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[5] BIOLOGICAL SOURCES OF METAL LOTH IONEI N 39[ 5 ] L a r g e - S c a l e P r e p a r a t i o n o f M e t a l l o t h i o n e i n :

B i o l o g i c a l S o u r c e sB y M I L A N V A ~, ~K

In an imals , meta l lo th ione in (MT) i s mos t abundant in parenchyma-tous t i ssues , the highest concentrat ions being found in kidney and l iver .How ever , their occurrence and biosynthesis have been d oc um en ted also inm an y other t i ssues and cell types . The absolute a m oun ts p resent in di ffer-en t species and tissues are h ighly variable, reflecting effects o f differences inage, s ta te of develop men t , die tary regime, an d other , n ot y et fully identi f iedfactors . 1 Thus, in hu m an and equine kidne y and l iver, wh ich are r ichnatural sources of MT, conc entrat io n can vary over a factor of 10 or m ore(Tab l e I2 , 3 ) . The biosynthesis of MT can be increased by heavy metaladm inistra t ion to labo ratory animals, i .e., rat an d rabbit, yielding abo ut10 mg of protein/g wet weight of t issue (Table I ) . Ca dm ium is the best M Tinducer , fol lowed by zinc. Liver is the mo st co m m on ly used source of MT ,because the MT isoforms are wel l character ized and good yields are ob-tained.

M e t a l l o t h i o n e i n I n d u c t i o n b y C a d m i u m a n d Z i n cIn m ale New Zea land White rabbi ts or Sprague-Dawley ra ts , the bio-synthesis of M T is increased by subcutane ous inject ion o f a chlor ide salt of

the appropr ia te m etal . The origina l p rocedure of Kim ura for cad m iuminjection into rabbits (21 injections ove r the t im e period o f 7 weeks)4 ma ynow be shor tened to 9 injections, 3 t im es per w eek, of sterile fi l tered CdC12(0.1 M ) in 0.15 M NaC1 at a dose of 1 m g/k g body weight. Wh ile an evenshorter t ime protocol (six injections) results in approximately 10% loweryield of M T, the use of higher levels of Cd dosing (2 m g/kg bo dy weight) orlonger induct ion per iods (more th an 3 weeks) resulted in increased rabbi tmortal i t ies. For rats, a dose of 1 mg/kg body weight injected on the f irstday, increased to 3 mg /kg bod y weight on three subsequ ent days , is used. Inspi te of the cadmium induct ion, Cd7-MT has never been isolated f rommammal ian t i s sues . The h ighes t cadmium conten t ob ta ined was about 5mo l Cd /mo l of p ro tein , the rem ainder be ing z inc.l j . H. R. K~gi and Y. Kojima, Experientia, SuppL 52, 25 (1987).2 j . H. R. K/igi, S. R. H imm elh och , P. D. W hanger, J. L. Bethune, and B. L. Vallee, J. BioLChem. 249, 3537 (1974).3 M. Su tter and M. Va~tk, unp ublishe d (1984).4 M. Kim ura, N. Otaki , and I . Imano, Experientia, SuppL 34, 163 (1979).

Copyright 1991 by Academic Press, Inc.METHODS IN ENZYMOLOGY, VOL. 205 All rights of reproduction in any form reserved.

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4 0 ISOLATION OF METALLOTHIONEINS [5 ]

TABLE IBIOLOGICAL SOURCES OF METALLOTH1ONEIN

Concentration MetalTissue [mg MT/g (wet weight)] a indu ctio n b Ref.

Hu ma n liver 0.1 - 15 - - 2Hor se liver 0.1 - 10 - - 2Horse kidney C 0.5- 10 -- 2Rabb it liver 13 Cd 3Rat liver 10 Cd 3

a Both electrophoretically different isof orms MT- I andMT-2 are considered.

~' Metal ind uct ion as described below.' Isolated from kidney cortex.

The induc t ion of MT by z inc is the m etho d o f choice if a m eta l -homo-geneous Z n-M T form is required. Since z inc is far less toxic than cad miu m ,m uc h larger am ou nts o f this m etal mu st be adminis tered. For rabbits, aproced ure s imilar to that d escr ibed for ca dm ium induct ion (see above) isemployed, the only except ion being a dose of 10 mg Zn/kg body weight .The y ield of Zn -M T is usua lly 20 -30 % lower than tha t o f cadm ium- in-duced Zn,Cd-MT (Table I) . For rats, no established protocol for a large-scale preparat ion of Zn -M T exists . In a l l induc t ion protocols the an imalswere sacrif iced on the day following the last injection and the l ivers weres tored a t -70 . I t should be no ted , moreover , tha t meta l -homogeneousZn -M T can a lso be genera ted f rom Zn,C d-M T by the meth od of meta lrecon sti tut ion as described elsewhere in this volum e. 5

C o m m e n t sBecause of the large varia t ion of M T concen trat ions in t i ssues wi thou tmetal in duct ion (Table I ), i t is advisable to es t imate thei r MT con tent pr iorto a large-scale pro tein isolation. Fo r this purp ose th e first prep arativ e step,up to and including the second alcohol precipi ta t ion s tep and pel le t lyo-phil ization, as described later in this volume,6 should be per formed on25 g of t issue. T he crud e fraction is subsequ ently take n up in a kn ow nvo lum e (about 2 ml) o f 20 m M Tris-HC1 buffer, pH 8.6, an d th e zinc,cadmium, and copper conten t de te rmined by f l ame a tomic absorp t ion5 M. Va ~k, this volume [54].6 M. V a~k, this volu me [6].

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[6] STANDARD ISOLATIONPROCEDURE 41spectroscopy. 7 At this preparat ive s tage the m etal ions are p redo min ant lybound to MT; therefore , the determined metal content is a reasonablemeasure o f the M T concent ra t ion present . Al though l iver MT s f rom adul tsusual ly contain a lmost exclusively z inc ions , the presence of large am ou ntsof copp er can som etime s be encountered. A special isolat ion procedu re isrequired to pu r i fy the oxygen-sensi t ive Cu(I)-M T. 87 M. C zupry n and K. H. Fa lchuk , th i s vo lu me [47].8 R. J . S tocker t , A . G. More l l , and I . S te rn l ieb , th i s vo lum e [34] .

[ 6] S t a n d a r d I s o l a t i o n P r o c e d u r e f o r M e t a l l o t h i o n e i nBy M I L A N V A ~ A K

As in the prepa rat ion of any metal loprotein , the presence o f adven-t i t ious metal ions should be kept to a minimum. All solut ions should bepassed through a Chelex 100 colu mn (Bio-Rad, Ric hm ond , CA ) to rem ovemeta l con taminants and labware rendered meta l f ree by ac id wash ing)The large-scale isolat ion and purif icat ion of metal lothionein (M T), a cyto-solic protein, involves alcohol precip itation followed by size-exclusion andanion-exchange chromatography. This procedure is used to isola te MTfrom hum an, ra t, and rabbi t l ivers 2 and is basical ly s imilar to that pub-lished b y K~igi et al. for hu m an liver, and horse l iver and kidn ey M T. 3,4 Alloperat ions w hen not otherwise indicated are carried ou t a t 4 .

M e t a l l o t h i o n e i n I so l at io n f r o m R a b b i t L i v e rStep I. Preparation o f Crude Fraction. Fresh of half-frozen liver from

rabbi ts in which the M T concen tra t ion has been increased by zinc orcad miu m ind uc t ion : a re cu t in to smal l p ieces and hom ogenized in por -t ions o f 250 g in a l - li ter Waring blend or (30 sec) with 500 ml o f 20 m MTris-HCl buffer at 4 . The hom ogen ate is p laced in a 4- li ter beak er im-mersed in a water / ice ba th and equi pped with an effic ient s ti rr ing arrange-ment . An equa l vo lu me of a 96% e thanol /ch loroform so lu t ion (1 .05 :0 .08 ,

B. Holmquist , th is ser ies , Vol. 158, p . 6 .2 M . S u t t e r a n d M . V a ~ k , u n p u b l i s h e d ( 19 84 ).3 j . H . R . K~g i , S . R . H im me lho ch , P . D. Wha nger , J . L . Be thu ne , and B. L . Va l lee , J . Biol.Chem. 249 , 3537 0974) .4 R. Btih ler and J . H. R. K~igi , FEBS Lett. 39, 229 (1974).

M . V a ~ k , t h i s v o lu m e [ 5] .

Copyright 1991 by AcademicPress, nc.METHODS IN ENZYMOLOGY, VOL. 205 All rightsof reproduction n any form reserved.