Purification and properties of α-actinin from rabbit skeletal muscle

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..xxt~ .MARVIN 11. STR()MI.II¢ l)cpartmcnt of Z~ioche,nist O, and I¢iophyslcs, lo~,,a State I :nt;,crsily, .4 m,'s, Iowa 5oo.lo ( 1 '.S..-I.) {Received J u l y zSth, xge,9)

FA.T.MM A Rh"

The ()-S a-actinin species can be purified from a Pls-o.s a-actinin fraction by l)EAF.-cellulose clmmlatography. The resulting P1-,-2-, (I)EAE) fraction is eluted as a single peak upon rechromatography on 4(~o agarose or l)EAE-cellulose colunms, although the rechronmtography on DEAE-cellulose removes a very small amount of aggregates from the P15-~.~ (DEAE) fraction. Sedimentation diagrams of the P,..., .,~ (1)EAE) fraction show that approx. 85°;, of the protein in this fraction sediments with an s:20.,.-~ 0.23 and about IO-IS°'{~ sediments with an observed s value of 9.I. "l'he ~).I-S component may be an aggregate of the 6.2-.q species. The P~,~-,,a (I)EAE) fractitm exhibits 2-3-fold higher specific activity in the turbidity assay of ,t-actinin activity than the original Plc,-2.s fraction. Amino acid composition of the Pls- ',s (DEAE) fracticm is clearh: different from the amino acid composition of actin, demonstrating that a-actinin is a separate protein ccmq)onent of the myofibril and is not sinlply all unusual form of denatured actin. These results also show that the 6-S a-actinin species does not exhibit marked aggregating tendencies and that the large aggregates prevalent in earlier a-actinin preparations were probably due to the presence of denatured re'tin in these preparations. By using purified a-actinin, it was shown that the stoichiometrv - f the a-actinin- F-actm interaction is o.4I parts of Pv~-.,a (DEAE) to i part of l:-actin. This corresponds to a molecular rati¢~ of ,ne a-actinin to ten (i-aetin monomers. "fropomyosin and a-actinin compete fi~r the same or closely located binding situs ~m actin, but at o:, ¢t-actinin appears able to displace tropomyosin from l:-actin. The presence of a-actinin was demonstrated in low-ionic-strength extracts , f ,
• J o u r n a l P a p e r No. J-~L~I7 ~1 t h e I o w a A g r i c u l t u r e a n d H o m e E c o n o m i c s 1Lxpcriment S t a t i o n , P r o j e c t x549. T h i s p a p e r is t a k e n f r o m a d i s s e r t a t i o n b y 1(..M. t¢.¢mso.~ s u b m i t t e d t~, I o w a S t a t e I ; n i v c r s i t y in p a r t i a l f u l t i l h n e n t of t h e r e q u i r e m e n t s t(w t h e t'h. 1). degree. "" l ' r c s c n t a d d r e s s : Musch' l~iochemistry ( ; r o u p , t 2 4 A n i m a l F,cicnces I . a b o r a t o r y , I ' u i v e r sity o f Illinois, l ' r b a n a , Ill. (~tSoX. "'" P r e s e n t a d d r e s s : D e p a r t m e n t of l:t~od ('hcllllStl'y, ()ch~lllOli)izu I.'nivursity, l h m k y o - K u , l'~,k y~,, J a pan. lliochim, l l i , , p h w . Acta. e~)o I J~U~ ) ~,qt, 3w.~

ct-ACTININ PURIFICATION AND PROPERTIES

297

Z-line constitutes about 6% of the dry mass of the myofibril, but a-actinin makes up only about I ° o of the myofibrillar protein, the Z-line is probably composed of substances in addition to a-actinin.

INTROI)UCTION

Investigations on the role of a-actinin in muscle have been hampered by lack of homogeneity in the usual a-actinin preparationsa, 2. GOLL and co-workers1, a and MASAKI et al. 4 have reported evidence that suggests that a-actinin is located in or next to the Z-line in striated muscle. Moreover, STRO.~.IER and co-workersS, ° flmnd that a protein fraction salting out between o and 4o% (NH4)2SO 4 saturation, a range in which a-actinin also salts out, would reconstitute Z-lines in Z-line-extracted fiblils. "l'hese results imply that a-actinin has a structural role in muscle, acting to bind actin filaments from adjacent sarcomeres across the Z-line 1. Since structural proteins often exhibit pronounced aggregating tendenciesT, 8, it is not clear whether the large aggregates that GOLL ct al? and NoxO.~t:~A n have described in ~t-actinin preparations ,nade according to EBASHI AND EBASHI~ o r SERAYDARIAN t'l al. ~° are merely aggregated forms of the active 6-S a-actinin component, which is inactive in the aggregated statO ,1', or whether they represent other protein species such as denatured (;-actin a. Attempts to puri~" conventional a-actinin preparations'a, 1° have met with onh" limited success, probably because these preparations contain only 5-IO~'o of the active ~-S a-aetinin species ~,~a. Recently, wO a have succeeded in preparing an initial a-actinin extract that contains 25 -3o°'0 of its protein as the 6-S a-actinin component and which exhibits 5 6 times higher specific activities in the ATPase and turbidity tests for a-actinin activitva, ~°. The present communication reports the further purification of this new a-actinin extract, which we have termed the Pas-2s fraction, and describes some properties of purified a-actinin. A preliminary report of this work was given at the I969 Biophysical Society meetings a4. MATERIALS AND METHOI)S

Protein preparations

Procedures fl)r the preparation of actin, myosin, reconstituted actomw~sin (I part of actin to 2 parts of myosin, by wt.), and the Pls-.,5 fraction have been described iu a preceding paper la, "Partially-purified a-actinin" was prepared as described by SERAYDARIAN et al. a°. In a few experiments, the procedure previously described for preparation of tile P15-2~ fraction~3 was modified by adding enough I M Tris-acetate buffer (pH 8.5) to the swollen myofibrils after 64-7,o h at 2 ° to make the final Trisacetate buffer concentration approx. IO mM at pH 8.5. This caused some shrinkage of the swollen myofibrils but did not result in any rebinding of the extracted a-actinin back onto the myofibrillar residue. Consequently, the swollen myofibrils could be separated from the a-actinin-containing supernatant by sedimentation at I4 ooo ;,: g for 6o rain. The sedimented myofibrils were washed once by suspension in Io mM Tris-acetate buffer (pH 8.5) followed by sedimentation at 14 ooo ×g for 0o rain. The combined supernatants were then salted out between o and 3o% (NH4)2SO4 saturation to produce a P0-ao ~t-actinin fraction. This modified procedure made it Biochim. tliophys. Acta, zoo ( i97 o) 290 31

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R. ?,1. RI)BSON t'[ oil.

morc c~mvcnient t~ cxtract and ~cdiment large quantities ~1 m\'~tibrils and also resulted in ab~,ut a 3 ° 5o",, increase in total yield of the b-S ~e-actmin species, ttowever the Po a0 fracti, m ~fften did not contain as high a Im~p~rtion of the (~-S species as did the PI:, ..a.',frm'tion, and the modified procedure was used only when large quantities of purified ~e-actinin were needed. "l'rop~m~yosin was prepared according to the method of F.lsasm ¢'l al. 15. The P40--7.-, fraction from the crude mvofibril extract (ref. I3, Fig. e) served as starting material fl~r tropomyosin preparation. Betk)re use, all tropomyosin preparations were examined fl~r homogeneity in the analytical ultracentrifuge. ('olum~z chromatography l)EAE-cellulose c h r o m a t o g r a p h y was perfi~rmed by using "Cellex D", exchange capacity of o. 9 mequiv/g, obtained from Bio-Rad Laboratories, llichmond, Calif. The cellulose was prepared as recommended bv the supplier, equilibrated with five changes of o.0z M Tris-acetate buffer (pH 7-5) at z , and placed in a column under gravity until the cellulose had settled; the column was then packed under one. pound of N., pressure. Protein samples, dissolved in o.oz M "fris-acetate buffer (pH 7.5), were applied to the column with the aid of a Sephadex sample applicator and eluted with an o-5o0 mM K('I column gradient. Flow rate was regulated by use of an I,KB Recychrom peristaltic pump. A n y material retained on the colunm after the KC1 gradient was eluted with o.5 M NaOH. KC1 concentration in the fractions was determined by titrating C1 with o.1 M AgNOa, using K.,CrO 4 as an internal indicator. Preparation of Sephadex G-zoo and agarose and pouring of molecular exclusion columns was done according to directions supplied by I~harmacia Fine ('hemicals, Piscataway, N.J. Sephadex flow adapters were used %r automatic sample application. Ascending c h r o n m t o g r a p h y was used, and uniform flow rates were obtained with an I . K B Recychrom peristaltic pump. Absorbance of the eluant was monitored at 28o nm, and fractions constituting a single peak were pooled and concentrated either by salting-out with (NH~)zSO 4 or by use of a Diaflow M,~del 4Ol or biodel 5 o ultrafiltration cell (Amicon Corp., Lexington, Mass) and a l.;M-Io ultrafilter membrane. After concentration, extracts were dialyzed against I m3,l KH('Oa for 2 4 48; h and then clarified at 18 3 ooo x g for 3o rain. Z-lira' extraction and rcconstitution Z-line extraction was done by a slight modification of tile method of.qxRoxu.:1~, ~. Rabbit psoas muscle, which had been glycerinated bv Huxley's method and stored at -.,o: for at least 3o days in the glycerol solution w~us teased into thin bundles of myofibrils and suspended in 2 mM Tris buffer (pH 7.6)-(~ mM 2-mercaptoethanol at 2 . After I h, the fibrils were transferred to fresh "Dis-naercaptoethanol solution and stored for lO-12 days at 2:. -l'he supernatant solution was decanted, clarified at lO 5 ooo × g fi~r I h, and the clarified solution concentrated bv using a Diaflo ultrafiltration cell and a UM-Io membrane. A portion of the concentrated solution was further fractionated with (NHa)2SO 4 into a P0-z0 fraction (11. 4 g (NH4)2SO a per ioo ml) and P',0-40 fraction (an additional I2.9 g (NH4),,SO 4 per IOO ml to the P0-z0 supernatant). The precipitates were dissolved in o. 5 mM KHCOs- 7.I 3 mM 2-mercaptoethanol, dialyzed against i mM K H ( ' O a for 24 h and clarified at 4 ° ooo × g for 3o rain. I¢~,,ch~m. I¢to/~hvs...tcta. zoo (m7 o) zgt~-?,t,s

( I - A C T I N I X P U R I F I C A T I ( ) N ANI) P R O P E R T I E s

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Z-line reconstitution and sample preparation and staining fl~r electron microscopy were done as described by STI~OMF.I¢and co-workersr', e. Sections were examined in an RCA EMU-4 electron microscope.

Other procedures Assavs fi~r ~t-actinin activity were done by using the ATPase and turbidity tests as described by ARAKaWA et al. aa. The relative proportion of 6-S rt-actinin species was estimated by using activity in the ATPase and turbidity tests together with area under the O-S peak in schlieren diagrams from the analytical ultracentrifuge. Protein hydrolysis, amino acid analysis and analytical ultracentrifugation were done as described previously la. Protein concentrations were measured by the biuret method an as modilied by Rousox ctal. 17, or by the Folinl-Lowry procedurO ~ as modified by (;O1.I. ¢'1

al. 1".

RESUI.TS

Pur(fication of Pls-'zs Molecular exclusion chromatography. Since the most obvious contaminant in the Pas-2~ fraction was a group of large aggregates that sedimented without forming ;.mv visible boundary in the analytical ultracentrifuge 13, molecular exclusion chromatography was the first method tried for purification of the 6-S ¢~-actinin species in this fraction. Preliminary experiments showed that the O-S species was ahnost totally excluded from Sephadex (;-2<)0, and even 8°.g agarose columns failed to provide adequate separation between the large aggregates and the 6-S a-aetinin species, l.ong (18 4 era) 4°o agarose columns, however, did effect some separation between the large aggregates which appeared with the void volume of the colunm, and the 6-S ~z-actinin c o m p , n e n t which was observed only in the last peak to emerge from the column 0.E

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I:ig. ~. E l u t i o n p r o f i l e o f Pan-2s o n a 2. 5 c m y i83. 5 c m S e p h a r o s e 4 B (4°o a g a r o s e ) c o l u m n . T o t a l b e d v o l u m e w a s 899 ml. A 6 . 5 - m l s a m p l e , 15.3 m g p r o t e i n / m l , w a s a p p l i e d a u t o m a t i c a l l y . T h e e l u t i o n b u f f e r w a s 20 m M K C I - I o m M T r i s . a c e t a t e b u t t e r ( p l l 7 . o ) - I m M N a N a. T h e a s c e n d i n g tlow r a t e w a s 12. 4 m l / h a n d 6 . 2 - m l f r a c t i o n s w e r e c o l l e c t e d .

lCmchim, tliophys. A cta. 2oo ( t97 o) 2~#, 3 t 8

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(Fig. I). l'r(,tein t h a t e l u t e d with the v , i d v()lmne did n o t possess a n y (e-actinin a c t i v i t y in e i t h e r the :Vl'Pase ()r t u r b i ( t i t v tests a n d did n o t e x h i b i t a n y trace of a 6-.~ peak in the a n a l y t i c a l ultracentrifuge'. Pr()tein e l u t e d in the sec()nd pe;tk (area ])et w e e n the vertical ¢h)tted lines in I:ig. I) c ( m t a i n e d a considerabh" higher t)rot)orti(m , ~fthe ()-.q (e-actinin species t h a n the 1'1.~ ',s fraction t h a t had been a p p l i e d to the c o l u m n , a l t h o u g h some aggregates were still d e t e c t a b l e early in the s e d i m e n t a t i ~ m r u n , f the agarose-purified m a t e r i a l (l:ig. 2, l¢()ws a a n d b). P r o t e i n from the second peak also e x h i b i t e d higher specific activities in the A T P a s e a n d t u r b i d i t y tests t h a n the original P~.~_.,-, fracti()n. I)EA E-cclhdose chromatography. A l t h o u g h 4o.o agar~se c o l u m n s p r o v i d e d subs t a n t i a l p u r i f i c a t i o n of the Pv,-e:, fraction, it was necessary to use c o l u m n l e n g t h s of

(o)

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(re;n) after reaching 59780 rev.lm;n

Fig. 2. Sedilnentation of the l)l~ ~ fraction before and after l)urification by column chromatography. (a) P~s-aa befl>re purification, 3. i2 mg protein/ml in l oo mM NCl-2o mM Tris.acetate buffer (pH 7.o); (b) same samph, as in (a) but after purification by a 2. 5 cm × IS3. 5 cm Sepharose .tB c(dunm, 3. I2 nag protein,iml in lOO mM K('l-2o m.M Tris-acetate butler (pH 7.o): (c) l'la ,~sbelbre purdication, 3. l zmg prot~:in.ml in ioo mM KCI--,o mM Tris .acetate buffer (plt 7-5) ; (d) same sample as in (c) but after purification by a 2. 5 cm "~ 25 cm l)EAF.-cellulose column, 3.r- mg protein'ml in [oom.M b:('l 2atom Tris.ucetatebuffer (pH 7 . 5 ) . . \ l l r u n s a t 2o.o ~. I8o cm or m o r e a n d t() at)ply tile s a m p l e in less t h a n 0 ml v o l u m e to a c h i e v e sufficient s e p a r a t i o n b e t w e e n the large aggregates which a p p e a r e d in the w i l d v o l u m e , a n d the 6-.'-; c o n t a i n i n g peak t h a t e l u t e d later. Since less t h a n 3o°.;o of t h e a p p l i e d p r o t e i n could be recovered its purified 6-S m a t e r i a l , yields of the purified 0-S species were o f t e n 3o mg or less. ( ' o n s e q u e n t l y , D E A E - c e l l u l o s e c o l u m n s , to which m u c h larger s a m p l e loads could be applied, were tested to d e t e r n f i n e their a b i l i t y to p u r i f y t h e 6-S a - a c t i n i n species. Fig. 3 shows the t y p i c a l profile o b t a i n e d w h e n the Pls-',s fraction was e l u t e d fronl a D E A E - c e l l u l o s e c o l u m n b y a KCI g r a d i e n t . O n l y the fractions b e t w e e n the vertical d o t t e d lines in Fig. 3 possessed an), a - a c t i n i n a c t i v i t y in t h e A T P a s e a n d t u r b i d i t y tests or e x h i b i t e d a 6-S a - a c t i n i n peak in t h e a n a l y t i c a l u l t r a c e n t r i f u g e . A t p t l 7.5, this , - a c t i n i n - c o n t a i n i n g peak was a l w a y s e l u t e d from l ) E A E - ( ' e l l u l o s e c o l u m n s b e t w e e n 25o-3oo mM KC1. T h e n a t u r e of t h e p r o t e i n in t h e lliock i~n. 1~w p k y s . .4 eta, . o o ( i o 7o) -'9(~-31N

¢I-ACTININ PURIFICATION AND PROPERTIES

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Fig. 3. Elution profile of P~5-~b on a 2.5 cm x 25 cm l)EAE-cellulose column. A 4oo-mg sample was applied to the column and a KCI gradient used for initial elution. Tightly b o u n d protein was eluted with 0. 5 M N a O H . Flow rate was 18 ml/h and 6.o-ml fractions were collected. Material in the peak between the vertical dotted lines is hereafter referred to as the P~5-25 (DEAE) fraction. Fig. 4. Effect of Pts-a5 and P~5-zs (DEAE) on Mg2+-activated superprecipitation of reconstituted actomyosin. Conditions of t u r b i d i t y assay: I mM MgC12, I mM A T P , o.o 5 mM CaCI v IOO mM KCI, 2o mM T r i s - a c e t a t e buffer (pH 7.o), 0. 4 mg a c t o m y o s i n / m l , actinins indicated as percent of a c t o m y o s i n present, 26 °.

long, broad peak eluted at lower KC1 concentrations was not completely elucidated, although the first part of tile broad peak (lOO-15o ml of tile KC1 gradient) possessed phosphorylase activity. ARAKAWAet al. ~3 have described the presence of phosphorylase in crude a-actinin fractions. A large part of the P~s-25 fraction was very tightly bound to the DEAE-cellulose at pH 7.5 and could not be eluted even with 2 M KC1. This material was eluted with 0.5 M NaOH, and examination showed that it consisted of large aggregates that sedimented with no visible boundary in the analytical ultracentrifuge. These aggregates did not possess any a-actinin activity in either the ATPase or turbidity tests. As measured by the amount of protein eluted between the vertical dotted lines in Fig. 3, the 6-S species usually accounted for approx. 3 o°/o of the original P15-2~fraction. The P15-25 (DEAE) fraction (concentrated from fractions between the vertical dotted lines in Fig. 3) exhibited a substantially higher specific activity in the turbidity assay than the original Pa5-25 fraction (Fig. 4), which was already 5-6 times more

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Fig. 5. Percent increase in reconstituted a c t o m y o s i n A T P a s e caused by P~5-2s (I)EAE) at v a r i o u s KCI concentrations. Final conditions: i mM ATP, I mM MgCI 2, 0.05 mM CaCI 2, KC1 as indicated, 2o mM T r i s - a c e t a t e buffer (pH 7.o), o.2 mg a c t o m y o s i n / m l , 25.o °. At each KC1 concentration, various a m o u n t s of l>ts-~5 (DEAE) were added up to 30% of the a c t o m y o s i n present. The level of Pla-~5 (DEAE) t h a t caused the greatest increase in specific activity of the MgZ--modified a c t o m y o s i n A T P a s e was used to calculate the percent increase over the activity of the control actomyosin.

Biochim. Biophys. Acta, zoo (I97 o) 296-318

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active than " p a r t i a l l y puriticd t~-a( t r a i n " RJ. vcf. I ~ ) . 1'1:,_25 (DEAl'2) a d d e d t,, lhc e x t e n t of i o ° , of the a c t o m x o s i n present cattscd an ;thn~,st i m m e d i a t e t u r b i d i t \ resp(mse at IOO mM KC1, after which thv actomy,:sin quickly underwent a "cluml)in f ' process and settled to the bott(ml of the c u v c t t c la. Fig. 5 sh,~ws the ct~ect of I'~,,__..:, (1)EAE) on the A T P a s e a c t i v i t y of an actonlvcsin snq~cnsion at various KC1 concentrations. A peak in percent a c t i v a t i o n is e v i d e n t betwet.n IOO-I') 5 mM 1,7,('1; this p a t t e r n of a c t i v a t i o n is similar to the ..Vl'Pase a c t i v a t i o n p a t t e r n e x h i b i t e d by the Px~-',r, fracti°nlS. C o n t r a r y to the t u r b i d i t y results, the Pla-'.,~ (1)EAE) fraction exh i b i t e d only" a slightly higher specific a c t i v i t y in the . \ T P a s e assav t h a n the original Pls- 25 fraction. S e d i m e n t a t i o n d i a g r a m s of the Plr,-,,s fraction before and after l ) E A E - c e l l u l o s e c h r o n a a t o g r a p h y (Fig. 2, Rows c and d) sh,)\ved t h a t a r e m a r k a b l e purification of the 6-S a-actinin species can be achieved b y DEAE-cellulose columns. Peak sizes in the flmr s e d i m e n t a t i o n diagralns shown in Fig. 2 can be c o m p a r e d d i r e c t h ' since all runs were done at the same protein c o n c e n t r a t i o n and p h a s e - p l a t e angle. The most striking p r o p e r t y of D E A E - c e l l u l o s e is its a b i l i t y to v i r t u a l l y eliminate the large aggregates in the original P1.~-e~ fraction. This is i n d i c a t e d b y the absence of a leading edge in the Pl,~-2a ( D E A E ) s e d i m e n t a t i o n p a t t e r n . A small, faster s e d i m e n t i n g peak (Jr q.i S (average of eleven samples) can be seen in the Pla -a~ ( D E A E ) fraction (Fig. 2, R o w d). The n a t u r e of this 9. I-S c o m p o n e n t is not known, b u t several lines of indirect evidence suggest t h a t it is an aggregate of the 6-S c~-actinin species. These results show t h a t D E A E - c e l l u l o s e c h r o m a t o g r a p h y was more desirable t h a n 4°4, agarose c h r o n a a t o g r a p h y for purification of the ()-S tt-actinin species because: (I) much less time was required to elute the 6-5 species from cellulose colunms (approx. 3o h) t h a n from the long columns necessary for agarose purification (approx. 7¢) h), and (e) larger a m o u n t s of protein could be applied to the l ) E A E - c e l l u l o s e columns t h a n to the 4 % agarose columns. Our experience has shown t h a t up to 8oo-~oo mg of the 111~_~ fraction can be applied to a 2. 5 × 25 cm D E A E - c e l l u l o s e column w i t h o u t an3: noticeable undesirable effects on resolution of the 6-S species. W h e n the I)l:,_~.r, ( D E A E ) fraction was r e e h r o m a t o g r a m m e d on a long 4 %

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F'ig. 6. Elution profile of l'ls_~ (DEAE) on a 2. 5 cm ,'< I82.o cm Scpharose 4 B (4~";, agarose) column. Total bed volume was 893 ml. An It.7-ml sample, 11.6 ing protem/ml, was applied automatically. The elution buffer was 2omM KCI-IomM Tris-acetate buffer (pH 7.5). The ascending flow rate was 1 r. 3 ml/fi and 5.6-ml fractions were collected. lliochim. Hiuphys..4 cta, 2oo ( 197o) 29~ -31 ,~

303

( I - A C T I N I N P U R I F I C A T I O N AND PROPERTIF.S

agarose colunm, the elution profile shown in ]rig. 6 resulted. Comparison of this elution profile with the elution profile of the original P15-25 fraction from a similar column (Fig. I) confirms the conclusion that I)EAE-cellulose removes the large aggregates which are contained in the P15-2,~ fraction and which are eluted with the void volume of 4,o.,, agarose columns. The [~15_o.~(I)E/\E) fraction elutes from 4°,o agarose columns as a single symmetrical peak and at the same point in the elution profile that the 6-S species was fimnd in the elution profile of the original P15-2.5fraction (cf. Fig. I). llechronmtograt)hy of the P15-25 (DEAE) fraction on DEAE-cellulose (Fig. 7)

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Fig. 7. E l u t i o n profile of l'x~-~5 (DI.'AE) w h e n a p p l i e d t o a s e c o n d 2. 5 c m x "5 c m I ) E A E c e l l u l o s e c o l u m n . A 7 1 . 9 - m g s a n l p l e w a s a p p l i e d to t h e c o l u m n , a n d a KC1 g r a d i e n t u s e d for i n i t i a l e l u t i o n . T i g h t l y - b o u n d p r o t e i n w a s e l u t e d w i t h o. 5 M N a ( ) H . F l o w r a t e w a s 19.S m l / h and 6.6-ml fractions were collected.

produced a single sharp peak eluting at 25o-3oo mM KC1, tile same KCI range at which the 6-S species was eluted when the original PI~-2s fraction was applied to DEAE-eellulose columns (cf. Fig. 3). Further etution with 0.5 M NaOH caused the appearance of a second small peak in tile elution profile of the P15-25 (DEAE) fraction. This suggests that rechromatography on DEAE-cellulose may eliminate a very small amount of aggregates which are still present in the P15-25 (DEAE) fraction and which are not resolved by rechromatography on 4°;~ agarose. This conclusion is supported by sedimentation diagrams of the Pls-2s (DEAE) fraction after rechronlatography on 4 % agarose or DEAE-cellulose colunms (Fig. 8). There was no evidence of rapidly., sedimenting species in material rechromatogranmled on DEAE-cellulose (Figs. 8c and 8d), but a very small amount of rapidly-sedimenting material was evident early in the run on the material rechromatogrammed on Sepharose 4 B (Figs. 8a and 8b). It is also obvious from the diagrams shown in Fig. 8 that sedimentation in low ionic strength solvents, such as I mM KHCOs, causes "self-sharpening" effects on the sedimentation diagrams. The reasons for these effects have been discussed by SCHACHMAN~°. In our experiments, IOO mM KCI was routinely added to danlpen out "charge effects", and the runs shown in Fig. 8 were done simply to afford comparison with reports in the literature describing sedimentation of a-actinin in low ionic strength solventsl0,n#

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Biochim. lliophys. Acta, 2o0 (197o) 296 3x8

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Time (mln) a f t e r reaching 5g 780 rev./mln l:ig.tS. S e d i m e n t a t i o n o f PI~-:~ (]_)I'LkEI a f t e r r c c h r o m a t o g r a p h y on a -'.5 cm × z82.o cm Sepharosc:.iB or a 2.5 cm ~" 25 cm Dl.'Al.:-cel]ulosc column. (a) P1s-sa ( D E A E ) a f t e r r e c h r o m a t o g r a p h y on a.Sepharose 4 B column, 3.o mg protein/ml in I mM KHCO3; (b) P,s-~s (DEAE) after rechrom a t o g r a p h y on a Sepharose 41¢ colunm, 5.oo mg protein/ml in too mM KC1-2o mM T r i s . a c e t a t e buffer (ptl 7.5); (el I>l~-a5 (DEAl.:) after r e c h r o m a t o g r a p h y on a second DEAF-cellulose column, 2.50 mg protein/n:l, i mM KHCO:~; (d) t~t: 2s ( I ) F A E ) after r e c h r o m a t o g r a p h y on a second DFAE-cellulose column, 5.oo nlg protein/nil, IOO mM KCI-2o mM T r i s . a c e t a t e buffer (pH 7.5). All r u n s done at 20.o ~.

Concentration dependence of the sedimentation coefficient of the 6-S peak in the P:5-25 (DEAE) fraction is shown in Fig. 9. The line in Fig. 9 is a least squares plot and extrapolates to an s°.,0,,,, of 6.23 S. P15-25 (DEAE) sedimentation is only slightly concentration dependent (regression: I/S = o.16o6 t- o.ooo915 (concentration); o.oo915 significantly different from zero at o.o5 probability level). These findings agree closely with those of GOLLet al. 1 who reported an S°2o,~,of 6.28 S for their Z-protein, a purified fi~rm of a-actinin obtained by brief tryptic digestion of myofibrils. A sample of partially purified a-actinin made according to SERAYDARIANet a l ? ° was also subjected to DEAE-cellulose chromatography (Fig. IO). The only fractions

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Biochim. Biophys..4cta, 200 (x97 o) " 9 6 - 3 t 8

a-ACTININ PURIFICATION AND PROPERTIES

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l:ig. lo. Elution profile of partially purified a-actinin t° on a 2. 5 cm × 25 cm DEAE-cellulose column. A 27o-mg sample was applied to the column and a KC1 gradient used for initial elution. Tightly b o u n d , p r o t e i n was eluted with o.5 M N a O H . Flow rate was z 8.6 ml]h and 6.2-ml fractions were collected.

in the elution profile that possessed any a-actinin activity or that exhibited any 6-S peak in their sedimentation diagram were eluted in a small peak between 250-300 mM KCI (peak between the vertical dotted lines in Fig. IO). The protein contained in this peak constituted about 5% of ttie total protein applied to the column, thus confirming Goll's finding x that all the a-actinin activity in partially purified a-actinin could be isolated in a tryptic fraction constituting only 6% of the total original protein. The protein in the peak between the vertical dotted lines in Fig. IO possessed much higher specific activities in the ATPase and turbidity tests and exhibited a much higher proportion of the 6-S species (approx. 70% as measured by area under the peak) than did the original partially purified a-actinin preparation. However, even after DEAEcellulose purification, the DEAE-cellulose-chromatogrammed partially purified aactinin was not as active in the ATPase and turbiditv assays as the P15-~5 (DEAE) fraction. This is probably due to inability of a single passage through a DEAE-cellulose column to remove the aggregates from partially purified a-actinin preparations as completely a.s from the P15-~5 fraction, which had a lower proportion of aggregates initially, and to the fact that partially purified a-actinin is extracted at room temperature wherea~s the Px5-25 fraction is extracted at 2 ° and has therefore had less exposure to denaturing conditions.

Properties of purified a-actinin Amino acid composition. The amino acid composition of purified a-actinin, i.e., the P15-25 (DEAE) or Px~-25 (DEAE-cellulose and Sepharose 4 B) fractions, is compared to the amino acid composition of Z-protein x, a-actinin 1°, and actin 2~ in Table I. It is somewhat difficult to compare these tabulations in detail since the results come from so many different laboratories, but by assuming the analyses to be technically comparable and by accepting only differences of z5°/0 or larger, purified a-actinin is higher in arginine, glutamic acid, aspartic acid, alanine and leucine and lower in threonine, proline, glycine, isoleucine and tyrosine than EBASHI'S e-actinin. The differences for threonine, glutamic acid, isoleucine, leucine and tyrosine are partiBiochim. Biophys. Acta, 200 (197 o) 296-318

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" P u r i f i e d a - a c t i n i u is t h e l'aa 2s f r a c t i o n p u r i f i e d b y 1 ) E A l - - c e l l u l o s e o r b y l ) E A l : ; - c e l h f l o s c f o l l o w e d b y St, p h a r o s e 4 B. M e a n s p l u s or m z n u s s t a n d a r ( i e r r o r o f a n a l y s e s o n f o u r d i f f e r e n t p r e parations. "" } ; r o i n (]OI,L t't al. 1. ' ' ' l"rolll EIIA,qH1 AND |'~BASIII ~. * F r o m ( ' : \ R S T I " N 22. F i g u r e s a r c m e a n s pl~ts o r ~ i n u s .~.17..

cularlv marked. Compared to actin, purified a-actinin is higher in histidine, arginine, aspartic acid, glutamic acid, alanine and leucine and lower in threonine, serine, glycine, isoleucine and tyrosine with the differences in thre(}nine, glutamic acid, isoleucine, leucine and tvrosine being particularly evident. Thus, purified a-actinin clearly differs from EBASm's a-actinin in xo of the z 5 amino acids listed and from actin in I I of the ~5 listed. It is evident from Table I that a gradation in amino acid composition exists from purified ~t-actinin to Z-pr{}tein, a purified %rm of a-actinin, to EBASm's a-actinin to actin. This trend in amino acid c~mq)osition is particularly evident for arginine, aspartic acid, threonine, glutamic acid, glycine, alanine, isoleucine, leucine and tvrosine and indicates that EBASm's early a-actinin preparations contained considerable denatured actin, as was suggested by GOLL et al?. Indeed, it is possible to nearly duplicate the amino acid comt~osition of EBASm's a-actinin preparation by assuming that this preparation contained only a-actinin and actin in a ratio of o.15 parts aactinin per o.85 parts actin (last column, Table 1). The agree.ment between this calculated amino acid composition and the actual composition presented by Ebashi is particularly striking for arginine, aspartic acid, threonine, glutamic acid, alanine, isoleucine, le.ucine, and tvrosine. Stoichiometrv of the ~-actinin--lg-actin interaction. Results of a study on the stoichiometry of the a-actinin-l:-actin interaction are shown in Fig. z I. In this study, a-actinin was added in increasing amounts to a series of tubes containing a tixed B i o c k i m . I ~ m p h y s . A~ta, . o o (t{~7o) 20{>- 3 x ~

(I-ACTININ PURIFICATION AND PROPERTIES

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Fig. i i . Stoichiometry of the a - a c t i n i n - F - a c t i n interaction. Conditions: v a r y i n g a m o u n t s of Pts-2~ ( D F A E ) were added to centrifuge tubes containing 2o mg F-actin. Final solution was loo m.M KCI-zo mM Tris-acetate buffer (pH 7.5). After t h o r o u g h mixing and standing at o ° for 3° rain, the t u b e s were centrifuged at 45 ooo rev./min for 6o rain, the s u p e r n a t a n t s decanted and analyzed for protein content. Protein concentration in s u p e r n a t a n t s from control t u b e s conraining only F-~ctiu was s u b t r a c t e d from these values. The corrected protein concentrations t h u s obtained were s u b t r a c t e d from protein c o n c e n t r a t i o n s in s u p e r n a t a n t s from a parallel set of tubes, handled identically b u t containing only Px5-25 (DEAl-), to obtain mg of P]s-zs (DV.At,2) nut combined with F-actin nor sedimented in the absence of F-actin. The dashed line represents the " e x p e c t e d " result if none of the a-actinin above a o.4x :x a-actinin to F-actin ratio was b o u n d to F-actin lint was left free in the s u p e r n a t a n t .

amount of l;-actin, and the mixtures centrifuged at 183 ooo × g fl)r I h to effect complete sedimentation of F-actin and the a-actinin-F-actin complex, but leaving unbound c,-actinin in the supernatant. Thus, up to the point that F-actin is able to bind (z-actinin, there should be no protein in the supernatant, but beyond this point, excess ¢~-actinin should appear in increasing amounts in the supernatant. Extrapolation of the amounts of protein appearing in the supernatant at these higher c~-actinin to F-actin ratios back to a supernatant protein concentration of zero gives the stoichiometrv of the (~-actinin-F-actin interaction. All previous attempts to determine the stoichiometrv of the a-actinin-F-actin interaction by this method have failed because up to 6o°.o of the protein in earlier a-actinin preparations sedimented in the absence of F-actinL However, only 5-1o% of the protein in our purified ~z-actinin preparations was sedimented in the absence of F-actin by centrifugation at 183 ooo × g for I h. The protein sedimented from a-actinin preparations in the absence of F-actin was subtracted from the total protein sedimented in the presence of l:-actin, although it is not excluded that the sedimented protein in ~-actinin l~reparations may also combine with F-actin if it were present; however, there is no explicit information on this point. It was also found that approx. 5% of the F-actin protein remained in the 18 3 0 0 0 × g supernatant when F-actin was sedimented bv itself; this amount was subtracted from the total mg of protein in the supernatant for each tube. However, as a result of this circumstance, there was ahvays a small amount of protein present in the supernatant solution, even at low u-actinin : F-actin ratios, and it was therefore possible to assay supernatant protein from tubes containing a-actinin :F-actin ratios below the saturation point. The results in Fig. I I show that l:-actin will bind all a-actinin added up to 41% of its own weight. Some additional binding of a-actinin occurs beyond this point,

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Fig. iz. l-fleet of s u p e r n a t a n t p r o t e i n not b o u n d by F - a c t i n or s e d i m e n t e d by i t s e l f (Fig. z I) on t h e MgZ+-activated s u p e r p r e c i p i t a t i o n of r e c o n s t i t u t e d a c t o m y o s i n , a. S u p e r n a t a n t s from t h e t u b e s c o n t a i n i n g o :o to o.3:1 a - a c t i n i n to E - a c t i n ratios, b. S u p e r n a t a n t s from t he t u b e s c o n t a i n i n g o.6o to i . o o : I ¢l-actinin to F - a c t i n ratios. F i n a l c o n d i t i o n s for t u r b i d i t y a s s a y : t mM ATP, i mM MgClz, o.o 5 mM CaC12, lUO mM KCI, zo mM T r i s - a c e t a t e buffer (pH 7.o), o. 4 m g a c t o m y o s i n / m l , 26~; c o n t r o l s c o n t a i n e d o n l y a c t o m y o s i n when present, s u p e r n a t a n t p r o t e i n s were a d d e d to t h e e x t e n t of 5'~, of t h e a c t o m y o s i n present. The p e r c e n t figures refer to t h e a - a c t i n i n to F -a c t i n r a t i o in th e t u b e from which the s u p e r n a t a n t o r i g i n a t e d .

since if all a-actinin above 41 °,o of the F-actin present had been left unbound, amounts of protein specified b y the dotted line in Fig. I I should have been observed in the supernatant. Apparently, a weak or "nonspecific" binding occurs above the 4I°..'o binding ratio. There is no information available on the exact nature of this binding ; it m a y be due to the presence of two kinds of binding sites on the F-actin filament or possibly to a weak interaction between individual a-actinin molecules. Both turbidity (Figs. I2a and I2b) and ATPase (Fig. 13) assays of the supern a t a n t protein confirmed the conclusion that all added a-actinin was bound below a-actinin :F-actin ratios of o.41. The supernatant protein from F-actin controls even

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Fig. 13. Effect of s u p e r n a t a n t p r o t e i n not b o u n d by F - a c t i n or s e d i m e n t e d b y i t s e l f (Fig. z I) on t h e MgS+-activated A T P a s e of r e c o n s t i t u t e d a c t o m y o s i n . F i na l c o n d i t i o n s for A T P a s e a s s a y : t m M ATP, T mM MgClz, o.o 5 rnM CaC12, 50 mM KCI, 2o mM T r i s - a c e t a t e buffer (pH 7.0), o.2 mg a c t o m y o s i n / m l , 25 °. Control a c t o m y o s i n c o n t a i n e d onl y a c t o m y o s i n , o t h e r t u b e s c o n t a i u e d s u p e r n a t a n t p r o t e i n or Pl~-ss ( D E A E ) f r a c t i o n a d d e d to t h e e x t e n t of z o % of t h e a c t o m y o s i n pr esent. A b s c i s s a refers to t h e Pls-2s (DEAl,;) to F - a c t i n r a t i o in tile t u b e from which t h e s u p e r n a t a n t protein originated.

lliochim. Biophys. Acta, 200 (197 o) 296-318

309

¢I-ACTININ PURIFICATION AND PROPERTIES

appear slightly inhibitory in the turbidity assay (Fig. I2a) and as the ratio of excess a-actinin to unsedimented actin increases in the supernatant (6o, 7 o, 8o, I 0 0 o, /o c u r v e s in Fig. I2b), the activity of the supernatant protein approaches that of the Px.~-2~ (DEAE) fraction. Stoichiometry of the a-actinin-F-actin interaction in the presence of tropomyosi.. DRABIKOWSKI AND NOWAK 2 a n d DRABIKOWSKI el al. n have reported that the presence of tropomyosin prevents the gelation effect ofa-actinin on F-actin and have suggested that tropomyosin and a-actinin compete for the same or closely related binding sites on the actin molecule. Consequently, it was of interest to determine the effect of tropomyosin on the stoichiometry of the a-actinin-F-actin interaction. The method()logy used for this study was the same as that described in the preceding paragraph, except that now tropomyosin was mixed with F-actin befi)re the addition of purified a-actinin. Control tubes containing only tropomyosin and F-actin possessed very small protein concentrations (less than 7% ()f total protein in the tube) in their 183 ooo × g supernatant, indicating that virtually all of the added tropomyosin was bound to F-actin in the absence of a-actinin. The results shown in Fig. 14 indicate

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Fig. 14. Effect of t r o p o m y o s i n on the stoichionletry of the a - a c t i n i n - F - a c t i n interaction. ('onditions were the s a m e as described for Fig. l t except t h a t 6 mg of t r o p o m y o s i n (3o% of the F-actin present) were mixed with the F-actin prior to the addition ()f Pt5-25 (I)EAE). Final solution was ioo mM KCl-2o mM Tris .acetate buffer (pH 7..5).

that when a-actinin is added to F-actin in the presence of tropomyosin, unbound protein immediately appears in the supernatant. However, assays of the unbound protein in the ATPase and turbidity tests showed that up to a-actinin:F-actin ratios of o.4-o.6, the supernatant protein did not possess any a-actinin activity. Beyond this point, a-actinin activity could be detected in the supernatant. These results suggest that a-actinin and tropomyosin compete for the same or closely located binding sites on F-actin, and that at o °, a-actinin is able to displace tropomyosin from F-actin. DRABIKOWSKI AND NOWAR2 have previously found that the ability of Biochim. 1-1iophys. ,4cta, 200 (197 o) 296-3x8

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tx-~q~,unx~sin t~ al)(~lish t h e gelati~,n effect ( ) l . - m - t i n i n ~,n l"-actin was m u c h s t r ( m g c r ;tt 2 I t i t a n ,tt () . Tile l)lccisi(m (d t)lli e x i w r i n l e n t s , h()we\'{'r, (t()','s nt~t t)crlnit llS t() ( I e t e r m i n c w h e t h e r s(m~e (~-actillin, t~, tilt' e x t e n t o f 2 -3",, ()f t h e l : - m ' t i n l)rc-ont. m i g h t I)e b ( ) u n d I w t - r e a n y t r ( ) p o m y o s i n is disl~la('ed.

Relation of . - a c t i n i n to the Z-lira" T h i s s e c t i o n will d e s c r i b e t h e r e s u l t s of s o m e e x p e r i m e n t s d e s i g n e d to d e m ( ) n s t r a t e t h e p r e s e n c e o f m a c t i n i n in Z - l i n e e x t r a c t s , a n d t o s h o w t h e a b i l i t y o f . - a c t i n i n c . n t a i n i n g f r a c t i ( m s t o r e c o n s t i t u t e Z - l i n e s in Z - l i n e e x t r a c t e d fibrils. ~-.4 ctinin i~z Z-line extracts. STRO.XH,:R a n d c o w o r k e r s s,G h a v e d e s c r i b e d a m e t h o d for first e x t r a c t i n g Z - l i n e s f r o m glycerinated r a b b i t p s o a s fibers a n d t h e n b y i n c u b a t i o n

Time (mln) after reochirKj 59780 rev./rain l:lbI. 1.5..%edilncntation of Pn ~0 and 1'20 .=0 fractions from a Z-line extract of glycermated fibrils. l.'pper (wctl~e) cell: 6. w.~ m~ I' o 2n/ml in 1oo mM K('l-2o mM Tris.acetate buffer (pll 7.o); lower cell: 3-7-I m~ 1)~o .,.'ml in voo mM K(I zo m.M Tris.acetate buffer (pit 7.o) zo.(f.

w i t h c o n c e n t r a t e d f r a c t i o n s ()1 t h e e x t r a c t , r e c o n s t i t u t i n g t h e s e s t r u c t u r e s in t h e Z - l i n e - e x t r a c t e d fibrils. I f t h e s u g g e s t i o n t h a t (~-actinin is l o c a t e d in or n e x t to t h e Z-linea, t is c o r r e c t , S'rRo.~lI.:R's Z - l i n e e x t r a c t s s h o u l d o b v i o u s l y c o n t a i n a - a c t i n i n . I:ig. 15 s h o w s t h e s e d i m e n t a t i o n p a t t e r n s o f t w o (NH4)2SO 4 f r a c t i o n s , Pn-',0 a n d P,~0-~0, f r o m s u c h a Z - l i n e e x t r a c t . A l t h o u g h b o t h f r a c t i o n s a r e h e t e r o g e n e o u s a n d c o n t a i n a s u b s t a n t i a l a m o u n t o f r a p i d l y - s e d i m e n t i n g m a t e r i a l , a 6 . I - S p e a k is e v i d e n t in t h e P0-,,0 f r a c t i o n , a n d t w o p e a k s , w i t h o b s e r v e d ,s v a l u e s o f 0.I a n d 8.45 S, c a n e a s i l y l)e s e e n in t h e P20-40 f r a c t i o n . ( ' o n s i d e r i n ~ t h e p r o t e i n c o n c e n t r a t i o n s of t h e "I'AI~I,I: 11 E F F E C T O F I ) o - 2 u A N D P~0 40 I : R A ( " F I O N : ' ; R F ( ; I ) N , q T I T U TF.D A C'I'O.M YO,% I N

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( / - A C T I N I N P U R I F I C A T I O N AND I}ROPI¢RTIES

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I;ig. 16. E f f e c t o f 1'0 20 a n d P~0-40 f r a c t i o n s fronl a Z - l i n e e x t r a c t on t h e M g 2 " - a c t i v a t e d s u p e r p r e c i p i t a t i o n o f r e c o n s t i t u t e d a c t o m y o s i n , l : i n a l c o n d i t i o n s : I m.M A T P , I m M Mg('l 2, o.0 5 mM CaCI 2, i o o m M KC1, 2 o m M T r i s - a c e t a t e b u f t e r ( p t ! 7.o), o.. t n a g a c t o m y o s i n / m l , Po 2. a n d 1',o 40 i n d i c a t e d a s p e r c e n t o f a c t o m y o s i n p r e s e n t . 26 ~.

samples shown in Fig. I5, it appears that the proportion of 0-S species in tilt" Po-.2o fraction is similar to that in the P20-40 fraction. Since a-actinin salts out between 15 and 25°.~, (NH4)2SO 4 saturation as, a-actinin wouht be expected to I)e distributed nearly equally between the two fractions. Assays of the P0-2o and P'-,0 40 fractions for a-actinin activity in the ATPase and turbidity tests are shown in Table II and Fig. 16, respectively. When added in large amounts (3o°.o of the actomvosin present), both fractions clearly possessed tt-actinin activity in the turbidity test, with the P0-~o fraction having a slightly higher activity than the P',0-40 fracti~m. The fractions exhibited similar activity in the ATPase test (Table I I). These results demonstrate that, although the Z-line extracts are hetert:geneous, they do clearly contain a-actinin. The 8.45-S material in the P2o-~0 fraction was not examined in detail, but it is probably a dimeric form of phosphorylase. AICA~.aw:xet a l ) s have previously described the presence of phosphorylase in crude rt-actinin extracts. Extensive tests have shown that the 8.45-S component does not bind to F-actin and is inactive or even slightly inhibitory in the ATPase and turbidity assays for ,-actinin activity. Consequently, presence of the 8.45-S species in the P~.0.-40 fraction does not alter our conclusion regarding the presence of a-actinin in this fraction. Z-line rcconslil.ulion by ¢t-actinin-containing exlracls. ~TRO31ER t't al. a have reported that partially,-purified tt-actinin prepared according to the procedure of Sk'RAVI)ARIANet al. TM caused a large amount of binding in the l-band but resulted in no reconstitution of Z-lines when incubated with Z-line extracted fibrils. Since partially purified ct-actinin contains less than ~o°.',, of its protein as the 6-S a-actinin species we repeated STnOMER'S Z-line reconstitution experiments using the P]s en fraction in place of partially-purified (t-actinin. Figs. I7a and 17t) show the ultrastructure - f glycerinated rabbit psoas nmscle before and after Z-line extractionS, n. Fig. ISa shows the fibrils seen in Fig. i7b after incubation with a P2s-40 fraction from an EI~ASH¢a extract fi)r 04 h in IOO inM KC1 at O'. The P23-40 fraction is po()r in ¢,-actinin but rich in phosplu)rylase]:L The only evident fi.~ature of this interaction is the appearance of some small tufts of material bound in the lateral third of the I-band, or alternatively, in the middle of the thin filament. Even this slight degree of binding may originate from the small amount of ()-S a-actinin species which n,)rmallv conta]hochtm. Btophys, ,4eta, 2oo (197 ° ) 206 31S

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314

R.M.F.OB~ON Ct gll.

l,ig, i,~. Structure of Z-line-extracted rabbit psoas fibers after i,)cubation with a P.aa ~0 fraction from all t':BASHI~ extract (a) ()r the l>~s-2.,, fractionla (I)). Incubation was d o n e for (),t h ill IOO nl.~,l I{.CI, 5 mY,l .Mg('l,z, 5 mM t'a('12, - m M Tris-HC1 butter (pH 7.6), ~ mM 2-mrrcaptoethanol, o . (i. 3 nlg [>.2a-.10/llll illld Io.o nlg Pl~ "2,'~/fill w e r e llSed ill the reconstitution media, c. Froln the same sample shown in b, this micrograph shows a cross section through the reconstituted Z-line area. The mvotibrillar profile at the left of center shows the characteristic basketweavc pattern. At the right edge of the right profile (arrow), the Imsketweave is oriented at the usual 45' angle to the square lattice seen in the remainder of the mvotihril. T, tufts, ('B, crossbrid.~cs, Z, Z-lines. a. >, 21 4(~o. b. . 3" 5.t °. c. > ~2 5oo. m i n a t e s tile P',a-4o f r a c t i o n , s i n c e o t h e r t e s t s s h o w e d t h a t p h o s p h o r y l a s e w a s m~t b o u n d b y e i t h e r a c t i n or m y o s i n , l . a c k of s i g n i f i c a n t r e c o n s t i t u t i o n b y t h e P~a-4o f r a c t i o n i n d i c a t e s t h a t Z - l i n e r e c o n s t i t u t i o n is specific for Z - l i n e p r o t e i n s . Fig. I 8 b s h o w s t h e fibrils s e e n in Fig. I 7 b a f t e r i n c u b a t i o n w i t h t h e Plr,-.,:, f r a c t i o n la for ~)4 h in I o o m M KC1 a t o . "File m o s t o b v i o u s effect o f i n c u b a t i o n w i t h t h e Pie,-,_,., f r a c t i o n is a g r e a t l y i n c r e a s e d d e n s i t y in t h e e n t i r e I - b a n d . Most of t h i s d e n s i t y is d u e t o a l a r g e n u m b e r o f c r o s s - b r i d g e s (CB) b e t w e e n a d j a c e n t t h i n t i l a m e n t s . I n a d d i t i o n , a s m a l l a m o u n t o f m a t e r i a l is b o u n d as t u f t s . T h e r e is also s o m e e v i d e n c e o f i n c r e a s e d d e n s i t y in t h e Z - l i n e r e g i o n w h e r e , p r i o r to r e c o m b i n a t i o n , t h e Z - l i n e s had been completely extracted. A moderate reconstitution of Z-lines has occurred. I f t h e i n c r e a s e d d e n s i t y d u e t o c r o s s - b r i d g e s in t h e l - b a n d w e r e a b s e n t , t h e d e g r e e o f Z - l i n e r e c o n s t i t u t i o n w o u l d b e e v e n m o r e e v i d e n t . S u p p o r t fi~r t h i s s t a t e m e n t is p r o v i d e d in Fig. iSc. A cross s e c t i o n t h r o u g h t h e r e c o n s t i t u t e d Z - l i n e a r e a f r o m t h e s a m e s a m p l e s h o w n in Fig. i Sb c l e a r l y d e m o n s t r a t e s t h e b m s k e t w e a v e p a t t e r n c h a r a c t e r i s t i c of t h e Z - l i n e 6. O r i e n t a t i o n o f t h e w e a v e a t tile r i g h t e d g e o f t h e r i g h t profile ( a r r o w ) is itt a 45 ~: a n g l e t o t h e s q u a r e l a t t i c e in t h e r e m a i n d e r of t h e mvofil)ril.

Hi,,chi*H. Ilt,~phx's..'Iota. _,(~o (t~7o) 2q¢~-3 l.'~

¢I-ACTININ PURIFICATION AND PROPERTIES

315

DISCUSSION

The results of this study show that DEAE-cellulose column chromatography is a very effective and convenient method for purification of the 6-S a-actinin species. Starting with the P15-2s fraction, it is possible to prepare 15o-3oo mg of tmrified ~t-actinin bv a single pass through a 2.5 cm × 25 cm DEAF-cellulose column. Purification is much more difficult if partially purified c,-actinin ~° is the starting material, since this preparation contains less than IO°o of its protein as the 6-S ~>actinin species. The purified ~t-actinin elutes as a single, symmetrical peak upon rechrnmatography on a 4°,~, agarose or a DE:\E-cellulose column, although the rechromat.graphy on DEAE-cellulose removes a very small amount of aggregates from the purified preparations. Measurements of area under the 6.2-S peak in schlieren diagrams from the analytical ultracentrifuge and estimation of the amount of protein bound by F-actin suggest that DEAE-cellulose-purified ~t-actinin contains more than 850,,, of its protein as the 6-S a-actinin species. Schlieren diagrams of purified tt-actinin indicate the presence of a small q.I-S peak (about 5-1o°.o of total protein) in addition to the main 6.2-S peak. This ct.I-S species is possibly a salt-induced aggregate of the 6.2-S species since: (I) it is more prominent in DEAE-cellulose-purified material, which is eluted at 25o 3oo mM K('I, than in 4°.,, agarose-purified material, which is eluted at 2o mM KC1; (2) it is eluted from DEAE-cellulose and 4~o agarose colunms at the sanle time as the 6.2-S species; (3) it is not evident in sedimentation diagrams of purified t>actinin in I mM K H('Os; (4) like the 6.2-S species, the 9.I-S material is bound by F-actin. These properties of the 9.I-S species also clearly show that it is not phosphorylase which is present in crude tt-actinin solutions; moreover, purified a-actinin solutions have no detectable phosphorylase activity. The successful purification of the (>S ~t-actinin component nlakes it evident that the 6-S species does not exhibit the marked aggregating tendencies characteristic of many structural proteins:, 8, and that the large aggregates prevalent in crude tt-actinin solutions are not aggregated a-actinin, as was suggested by Nogo.stvRa n, but instead represent other contaminating protein species. Comparison of the amino acid analyses of purified a-actinin with those of the earlier crude tt-actinin solutions clearly shows that much of this contaminating protein is denatured actin. This accounts for the many similarities that EBASHI A,XI) EBASHI9 observed between their a-actinin preparations and denatured G-actin. The striking differences in amino acid composition between purified a-actinin and actin also clearly demonstrate that ttactinin is a new protein component of the myofibril and is not simply an unusual form of denatured actin, a question that could not be definitely answered before now. This conclusion is substantiated by the finding that our purified ~z-actinin preparations do not contain 3-methylhistidine, an amino acid which is found in both purified actin 2a,24 and myosin24, 25. The availability of purified a-actinin also makes it possible to ascertain the stoichiometry of the a-actinin-F-actin interaction. BRISKEr el al. 2~ had earlier determined this stoichiometry as o. 9 parts a-actinin to I part of actin, but as pointed out by GOLL et al. ~, the evidence for this determination was misleading. Neither GoI.L et al. ~ n o r DRABIKOWSKI AND .NOWAK2 were able to determine the stoichiometry of the a-actinin-t--actin interaction, due in large part to the tremendous heterogeneity Btochim. Bzophys. A cta. 2oo ( i~,~7o) ,qtJ 31

316

R.M. ROBSON el al.

of crude a-actinin preparations. Our studies with imrified a-aetinin show that F-actin is able to quantitatively bind a-actinin up to o.4x parts of a-actinin per I part of actin. Past this o.4x : i ratio, F-actin binds only part of the added a-actinin. The proportion of a-actinin bound past the o . 4 I : r ratio varied in different experiments, suggesting that this binding was of a weak or nonspecific kind. By assuming a molecular weight of 45 ooo daltons fi~r the G-actin subunits 26 in F-actin, a molecular weight of 18o ooo daltons fl~r the 6-S ~-actinin species (R. M. ROBSON, unpublished observations), and that each G-actin subunit hms one primary binding site fl~r ~z-aetinin, it can be calculated that the o.41:I stoichiometrv corresponds to a molecular binding ratio of I molecule of a-actinin per every IO G-actin subunits. Since there are approx, t3 subunits per chain in one complete turn of the F-actin helix 2v, this ratio suggests that (i-actin subunits are arranged along the F-actin filament in such a fashion that their a-actinin-binding site is available only once (luring one complete turn of the F-actin helix. Our studies also clearly show that a-actinin and tropomyosin compete fl)r the same or closely related binding sites on the F-actin filament. Since in vivo, tropomyosin appears to be located along the entire length of the thin filament28, e~ whereas aactinin seems to be restricted to the Z-line area, i.e., one end of the thin filament a,a, it is possible that tropomyosin acts i~l vivo to prevent binding of a-actinin along the length of the thin filament. By assuming a thin filament length of I/~ and by postulating that one inoleeule of a-actinin interacts with each of the flmr strands suggested to extend from the Z-line "end" of the thin filament a°, it is possible to calculate that the a-actinin-Ig-actin stoichiometry in vivo would be about o.04:I. This is approximately the same magnitude as the experimental error in our experiments on the a-actinin tropomyosin-F-actin interaction. Consequently, we cannot eliminate the possibility that l"-actin bound about 4% of its weight of ~t-actinin before the a-actinin began to displace tropomyosin. The studies of Z-line extracts clearly demonstrate that a-actinin is present in these extracts. However, this does not prove that this a-aetinin originates from the Z-line. l:urther evidence on this latter point was obtained when it was shown that the 1'~,~_,,s flaction, which consists largely of a-actinin together with some denatured aetin, will cause nloderate reeonstitution of Z-lines in Z-line-extracted fibrils. The Pls-2~ fraction does n , t contain tropomyosin, thus confirming the conclusion of S'rRO.m.:R and co-workers ~,e that Z-lines can be reconstituted in Z-line-extracted fibers by fractions that are devoid of tropomyosin. The most obvious effect of the Pls-2s fraction on Z-line-extracted fibrils was fl~rmation of nunlerous cross-bridges in the l-band. This suggests that the technique fl~r Z-line extraction also caused considerable extraction of tropomyosin along the length of the thin filament, thereby making sites available for binding ofa-actinin. It is obvious that incubation with the I'ts_25 fraction causes much more binding in the Z-line region of Z-line-extracted fibrils than was previously flmnd when such fibrils were incubated with partially purified a-actinin (ref. 6). Moreover, we have observed that the P15-2s fraction causes Z-line reconstitution at nmch lower protein concentrations than were necessary when the P0-40 fraction of the Z-line extract was used in the studies of STROMER and co-workers~,e. It is not clear whether the difference in ability of the P~5-:5 fractions and partially purified a-actinin preparations to cause reconstitution of Z-lines in Z-line-extracted fibrils is due only to the higher proportion of 6-S a-actinin species contained in the Hiochim. t l m p h y s . .4 eta. 2ot} (l~Uo) -'0~ 31S

( I - A C T I N I N P U R I F I C A T I O N AND P R O P E R T I F S

317

P15-25 fraction, or to the fact that the Px5-25 fraction was extracted at 2 ° in contrast to partially purified a-actinin which is extracted at 25 °, or to both these possibilities. Regardless of the cause, the finding that the Px~-25 fraction, which is rich in the 6-S a-actinin species, causes moderate Z-line reconstitution indicates a central role for a-actinin in Z-line structure. At this point, however, it does not appear that a-actinin is the only protein in the Z-line. Our yields suggest that a-actinin constitutes approx. I°,o of the total mvofibrillar protein in rabbit skeletal muscle. This is considerably less than the estimate of HUXLV:Y AXl) HANSON :~I t h a t 6?.o of the dry mass of the t a r o fibril is Z-substance. Hence, there must be substances in addition to a-actinin in the Z-line; possibly these substances act to limit the binding of a-actinin to the Z-line end of the thin filament, as suggested by S'rltoMV.R et al. 6. Because of the uncertainty concerning the composition of the Z-line, it is difficult to determine whether a-actinin has a purely structural role in musclO or whether the ability of a-actinin to accelerate the ATPase activity and turbidity response of actomvosin suspensions presages a m o r e d i r e c t role for a-actinin in the contractile process. AC KNOWLEDG."t,I ENTS

We are grateful to Joanne Temple and Jean Preston fi)r devoted and expert assistance and to Karen Schwarz for assistance with the manuscript. We are indebted to Dr. R. S. Adelstein, National Heart Institute, Bethesda, Md., who kindly assayed one of our purified a-actinin preparations for the presence of 3-methylhistidine. During this investigation, R.M.R. held a National Institutes of Health Predoctoral Yellowship 5-FI-GM-28 o41. N.A. was a recipient of a Visiting Professorship from a Training Grant UI-oIo43-o 3, National center for Urban and Industrial Health, U.S. Public Health Service.

l,~E F E 1¢EN(" F S I I). E. (;OLL, W. F. H..M. I~'IOMMAERTS, 31. K. |¢EEI)Y ANI) |{. SERAYDARIAN, Biochim. Biophys. Acta, 175 (I969) I74. 2 Vf. I)RABIKOWSKI AND t~.. NOWAK, Iguropean J. Biochem., 5 (I968) 2o9. 3 l_). |.~. (;OLL, $,V. F. l i . M..~,'IOMMAERTS AND K. SERAYDARIAN, Federation Proc., 26 (1967) 499. 4 T. •IASAKI, M. ENDO AND S. EBASHI, J. Biochem. Tokyo, 62 (1967) 63o. 5 .",I. H. STRO.',tER, D. J. HARTSHORNE AXD R. V. RICE, J . Cell Biol., 35 (I967) ('23. (i .x,l. H..'qTROMER, l-). J. HARTSHORNE, H..~,'IL'ELLER AND I{. V. I{Icr,', .f. Cell Biol., 4 ° (x969) 167. 7 (;. LENAZ, •. ];. HAARD, A. 1.AUWERS, l.). W. ALLMAN AND l). |~. (;REEN, AYch. Biochem. Biophys., 12~)(i968 ) 746 . 8 l{. 1'.]..~TEI'HENS, J. 31ol. Biol., 33 ( i 9 6 8 ) 517. 9 S. I"BASHI AND t ¢. }']BAStII, J. Biochem. Tokyo, 58 (1905) 7IO |'~. SFRAYDARIAN, |{. J. BRISKEY AND W. |:. 1t..M. MOM.MAERTS, Biochim. Btophys. Acta, I33 (t967) 399. I I Y. NONOMURA, J. Biochem. Tokyo, 61 (1967) 796. 12 V',:. I)RABIKOWSKI, Y. NONOMURA AND t{. MARUYAMA, J. Biochem. Tokyo, 63 (]968) 761. 13 N. ARAKAWA, ]{..~|. R o r ~ s o x AXD I). E. (;OLL, Biochtm. Biophy.~..4eta, 200 (I97o) 284. 14 D. 1'.'. GOLL, R. M. |~OBSON, 3[. II..~TROMER AND X. ARAKAXVA, Bzophys. J., 9 (i96Q) A i 2 . 15 S. EBASHI, A. KODAMA AND F. EBASHI, J. Biochem. Tokyo, 64 (I968) 465 . I 6 A. G. GORNALL, C. T. BARDAWILL AND M. M. DAWD, J. Biol. Chem., ~77 (I949) 75 I. r 7 R. 31. ROBSO.X', 1-). E. GOLL AND M. J. TF.MPLE, Anal. Biochem., 24 (I968) 339. 18 O. H. LOWRY, N. J. ROSFmROUGH, A. I.. |:ARR AND R. J. I~ANDALL, J. Biol. Chem., 193 (195 I) 265. 19 O. I".. (~OLL, "~V. G. HOEKSTRA AND R. V',r. BRAY, J . Food Sci., 29 (1964) 608.

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R.M. ROBSON et al.

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Press, New

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