Separation of metallothioneins in rat liver, kidney, and spleen using SW and Sephadex columns

ANALYTICAL

107.

BIOCHEMISTRY

Separation

75-85

( 1980)

of Metallothioneins in Rat Liver, Kidney, and Spleen SW and Sephadex Columns

K~zuoT.Suzurc~.

TAKAKO MOTOMURA,YUKOTSUCHIYA,AND

Received Metallothioneins spleen

were

and separated

used as a gel permeation concentration of metals flame same

atomic elution

other both

column in eluate

it was explained ation of hydroxyl

by the cation groups of

plates. retention

The

3000

and

’ Abbreviation

were apparently

HI-C-AAS, absorption

1980

except

G-75

rat

columns.

a high-speed determined Metalloproteins for the separation

liquid

also

exchange chromatographic polyhydroxylated gel

ofeluting resolution

buffer solution of metallothioneins.

high-speed spectrophotometer:

liquid

was concluded

kidney.

and

column

was

chromatograph and the by directly connecting to a

were generally of metallothioneins

property materials at

separated into the isometallothioneins eluated as a single peak due to shortage

liver,

The former

several unidentified zinc proteins. Slower elution were observed on a SW column for neutral and acidic

pH value times and

used:

MITSURU YAMAMURA

in cadmium-exposed

Recently we have explored a new analytical method for the separations of metallothioneins and suggested that the method can be applicable as an analytical method for genera1 metalloproteins (1). The new analytical method consists of a combination of a high-speed liquid chromatograph equipped with a gel permeation column and a flame atomic absorption spectrophotometer (HLCAAS).’ The outlet of a column was directly connected to a nebulizer tube of a flame atomic absorption spectrophotometer for simultaneous and continuous determination of metal concentration in eluate. A gel permeation column (TSK GEL SW 3000) used for the demonstration for separations of metallothioneins was indicated to have both gel filtration and cation exchange chromatographic properties when an alkaline buffer

chromatograph-atomic SIN, signal/noise.

4.

Sephadex

equipped with was simultaneously

absorption spectrophotometer. orders on the two columns and

metallothioneins proteins were

metalloproteins

on SW

metallothioneins of metallothioneins

February

Using

into

at the iso-

and poorer resolution buffer solutions, and

caused alkaline

on a Sephadex in the numbers to be a main

eluted

factor

by the dissocipH. Although column. the of theoretical which

affects

solution was used as an eluting buffer solution. As a result, metallothionein in rat liver supernatant obtained after injection of cadmium was analyzed as the two isometallothioneins within an hour at a concentration of less than 1 pug Cd or Zniml for an application of a l-ml aliquot. The new analytical method was applied to characterize metallothionein dimers which are observed as minor components when tissues accumulate cadmium in a large quantity (2). and also to characterize kidney metallothioneins induced by injections of metallothionein or earthworm cadmium-binding proteins (3,4). The present study was intended to compare the relationships for separations of metalloproteins between SW and Sephadex columns. At the same time, this study also focused on getting an insight into the mechanism of why a SW column has not only gel filtration but also cation-exchange chromatographic properties, and to find a better

76

SUZUKI TABLE CONCENTRATIONS

OF

I MEIAIS

IN TISSUE

SUPERNATANTS

Metals Tissues

Zn

Cd

Cu

Fe

Ca

Mg

Liver Kidney Spleen

14.2” 5.4 3.1

21.7 11.1 2.2

1.3 7.7 0.3

32.0 9.2 96.0

1.6 4.9 2.7

24.6 24.0 23.0

ET AL

length uv detector (Altex Model 152) and a conductivity meter (M & S Instruments Inc., Model CD-35 M II), respectively. Fractions (5 ml) were collected and concentrations of metals were determined by flame atomic absorption spectrophotometry (Hitachi Model 170-50 A) in each eluate. Gel Prmzeutiotl s w CoIlrttltl

ChrotncrtogrL1pIl?’

ot1 (I

The outlet of a high-speed liquid chromatograph (Toyo Soda HLC Model 803 A) equipped with a gel permeation column (TSK GEL SW 3000, Toyo Soda, 21.5 x 600 procedure for the separation of metallothimm with a precolumn (21.5 x 100 mm)) oneins into isometallothioneins. was directly connected to a flame atomic absorption spectrophotometer (Hitachi Model MATERIALS AND METHODS 170-50 AI. The tissue supernatants were applied in a I-ml or 500-~1 portion and eluted with 50 mM Tris-HCI buffer solution (pH 8.6 at 25°C. dissolved gas was removed at Cadmium chloride was injected subcuta80°C under reduced pressure) containing neously into female rats of the Wistar strain 0.1% sodium azide at a flow rate of 3.7 (mean body weight 298 g) 12 times over 18 mlimin. Molecular absorbances at 254 and days (total amount of injected cadmium, 1.5 280 nm, and atomic absorbance of one of the mg Cd/rat). The animals were sacrificed by metals (Cd. Zn, Cu, Mg. and Fe) or exsanguination 10 days after the last conductivity (mmho) were recorded on a injection. The livers, kidneys, and spleens Model PC-3 were pooled and homogenized using a three-pen recorder (Rikadenki Teflon homogenizer in 3 vol of 0.1 M with pen gap adjustment memory). Tris-HCI buffer solution (pH 7.4 at 25°C) containing 0.25 M glucose under nitrogen RESULTS gas. The homogenates were centrifuged at 170,000,~ for 60 min. The concentrations of Figure 1 shows typical elution profiles of metals in each supernatant were determined representative metals in cadmium-exposed after dilution with doubly distilled water by rat liver supernatant on a Sephadex G-75 flame atomic absorption spectrophotometry column. Three cadmium peaks were obwith background correction (Shimadzu served: the main cadmium peak, metallothiModel 640-12). The data are listed in onein. was eluted at tube 58 and accomTable 1. panied by zinc with absorbance at 254 nm but not at 280 nm. A small cadmium peak at Scphades G-75 Colutm Chron~cltogrnpll? tube 48 was assigned to be metallothionein The tissue supematants (4 or 8 ml) were ap- dimers which were observed when tissues cadmium in a large quantity plied to a Sephadex G-75 column (2.6 x 90 accumulated cm) and eluted with 1 mM Tris-HCI buffer (2). The third cadmium peak which was solution (pH 8.6) at a flow rate of 62 or 22 bound nonselectively to high molecular ml/h. Molecular absorbances at 354 and 280 weight proteins was eluted at the void volume. Iron was mostly eluted at the void nm and conductivity were continuously volume along with a small peak at tube 36; monitored using flow cells by a dual wave‘1 &ml

supernatant.

SEPARATION

OF

77

METALLOTHIONEIN

i ty

FIG. aliquot

I. Gel filtration ofcadmium-exposed

and eluted sorbances

chromatograms rat liver

with 50 mM Tris-HCI at 254 and 280 nm and

were determined metallothionein

in each fraction.

eluate

of rat supernatant

buffer solution conductivity by

atomic

liver supernatant was applied

on a Sephadex to a Sephadex G-75

G-75 column. column (2.6

(pH 8.6). Five-milliliter fractions were were continuously monitored. Concentrations absorption

those were tentatively assigned to be ferritin and hemoglobin, respectively. On the other hand, magnesium was mostly eluted as free ion or low molecular weight complexes. Substances eluting slower than conductivity peak at tube 88 were observed, and can be explained by hydrophobic interactions. Figure 2 shows gel permeation chromatograms of representative metals in cadmiumexposed rat liver supernatant on a SW 3000 column. Cadmium and zinc bound to metallothionein were separated into two peaks: metallothionein I at a retention time of 39.6 min and metallothionein II at a retention time of 37.2 min ( I ). Iron peaks at retention times of 22.6 and 28.8 min correspond to ferritin and hemoglobin peaks on a Sepha-

ypectrophotometry.

The

arrow

A 4-ml x 90 cm)

collected. Abof metals indicates

the

dex G-75 column (Fig. 1). Zinc- and coppercontaining peaks at retention times of 30.8 and 3 1.O min correspond to the peak at tube 42 on a Sephadex column, which was tentatively assigned as superoxide dismutase ( I). Four unidentified zinc peaks were observed at larger retention times than metallothionein peaks. Those peaks were found to originate from high molecular weight zinc proteins (which were eluted around at tube 33 in Fig. I). The zinc proteins were unstable to heat treatment (80°C for 10 min) except for one at a retention time of 44.0 min. Magnesium did not give a reliable elution profile. Figure 3 shows typical elution profiles of representative metals in cadmium-exposed nit kidney yupernatant on a Sephadex G-75

78

SUZUKI

Retention

Tim

ET AL

( min )

FIG. 2. Gel permeation chromatograms of rat liver supernatant on SW 3000 column. A I-ml (0.5 ml for Cd) aliquot ofcadmium-exposed rat liver supernatant was applied to a high-speed liquid chromatograph equipped with a TSK GEL SW 3000 column (21.5 X 600 mm with a precolumn (21.5 x 100 mm)) and eluted with 50 mM Tris-HCI buffer solution (pH 8.6 at 25°C. containing 0.1% NaN,) at a flow rate of 3.7 mlimin. Molecular absorbances at 254 and 280 nm and one of the atomic absorbances (Fe. Zn. Cd, Cu, and Mg) or conductivity were continuously monitored using a three-pen recorder. Molecular absorbances and conductivity (mmho) were recorded at arbitrary units. Concentrations of metals were recorded by setting a detector of an atomic absorption spectrophotometer at a concentration of 0.1 &ml as indicated by individual bars.

Figure 4 shows gel permeation chromatosupernatant (Figure grams of representative metals in cadmiumwas low and that of copper was high in the exposed rat kidney supernatant on a SW metallothionein fraction as already reported 3000 column. Striking differences were obt-5). Iron peak at the void volume (ferritin) served for the three metals in the kidney was lower and calcium peak in the low mo- metallothionein fraction from those in the lecular weight fraction (free ion and low mo- liver metallothionein fraction. Although liver lecular weight complexes) was higher than metallothionein isolated on a Sephadex those in liver supernatant. G-75 column was separated only into two column. In contrast

to the profiles I), the amount

in liver of zinc

SEPARATION

OF

79

METALLOTHIONEIN

/

2.

T.

30

40

50

60

70

in the

3. Gel filtration of cadmium-exposed legend

of Fig.

chromatograms rat kidney 1. The

arrow

indicates

Retention 4. Gel permeation of cadmium-exposed with a SW 3000

80

the

100

90

of rat kidney supernatant supernatant was applied

nm

I

110

120

on a Sephadex G-75 column. A 4.ml to a Sephadex G-75 column as indicated

metallothionein

III

FIG. aliquot equipped

280

r’

Number

Fraction FIG. aliquot

.,

,I

-8

Time

fraction.

I II 39.6

(

min

)

chromatograms of rat kidney supernatant on a SW rat kidney supernatant was applied to a high-speed column as indicated in the legend of Fig. 2.

3000 liquid

column. A I-ml chromatograph

80

SUZUKI

ET

AL

Fe ‘254

-.

to

09 -CT - sbr-70 Fraction

FIG. aliquot cated

5. Gel

filtration

chromatograms

of cadmium-exposed in the legend of Fig.

rat spleen 1. The arrow

280

/

of rat spleen supernatant indicates

peaks, kidney metallothionein isolated on a Sephadex G-75 column was separated into three peaks on a SW column as observed for kidney supernatant: Isometallothionein peak at a retention time of 39.0 (Cu), 39.4 (Cd), or 39.6 min (Zn) which corresponds to metallothionein I was biggest and isometallothionein peak at a retention time of 36.6 (Cu). 36.8 (Cd), or 37.2 min (Zn) which corresponds to metallothionein II was smaller than that of metallothionein I (the three metals had to be eluted at the same retention time when the conditions were strictly controlled or when the three metals were determined simultaneously). A third peak at a retention time of 42.6 (Cu) or 43.0 min (Cd, Zn) was observed other than the two isometallothionein peaks. Although the third peak has never been observed in the liver metallothioneins induced by cadmium loadings, it has been observed in the kidney metallothioneins induced by cadmium loadings. An in \sitw study indicated that the third peak was observed by replacement of zinc and/or cadmium in liver metallothionein with cupric ion (unpublished observation).

lo

nm nm

40 --r-c-07

Number supernatant was applied the metallothionein

on a Sephadex

G-75

column

A 4-ml

to a Sephadex fraction.

G-75

column

a9 indi-

An intermediate chromatogram between a typical liver metallothionein profile and a typical kidney metallothionein profile was observed when kidney metallothionein was induced by injections of cadmium-thionein (3) and earthworm cadmium-binding protein (4). Magnesium in the kidney supernatant did not give a reliable profile as also observed for liver supernatant. Although spleen is an important organ for immune responses upon cadmium loadings, metallothionein in spleen has not been studied in detail probably due to a low accumulation of cadmium in spleen and due to the small organ size of spleen (6). The amount of cadmium found in spleen supernatant was about l/lOth (wet weight ratio) that found in liver supernatant and the cadmium was mostly found in the metallothionein fraction (Fig. 5). Zinc and copper in the metallothionein fraction were low in amount in comparison with the amount of cadmium. The amount of iron in spleen supernatant was extremely high and the metal was found to be present as ferritin and hemoglobin. Gel permeation chromatogramsof cudmium-

SEPARATION

Retention

OF

81

METALLOTHIONEIN

Time

(

min

j

FIG. 6. Gel permeation chromatograms ofrat spleen supernatant on a SW 3000 column. of cadmium-exposed rat spleen supernatant was applied to a high-speed liquid chromntograph with a SW 3000 column as indicated in the legend of Fig. 7.

exposed rat spleen supernatant indicated that metallothionein in spleen supernatant was separated into two isometallothioneins as observed for liver supernatant. The ratio of metallothionein I to metallothionein II was at an intermediate between those of livei and kidney metallothioneins. An elution profile of magnesium in spleen supernatant was again not reasonable and most of magnesium in the low molecular weight fraction on a Sephadex G-75 column was not eluted on a SW column. The following experiments were performed to get an insight into the mechanism of why metallothionein is separated into isometallothioneins in a cation exchange mode at alkaline buffer solution; why metallothionein I of higher isoelectric point is eluted slower than metallothionein II of lower isoelectric point. First, the effects of pH values of elut-

A I-ml aliquot equipped

ing buffer solutions on the separations into isometallothioneins were investigated. When the pH value of eluting buffer solution was decreased from 8.6 to 7.4 (50 mM Tris- HCI buffer solution, containing 0.1% NaN,,), retention times of isometallothioneins both in liver and kidney supernatants increased more than 5 min on a SW 3000 column (compare Fig. 7 with Figs. 2 and 4). In contrast to changes of retention times for metallothioneins, retention times of other proteins changed only slightly as sugested from retention times of the void volume and the slower eluting peaks than metallothioneins. Furthermore, the resolutions of isometallothioneins became poor when the pH value of eluting buffer solution was changed from alkaline to near neutral pH a\ observed in Fig. 7. When the pH value of eluting buffer solu-

82

SUZUKI

F~ti. as eluted

7. Gel

permeation

with

50 mM

chromatograms Tri-HCI

buffer

supernatants was applied to a high-speed indicated in the legend of Fig. 2 except 7.4 at 25°C) as an eluting buffer

of rat solution

ET

AL

liver

and

kidney

supernatants

( pH

7.4

at 25°C).

A I-ml

liquid chromatograph for using 50 mM Tri-

tion was further changed to weakly acidic pH (citric acid-phosphate buffer (7). pH 6.1. containing 0.1% NaN:,), no more separations of liver and kidney metallothioneins were observed on a SW 3000 column (Fig. 8) and metallothioneins were eluted at a slower rate than those eluted with alkaline and near neutral buffer solution. As the above experiment suggested that molecules of different isoelectric points are separated on a SW column in a cation exchange mode by ionized hydroxyl groups of gel materials at alkaline pH, cation exchange chromatographic properties of a Sephadex G-75 column (which is also packed with polyhydroxylated materials) were investigated as follows. Figures 9 and IO illustrate changes of isometallothionein ratios with eluate fractions on a Sephadex G-75 column as analyzed on a SW 3000 column. Elution profiles of the three metals related to metallothionein in cadmium-exposed rat liver supernatant were obtained using the same conditions as illustrated in Fig. I. A I-ml aliquot of the representative five fractions (tube numbers 58, 59, 62, 6S, and 66) was applied to a SW 3000 column and cadmium atomic absorption was continuously recorded as shown in Fig. 9. The faster eluting frac-

equipped HCI buffer

on aliquot

a SW of liver

3000 and

column kidney

with a SW 3000 column solution of lower pH

a, ( pH

tions (tube numbers 58 and 59) were rich in metallothionein I1 (isometallothionein of lower isoelectric point) and the slower eluting fractions (tube numbers 65 and 66) were rich in metallothionein I (isometallothionein of higher isoelectric point). The result suggested that metallothionein can also be separated into two isometallothioneins on a Sephadex G-75 column at alkaline pH. though the peak appears as a single peak due to shortage in the numbers oftheoretical plates. Figure IO illustrates the results obtained by using cadmium-exposed rat kidney metallothionein in which copper is the second metal instead of zinc in liver metallothionein. The kidney supernatant was eluted at a slower rate on a Sephadex G-75 column because of the application of two times the volume used for liver supernatant. A I-ml aliquot of the representative eluate fraction4 (tube numbers 70, 71, 74. 77, 78. and 79) was applied to a SW 3000 column and cadmium atomic absorption was continuously monitored. The faster eluate fractions on a Sephadex column (tube numbers 70 and 7 I ) were again rich in metallothionein II and the slower eluate fractions were rich in metallothionein I. The third peak which is observed when metallothionein is rich in copper

SEPARATION

OF

METALLOTHIONEIN

as observed in Fig. 4 was eluted at the slow est rate among the three peaks. Although the direct application of liver supernatant to a SW column and elution with acidic buffer solution (citric acid-phosphate buffer, pH 6.2) resulted in a sharp and single cadmium peak as illustrated in Fig. 8. the application of metallothionein obtained from a Sephadex G-75 column with acidic buffer solution resulted in broad cadmium peaks on a SW column. This is probably due to the instability of zinc in metallothionein at acidic pH for a prolonged procedure on a Sephadex G-75 column and further investigation was not performed to determine whether or not metallothionein is separated into isometallothioneins on a Sephadex column with acidic buffer solution.

. .

-30

for

40 Fraction

FIG. 9. Comparisons separations of

aliquot applied

50

with

50 rnM

70

of Sephadex and liver metallothionein.

rat

of cadmium-exposed to a Sephadex

eluted

60 Number

G-75

Tris-HCI

SW

columns A 4-ml

rat live]- supernatant was column (2.6 * 90 cm) and buffer

solution

tpH

8.6

at 25°C) at a Row rate of 62 ml/h. Five-milliliter fractions were collected. Metallothionein was eluted at around tube 62. A I-ml aliquot of tubes 58, 59. 62. 65.

and

66 on

the

Sephadex

G-75

column

was

applied

to a high-speed liquid chromatograph equipped with SW 3000 column (21.5 ti 600 mm with a precolumn (2 1.5 x 100 mm)) and eluted under the same conditions as described metallottiionein Liver

in the I and

legend

of Fig.

II.

respectively

2. I and

a

II indicate

Suwrnatant

DISCUSSION

Retention

Time

( min )

FIO. 8. Ccl permeation and kidney \upernatantx eluted with citrate-phosphate

chromatograms of rat live1 on a SW 3000 column a\ buffer solution I pH 6.2

at 25°C).

of liver

A

I-ml

aliquot

natants was applied to a high-speed graph equipped with a SW 3000 in the legend of Fig. 2 except phosphate buffer solution t pH elutiny huffer.

and

kidney

suprr-

liquid chromatocolumn as indicated for using citrate6.2 at 2YC) as an

Although elution profiles of metals and proteins on a Sephadex G-75 column were obtained by detecting concentrations of all metals and absorbances in each eluate for a single application of individual supematants. elution profiles of metals on a SW 3000 column were obtained by monitoring atomic absorbance of single metal for every application of the same supernatant. Elution profiles (on a SW 3000 column) of the same metal in different tissue supernatants were obtained on the same day using the same lot of eluting buffer solution. But elution profiles of different metals were recorded on different days using different lots of eluting buffer solution. Therefore. differences in re-

84

SUZUKI

ET

Fraction FIG.

10. Comparisons

of Sephadex

and

SW

AL

Number columns

for

separations

of rat

kidney

metallothionein.

An g-ml aliquot of cadmium-exposed rat kidney zupernatant (2.6 x 90 cm) and eluted with 50 mM Tris-HCl buffer solution

was applied to a Sephadex ( pH 8.6 at 25°C) at a flow

Five-milliliter

eluted

fractions

of tubes 70,71.74,77.78, I. II. and III indicate

were

collected.

and 79 was metallothionein

Metallothionein applied I. II,

was

at around

to a SW 3000 column as described and the third peak. respectively.

tention times of the same molecule recorded by monitoring atomic absorptions of different metals and molecular absorbances showed the reproducibility of the present analytical method (for example, small changes of retention times for isometallothionein peaks of cadmium, zinc, copper, and absorbance at 254 nm). The pH value of eluting buffer solution changes with temperature and the temperature of a column is not easy to control due to the use of flame atomic absorption spectrophotometer near the column. Retention times of metallothioneins are susceptible to the changes of pH value of eluting buffer solution as illustrated in Figs. 7 and 8. Changes of each 0.1 pH unit between pH 6.2 and

tube

74.

in the

G-75 column rafe of 22 ml/h. A I-ml legend

aliquot of Fig.

2.

8.6 caused the change of retention time at a rate of about 0.5 mini0.1 pH unit. A small change of pH value for every preparation of buffer solution may also have caused the small changes of retention times as observed in the present study. Although the details of the chemical structure of the SW gel material are unknown (columns are packed with microspheres of hydrophilic polymer gel which are coated on a silica gel), the present observation suggests that a SW column and a Sephadex column are same with respect to separation mechanisms; molecules of different molecular weights are separated by different elution rates through microspheres of hydrophilic polymer gels and molecules of different isoelec-

SEPARATION

OF

85

METALLOTHIONEIN

tric points are separated in a cation exchange mode by ionized hydroxyl groups at alkaline solution. The present results suggested that an application of an aliquot of metallothionein obtained on a Sephadex G-75 column may lead to different isometallothionein ratios from the genuine ratio when only a part of metallothionein fractions is pooled and applied to an ion exchange column; a faster eluting metallothionein fraction on a Sephadex G-75 column is rich in metallothionein II and a slower eluting metallothionein fraction is rich in metallothionein I. Therefore, the formet fraction gives lower. and the latter fraction gives higher ratios of metallothionein 1 to metallothionein II. A slower elution rate of copper-containing metallothioneins than metallothioneins with low or no copper on a Sephadex G-75 column was reported and it was explained by a more compact conformation due to replacement of zinc and/or cadmium by copper (8). The present results indicated that another explanation may be more suitable for the different elution rate: namely. the apparent slower eluting rate of copper-containing metallothionein on a Sephadex G-75 column is due to the low ratio of metallothionein I to metallothionein II and the presence of the third isometallothionein which is eluted at the slowest rate both on Sephadex and SW columns. Faster elution of liver metallothionein than kidney metallothionein on a Sephadex G-75 column can be explained as follows: liver metallothionein is a mixture of an almost equivalent amount of metallothionein I and II. On the other hand, metallothionein I1 is present as minor isometallothionein and the third isometallothionein is present in the kidney metallothionein. Therefore. kid-

ney metallothionein consists of slower eluting isometallothioneins than liver metallothionein on a Sephadex G-75 column. As we have suggested in the previous report (1). the detection limit for metallothioneins by the new analytical method (HLCAAS) depends on an atomic absorption spectrophotometer. The distribution profiles of metals presented in this report showed far better S/N ratios than those of previous ones. This is primarily due to the better S/N ratio and flame stability of the atomic absorption spectrophotometer used in the present study. Cadmium. zinc, and copper bound to metallothionein can be easily analyzed as isometallothioneins at a concentration of less than 0.3 pug/ml supernatant for an application of 1 ml of solution. Further study using an analytical column (7.5 % 600 mm) of SW 3000 revealed that cadmium in metallothionein was detectable as the two isometallothioneins with an application of 78 ng (0.25 nmol) Cd in 0.1 ml of solution. ACKNOWLEDGMENT We

thank

E;. Kuhota

Dr.

encouragement.

for

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K. T. (1980) A~tri. Biwirc,v~. 102, 31-34. and Yamamura, M. (1980)Bioc~lzrrn.

?.. Suzuki. K. T..

P/~trrrmrc~o/. 29. 689-692. 3. Suzuki, K. T.. and Yamamura. 4.

LPU. Suzuki,

Arclr. 5.

Suzuki,

5, 131-138. K. T.. Yamamura.

Etr~~iror~.

M. (1980) M..

Corrtorrr.

K. T. ( 1979)

Arc,ll.

and

To.rico/.. E~vif,w.

~,.I;c~I~/.

Mori. T. ( 1980) in press. (‘onttrrr~. T~,.ri-

c.01. 8, ‘55-168.

6. Amacher. Etl\‘irm.

D.

E..

Hc~trltll

7.

and

Ewing.

30. 5 IO-5

K. I.. (1975) ilr-r.11. 13. D. C.. Elliott. W. H., (1969) in Data for Bioed.. p. 484. Clarendon

Dawson. R. M. C., Elliott, and Jane\. K. M. (eds.). chemical Research. 2nd. Press, Oxford. 8. Suzuki, K. ‘I‘.. and Yamamura.

E~r~~i,z,~r. (‘c)uttrt,t.

M.

(1979)

Tc~\-/c~d. 8. 471 -485.

Arc,lr.