaikh & Lucis, 1971; Nordberg et al., 1972; Chen et al., 1975a), and horse (K~igi & Vallee, 1961; Pulido et al., 1974) and in horse kidney (K~igi & Vallee, 1961; Pulido et al., 1966; K~igi et al., 1974). Two forms of metallothionein also have been observed in sea lion kidney (Table 7) and liver. Sea lion form I metallothionein may have a lower content of Cd and Hg than form II because zinc (R. A. Rimerman & D. R. Buhler. unpublished results) or other metals also may be bound to the form I protein. Such is the case in the liver of the rabbit (Nordberg et al., 1972).

Linear correlations were found between the levels of Hg and Cd in sea lion tissues and in metallothionein (Fig. 4). This is consistent with the finding that Hg or Cd induce and subsequently bind to metallothionein in liver and kidney (Piotrowski & Balanowska, 1970; Piotrowski et a l , 1974; Chen et al., 1975a). Whether the levels of metallothionein in sea lion liver or kidney actually increase in response to increasing levels of Hg or Cd cannot be determined from the present data.

The high MW Hg- and Se-binding protein(s) from sea lion liver 1.4 x 106 0-min supernatant found in the void volume on Sephadex G-75 (Fig. IA) but separated on G-200 chromatography (Fig. 1B) may be similar to Hg-binding proteins found in rat liver or kidney IChen e t al., 1973; Chen et al., 1974; Pio- trowski ctal., 1974; Chen et al., 1975b). Injection of VSSe-labeled selenite in addition to Hg 2+ (Chen et al., 1974) or Cd 2+ (Chen et al., 1975a) in rats causes a shift of metal from the metallothionein region into high molecular weight proteins which contained VSSe. The presence of high levels of Se in marine mammal liver (Koeman et al., 1973) and in sea lion liver and kidney (R. A. Rimerman & D. R. Buhler, unpublished results) may be related to the high proportion of Hg

52 S.S. LEE et al.

and Sc bound to these high molecular weight pro te ins (Fig. 1B).

Acknowledgements--We are grateful for the technical as- sistance of Ms. Valerie yon Tongeren, Judy McIntire, and Marilvn Henderson for doing metal analysis and to Drs. P. D. Whanger and T. L. Miller and Ms. Mary Rasmusson for valuable suggestions during the course of this work. This work was supported by grants No. ES-00887 and ES-00210, U.S. Public Health Service, National Institutes of Health. Manuscript issued as Technical Paper No. 4250 from Oregon Agricultural Experiment Station.

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metallothionein and the subcellular localization of mercury and cadmium in the california sea lion

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