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The modification of actomyosin by α-actin
309
BIOCHIMI@A ET BIOPHYSICA ACTA
BBA 3 5 o 5 4
T H E MODIFICATION OF ACTOMYOSIN BY a-ACTININ* I. A SURVEY OF E X P E R I M E N T A L CONDITIONS K. S E R A Y D A R I A N , E. J. B R I S K E Y * * AND VVT. F. H. M. MOMMAERTS
The Departments of Medicine (The Los Angeles County Heart Association, Cardiovasczdar Researcli Laboratory), and Physiology, UCLA School of Medicine, Los Angeles, Calif. (U.S.A.) (Received S e p t e m b e r ieth, 1966)
SUMMARY
The finding by EBASHI that the superprecipitation of actomyosin, as measured in a turbidimetric assay, requires a-actinin can be confirmed under specific conditions of ATP concentration and cationic composition. Under other conditions, however, including notably ionic strengths in the physiological range, the presence of a-actinin merely accelerates the turbidity change or is without influence. The ATPase activity of actomyosin is activated only little by a-actinin, only at low ionic strength. This study constitutes a survey of experimental conditions basic to the following publications.
INTRODUCTION
Recent contributions by EBASHI AND EBASHI1,2 on the subject of regulatory proteins affecting the interactions between actin, myosin and ATP, have led to new viewpoints in the interpretation of the control of these interactions as occurring in contraction and relaxation of muscle. Foreshadowed by the observations of PERRY AND GREY3 and WEBER AND WINICUR¢ on the variability of actin (giving rise to 'relaxing' and 'non-relaxing' actomyosins), by KATZ5 on effects of tropomyosin upon the turbidity changes in actomyosins, and indeed b y the early suspicion expressed by MORALES AND BOTTS6 on possible differences between 'natural' and 'synthetic' actomyosin, EBASHI AND EBASHI 1,2 have announced tile discovery of troponin*** conferring a calcium requirement upon actomyosin, and of a-actinin necessary for contraction to occur at all. In our extension of this work, we have confirmed the described properties of troponin, but have found the effects of a-actinin to be much more complex; we conclude that they are not to be interpreted in the indicated sense of identifying a-actinin as a required factor in the contraction process. " This series of three papers is dedicated to Prof. HANS H. ~VEBER at the occasion of his 7oth a n n i v e r s a r y , and in recognition of his continued pioneering investigation in the contractile protein s y s t e m of muscle. ** F o r the period 1965-1966, E. J. B. h a s been a Visiting Scholar from the D e p a r t m e n t of Meat a n d Animal Science, The U n i v e r s i t y of Wisconsin, Madison, Wisc., U.S.A. ** * According to a personal c o m m u n i c a t i o n by S. EBASHI (1965) the fraction of native tropomyosin t which confers calcium sensitivity to a c t o m y o s i n is being t e r m e d ' t r o p o n i n ' .
Biochim. Biophys. Acta, 133 (1967) 399-411
400
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
SCHEME I PREPARATION OF ~-ACTININ-FREE ACTIN W i t h m i n o r m o d i f i c a t i o n s from p a r t i a l d e s c r i p t i o n s in s e v e r a l m a n u s c r i p t s l , 2 , ~. A n i m a l s are gi ve n s o d i u m p e n t o b a r b i t a l (90 rag) a n d ~ - t u b o c u r a r i n e c h l o r i d e (1. 5 rag) pri or to e x s a n g u i n a t i o n . Muscles are excised i m m e d i a t e l y , i m m e r s e d in ice, a n d c o a r s e l y (2 mm) g r o u n d . All e x t r a c t a n t v o l u m e s up to S t e p X V I , for ~-actinin-free a c t i n p r e p a r a t i o n s are b a s e d on w e i g h t of a c e t o n e - d r i e d powder. I MUSCLE TISSUE (IO0 gin) (a) E x t r a c t w i t h 3.3 voh (O.15 M KH~PO4, o. 3 M KC1) (pH 6.5, o°). (b) Stir 15 rain a n d t h e n s l o w l y a d d 13. 3 vol. H 2 0 w hi l e s t i r r i n g . (c) S t r a i n t h r o u g h one l a y e r of g a u z e a n d squeeze.
FILTRATE
n RESIDUE
(use for m y o s i n A preparation)
FILTRATE
(a) W a s h t w i c e (IO m i n each) w i t h 3 vol. (o.o2 M KCI-o.2 mM N a H C O a ). (b) W a s h t w i c e (5 m i n each) w i t h 3 v o h H 2 0 . (c) S t r a i n t h r o u g h one l a y e r of g a u z e a f t e r e a c h w a s h a n d squeeze g e n t l y .
I I I RESIDUE
(discard)
I
(a) A d d I vol. H~O a n d l e t s t a n d 4 h a t 20 °. (b) F i l t e r ( W h a t m a n No. 4 paper), for 4 h, a t 20 °.
FILTRATE I V RESIDUE ( e x t r a c t A for a - a c t i n i n preparation) (a) A d d i voh i mM N a H C O 3 a n d l e t s t a n d 5 h a t 20 ° (b) F i l t e r for 4 h a t 20 °.
FILTRATE
V RESIDUE
( e x t r a c t B for a - a c t i n i n preparation)
FILTRATE (discard)
(a) A d d 2 vol. I mM N a H C O 3 a n d l e t s t a n d 2 h a t 20 °. (b) F i l t e r 2 h a t 20 °.
V I RESIDUE (a) A d d M KC1 to final concn, of o.oi M (I m l / I o o g). (b) L e t s t a n d i h a t 20 ° a n d filter for i h a t 2o ° .
FILTRATE (discard)
vii
RESIDUE
(a) A d d 2 vol. o . o i M N a H C O 3 a n d s t i r I h a t 20 ° . (b) F i l t e r 1-2 h t h r o u g h filter p a p e r .
FILTRATE
viii
RESIDUE
(discard) (a) A d d 4 voh I mM N a H C O 3 a n d s t i r I h a t 20 ° . (b) P a s s t h r o u g h gauze.
Biochim. Biophys..4cta, 333 (1967) 399-411
401
ACTOMYOSIN AND ~-ACTININ SCHEME I
(continued)
I FILTRATE
I X RESIDUE
(discard) (a) Repeat Step V I I I
FILTRATE
X RESIDUE
(discard) (a) Add 2 voh H20 and stir 3 ° rain. at 2o °. (b) Pass through gauze.
FILTRATE
X I RESIDUE
(discard) (a) Repeat Step X.
FILTRATE
X I I RESIDUE
(discard) (a)Add 3 vol. acetone at o °. (b) After 5 min pass through gauze.
FILTRATE
X I I I RESIDUE
(discard) (a) Add I vol. acetone at o °. (b) After 5 min pass through gauze.
FILTRATE
XIV RESIDUE
(discard) (a) Repeat Steps X I I and X I I I .
FILTRATE
XV RESIDUE
(discard) (a) Dry at room temperature.
X V I ACETONE-DRIED POWDER
(a) Extract with 0.2 mM ATP and 0.2 mM ascorbate (pH 7-9) and continue as described in Scheme II Step VI.
t METHODS
A ctin M u c h of t h e e x p e r i m e n t a l f o u n d a t i o n of t h i s w o r k rests u p o n t h e a v a i l a b i l i t y of p u r e a c t i n , n o t a b l y a p r e p a r a t i o n n o t c o n t a m i n a t e d w i t h ~-actinin. A l e n g t h y p r o c e d u r e d e v e l o p e d b y EBASHI AND MARUYAMA7 assures t h i s d e g r e e of p u r i t y , a n d we h a v e used, as s t a n d a r d s of c o m p a r i s o n , a c t i n s p r e p a r e d a c c o r d i n g to t h i s m e t h o d , w h i c h , w i t h s o m e m o d i f i c a t i o n s a n d e x t e n s i o n s is g i v e n in t h e f l o w s h e e t of S c h e m e I. B e s i d e s t h i s e l a b o r a t e p r o c e d u r e w e h a v e also, a n d i n c r e a s i n g l y , u s e d a m o d i f i -
Biochim. Biophys. Acta, 133 (1967) 399-411
402
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
cation of our original methodS, 9 by incorporating a precipitation step with 3-3 M KC1 (ref. 2) to remove ~-actinin. This method is given in the flowsheet of Scheme II. The KC1 precipitation is applied directly to the crude extract, and is followed by the ultracentrifugation step desirable to clear the extract (no polymerization occurs at this high ionic strength). This centrifugation results in the removal of some fatty material by flotation, and of some debris; no observable protein precipitate appears at this point, nor is any precipitate obtained b y 3-o-3.3 M KC1 at later stages in the preparation when the protein concentrations are higher. Since, furthermore, the same results are obtained with our routine actin preparations as with the elaborately purified standard, we conclude that the standard method of this laboratory, when properly executed, yields pure actin also according to this new criterion. However, this conclusion was drawn only after the experience gained in the course of this work; all experiments reported here were done with actin subjected to the additional KC1 step, i.e. with procedures outlined in Schemes I or II. Actin prepared b y any of our methods shows, after polymerization, one distinct hypersharp boundary during ultracentrifugal sedimentation at 20410 rev./min. SCHEME II PREPARATION OF PURE ACTIN Modification of the s t a n d a r d m e t h o d of this laboratory. Animals given s o d i u m p e n t o b a r b i t o l (9o mg) a n d D-tubocurarine chloride (I.5 rag) prior to exsanguination. Muscles excised immediately and coarsely (2 mm) ground. All procedures except drying conducted at o °. All e x t r a c t a n t volumes u p to Step VI are based on the weight of the muscle used for the first extraction. E x t r a c t a n t volumes, after Step V, are based on weight of acetone-dried powder. I MUSCLE TISSUE (IO0 g) (a) E x t r a c t w i t h 3.3 vol. G u b a - S t r a u b soln. (o.15 M K H 2 P O ~, 0. 3 M KC1) (pt-I 6.5). (b) Stir 15 min a n d t h e n slowly add 13. 3 vol. H 2 0 while stirring. (c) Strain t h r o u g h two layers of gauze and squeeze.
FILTRATE (use for m y o s i n A preparation)
Filtrate (discard)
I I RESIDUE (a) Add 4 vol. 0.05 M N a H C O a and e x t r a c t for 20 nlin while stirring. (b) Strain t h r o u g h two layers of gauze.
I I I RESIDUE (a) E x t r a c t with i vol. soln. (0.05 M N a H C O 3 and o.o5 M Na~CO3) tor io min and add io vol. 5" lO-5 M CaCI v (b) After IO rain strain t h r o u g h two layers of gauze and squeeze.
FILTRATE (discard)
IV ~ESIDUE (a) W a s h 3 times (5 min each) with 3 vol. each of cold acetone a n d pass t h r o u g h gauze after each wash.
Biochim. Biophys. Acta, I33 (1967) 399-411
4o3
ACTOMYOSIN AND ~-ACTININ S C H E M E II
(continued)
I V RESIDUE
FILTRATE
(discard) (a) S p r e a d on filter p a p e r a n d d r y at r o o m t e m p e r a t u r e . ~7I ACETONE-DRIED POVCDER
(a) E x t r a c t twice (3 ° rain each) w i t h 0.2 m M A T P a n d 0.2 m M a s c o r b a t e (pH 7.9) a t o (io vol. each). (b) Filter, u s i n g s u c t i o n in cold a n d c o m b i n e filtrates (wash p a p e r w i t h H 2 0 a n d A T P ascorbate)
V i i FILTRATE l (a) Centrifuge a t 7 8 o o o × g at o ° f o r 6 o m i n .
RESIDUE
(discard) i
V I I I SUPERNATANT
SEDIMENT
(discard) (a)Make 3-3 M KC1 u s i n g solid KC1. (b) H o l d IO m i n a t o ° t h e n c e n t r i f u g e at 20000 × g for io rain. (c) Filter at o ° u s i n g s u c t i o n (wash p a p e r w i t h H 2 0 a n d A T P - a s c o r b a t e ) . I X FILTRATE
(a) Dialyze a g a i n s t 33 vol. of 0. 3 m M Na]-ICO 3 for lO h. (b) C e n t r i f u g e at 78000 × g for 3 h.
4 SUPERNATANT
X PELLET
(discard) (a) R i n s e walls of t u b e w i t h H 2 0 of o °. (b) T a k e u p in io vol. of 0.2 m M A T P a n d 0,2 niM a s c o r b a t e (pH 7.9) (glass homogenizer). (c) Centrifuge depolynIerized actin at 78000 × g for 3 ° rain.
SEDIMENT
X I SUPBRNATANT
(discard) (a) B r i n g to o.I M KC1 a n d let polymerize. (b) Centrifuge at 78ooo × g for 3 h.
SUPERNATANT
XlI PELLET
(discard) (a) R i n s e wall of t h e t u b e w i t h H 2 0 of o °. (b) T a k e u p in 2 - 3 vol. of o.i M KC1 (final concn.). (c) Dialyze a g a i n s t large vol. of o.i M KC1 for 6 h. X I I I PURE ACTIN
Myosin Routinely, this protein was prepared by the method of this laboratory 1°. The final solution is in 0.5 M KC1. In the course of this work, a number of variations have also been studied, by preparing myosin according to SZENT-GY6RGYIn, AZUMA AND Biochim. Biophys. dcta, 133 (i967) 399-411
404
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
WATANABE12 and EBASHI AND ~ B A S H I 1. NO differences in myosin behavior were seen as a result of these modifications. A sensitive criterion for the purity of myosin, in terms of the presence or absence of actin or actomyosin, is due to KESSLER AND SPICER as modified by AZUMA AND WATANABE12. At a suitably selected ion composition, while the addition of ATP gives rise to no turbidity change in myosin, marked increases in turbidity are evident in the presence of 5-1o parts of actin to IOOOparts of myosin (w/w). All myosin samples tested were pure by this criterion, as well as by ultracentrifugal inspection according to MOMMAERTS AND PARRISH 14,15.
e-A ctinin Our preparation of this protein is derived from the procedures of EBASHI AND EBAStII 2, with an additional purification step, as shown in the flowsheet of Scheme I I I . The protein so obtained shows a single peak in the ultracentrifuge (Fig. I), but has not otherwise been extensively tested for purity.
Fig. i. U l t r a c e n t r i f u g a l s e d i m e n t a t i o n p a t t e r n s of purified ~-actinin. A p r e p a r a t i o n c o n t a i n i n g 2.25 m g p r o t e i n / m l in I m M N a H C O z w a s p h o t o g r a p h e d (2o°), 4, 26, 34, a n d 42 rain a f t e r r e a c h i n g a speed of 59 780 r e v . / m i n (a-d, respectively).
Actomyosin In order to maintain direct comparison with the experiments of EBASHI AND EBASI-II~, actomyosin was prepared b y mixing polymerized actin and myosin in the proportion 1:2 (w/w). Other proportions, when used, will be so identified.
A denosine triphosphatase For survey purposes, and in certain experiments of long duration requiring continuous observation, the electrotitrimetric method of GREEN AND MOMMAERTS~2 was used, b y means of the Radiometer pH-stat system. At the chosen p H of 6.8 (which was used in the majority of the turbidity and gel contraction experiments), close to 0.5 equivalents of acid are produced per mole of ATP split, and this is little dependent upon variations in the salt composition15; an initial and final phosphate determination serve to establish the equivalence ratio in each experiment. However, all crucial experiments aiming at the measurements of initial rates and at the accurate comparison of enzymatic activities were done b y phosphate analysis according to FISKE AND SUBBARow16. The assay temperature was 27 °.
Turbidimetric test for superprecipitation As practiced by EBASH117, a major test has been the recording of transmittance changes in actomyosin suspensions upon the addition of ATP. This is done in I cm × I cm glass cells of 4 cm height, in the optical path of the Beckman DU spectrophotometer (at 660 mt~ wavelength) in the Gilford arrangement for recording, at 27 °. The electrolyte medium will be specified in the description of individual experiments; Biochim. Biophys, Acta, 133 (1967) 3 9 9 - 4 I I
405
ACTOMYOSIN AND ~ - A C T I N I N SCHEME
22I
PREPARATION OF ~-ACTININ W i t h m i n o r m o d i f i c a t i o n s f r o m d e s c r i p t i o n s of EBASHI AND EBASHI 1 a n d EBASHI AND MARUYAMAT. All p r o c e d u r e s w e r e c o n d u c t e d a t o °. I COMBINED EXTRACTS A AND B (SCHEME I) (a) A d d 22. 5 g r e c r y s t a l l i z e d NH4SO4/IOO m l e x t r a c t . (b) L e t s t a n d a t o ° for 20 m i n . (c) C e n t r i f u g e a t 5000 x g for i o rain.
SUPERNATANT (contains troponin)
I I SEDIMENT (a) D i s s o l v e i n o. 5 vol. (on b a s i s of o r i g i n a l m u s c l e w t . ) i mM NaHCO~. (b) C e n t r i f u g e a t 2 o o o o × g for 3 ° m i n .
SEDIMENT (discard)
I I I SOPERNATANT (a) A d d i o g NH4SO4/IOO m h (b) L e t s t a n d a t o ° for 20 m i n . (c) C e n t r i f u g e a t 5000 × g for i o rain.
SUPERNATANT (discard)
I V SEDIMENT (a) D i s s o l v e s e d i m e n t in o. 4 vol. i m M N a H C O 3. (b) C e n t r i f u g e a t 2 o o o o × g for 3 ° m i n .
SEDIMENT (discard)
V SUPERNATANT (a) D i a l y z e a g a i n s t I m M N a H C O s for i o h. (b) C e n t r i f u g e a t 2 o o o o × g for i h.
SEDIMENT (discard)
SUPERNATANT (discard)
7 2 SUPERNATANT (Partially purified ~-actinin) (a) A d d KC1 t o 3.3 M. (b) L e t s t a n d a t o ° for 20 m i n . (c) C e n t r i f u g e a t 500o × g for IO rain.
V I I SEDIMENT (a) D i s s o l v e i n 0.32 vol. i m M N a H C O a. (b) D i a l y z e a g a i n s t i m M N a H C O a for IO h. (c) C e n t r i f u g e a t 2 o o o o × g for i h.
SEDIMENT (discard)
V I I I SOPERNATA•T ~-actinin (Steps VI and VII may be repeated). Purified ~-actinin.
Biochim. Biophys. Acta, 133 (1967) 3 9 9 - 4 i r
406
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
the protein concn, is 0.80 mg of actomyosin per ml of final suspension. As will be set forth in the subsequent paper, there is much uncertainty about the actual meaning of this measurement. However, with the given optical arrangement, our results appear to be identical with those of EBASH117 and other workers12, is. A number of precautions must be observed. According to EBASH117, the actomyosin must be in a sufficiently fine suspension, before the addition of ATP, to prevent it from settling during the observation time. Following t~NDO 19, the suspension is to be brought to low ionic strength in KC1 first, and the Mg 2+ is added last, just prior to the introduction of the ATP. Constant temperature is maintained in the cell holder and, in view of the work of Y a s u I AND WATANABE is we have examined the effect of stirring the contents of the cuvet by a magnetically rotated disc in the bottom; with fresh actomyosin, this is not advantageous. In our own experience, it is advisable to dilute the original actomyosin solution slowly with water in small portions, lO-15 volume percent at a time, with gentle mixing.
Protein concentration The amounts of actomyosin and other protein are determined b y the biuret method according to Weichselbaum-Kirk as modified by Beisenherz (see ref. i0), after calibration against a nitrogen analysis for the protein in question.
Treatment of protein fractions Unless stated otherwise, and indeed always in the experiments reported in these first three papers, all proteins needed in an experimental series are freshly prepared in the course of 2 successive days, and stored in closed vessels kept in crushed ice. The experiments are then performed within the next 2 days. RESULTS
The findings presented in this paper comprise a description of the basic experiment of EBASHI AND EBASI-II~ concerning the turbidity changes in a suspension of actomyosin with and without a-actinin, performed under a variety of circumstances, and further extended by simultaneous determinations of the ATPase activity. The influence of several variables will be given seriatim; until the effect of p H itself is discussed, all descriptions refer to experiments at p H 6.8 which was selected in order to remain in conformity with the work of EBASHI AND EBASHI2; it represents a favorable choice, being near the optimal range in some, and within a broad optimal plateau in other media. The temperature is 27 ° , but several crucial points were checked at other temperatures, including 18 ° as employed b y EBASHI AND EBASHI 2, without indications of differences other than with respect to reaction rates.
The effect of the KCl concentration Typical results for tile turbidity response are given in Fig. 2 from which it is seen that in the lower range of KC1 concentrations a-actinin always accelerates the increase in turbidity. None of the experiments used in the illustration duplicates EBASHI'S findings but as will be documented in the following paper, some further modifications of the medium do indeed give rise to his finding that a turbidity response m a y be pronounced in the presence, and not occurring in the absence of a-actinin t~iochim. Biophys. Acta, 133 (1967) 399-41i
407
ACTOMYOSIN AND 0~-ACTININ
also. Thus, the presented material shows that his result is not universal but restricted to very specific circumstances, and that otherwise the occurrence of superprecipitation, as far as indicated by the optical method, is merely accelerated by ~-actinin. At higher KC1 concentrations (Fig. 2d) there is no clear effect : ~-actinin actually lengthens the clearing phase, although it increases the turbidity rise once it occurs. 0.6 = - Actinin , ~ ) -
25mM
=oE "66 g
c
~
(c)
0.4 0.3
~ControI v
==-A
05
0.2
KCI
0.1
a - Actinin
~5 :6 23
IOOmM KCI
0.:
Control
0.1
~ ~ ~ Time (min)
ib
z
16
~
20 22 Time ( rain)
24
26
28
3b
Fig. 2. Effect of ~-actinin u p o n superprecipitation of r e c o n s t i t u t e d a c t o m y o s i n at various KC1 concentrations. Final concn.: KC1 as indicated; i inM MgCI2; i mM ATP; 20 mM T r i s - m a l e a t e (total) (pH 6.8); 0.8 m g actomyosin/inl; 0.24 mg ~-actinin/m]. Temp., 27 °.
2
~-
a - Actinin
5
I0
5
I0 15 20 150raM KCI
ft 5
10
15
15
20
5
I0
15
2'0
5
I0
15
20
Controt
Time [ min )
45
Fig. 3. Effect of 0~-actinin u p o n tile Mg2+-activated ATPase of reconstituted a c t o m y o s i n at various KC] concentrations. Final conch. : KC1 as indicated ; I mM MgC12 ; t m M ATP; 2o mM T r i s - m a l e a t e ( p H 6.8); actomyosin o.18 mg/inl; ~-actinin 0.054 mg/ml. Temp., 27 °.
Biochim. J~iophys. Acta, 133 (1967) 399-411
408
I~. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
The physiological ionic strength is above o. I, thus above the region of an acceleration b y a-actinin. The ATPase activities in a parallel series of experiments (Fig. 3) show an influence of a-actinin which is roughly parallel with the effects upon the turbidity change, but of small magnitude. The largest accelerations are found at low ionic strengths, but they amount to less than 50 % increase; at ioo mM KC1, the addition of a-actinin is indifferent (Fig. 3d), at 15o mM (Fig. 3e) this component becomes inhibitory. A similar stimulation of the ATPase activity has just been announced in a note by MARUYAMA 2°. Without further documentation it can be stated that identical results are obtained whether the actin has been prepared by our own procedure of Scheme I I (as in Figs. 2 and 3) or with an actin extensively purified as outlined in Scheme I. It can further be stated that the results are not affected b y the absence or presence of small amounts of CaC12 (e.g. 5"1o-5 M) or of EDTA, neither with respect to the ATPase activity or turbidity response as such, nor with regard to the effect of a-actinin upon these rates. Thus, all proteins are free of troponin, and the effect of c~-actinin is not related to, or influenced by, the effects of Ca 2+.
Tropomyosin We have also examined the reactions of actomyosin and a-actinin in the presence of tropomyosin. Under certain conditions 5, this protein can modify the turbidity response of actomyosin, notably by increasing the clearing phase at KC1 concentrations above o.I M. However, the effects of ~-actinin are not further modified by tropomyosin, present to the amount of one-fourth of the actin, regardless of whether the tropomyosin is added to the actin prior to its polymerization, or afterwards, or added only subsequently to the actomyosin.
Composition of the buffer A considerable amount of experimentation has been devoted to surveying the reactions in different buffers. Phosphate, Tris-maleate, Tris-acetate, and histidine buffers were included. Despite individual differences in the rates and degrees of the turbidity responses, the picture presented with respect to the effect of a-actinin is not modified by this additional material. Reactions in some of these media will be described in the following papers.
Effects of Mg 2+ and of A TP concentration While in the experiments so far both Mg 2+ and ATP were present in I mM concentration, some variations of this condition have been studied. Without Mg 2+ at 50 mM KC1 and I mM ATP, there is a very rapid turbidity response and an inhibition by a-actinin whereas with Mg 2+ at I or 2 mM the turbidity response b y itself becomes suppressed, but is now markedly accelerated by a-actinin. The ATPase is moderately activated by Mg 2+, and its acceleration b y ~-actinin becomes pronounced only in the presence of this ion. Variation of the ATP concentration, illustrated in Fig. 4 for I mM MgC12 and 50 mM KC1 modifies the turbidity response in the sense that increasing ATP concentrations suppress the rise in turbidity. The addition of a-actinin then promotes the turbidity rise; shown in Fig. 4 for I and 3 mM ATP only, this applied equally to the Biochim. Biophys. Acta, 133 (1967) 399-411
409
ACTOMYOSIN AND ~-ACTININ
intermediate concentrations. It will be observed that the experiments at 3 mM ATP in this figure provide an example of the validity of the basic experiment of EBASHI AND EBASHI 2, the same result can be obtained at 3 mM MgC12.
-6
ImM ATP ,'~'--"-~+ a - Actinin ~ _ _ 3 r n M
ATP ÷ (]-Aclinin
:t..5E
o ~0
I mM ATP
g h~
2.SmM ATP
~ll
3mM ATP f
4
6
8 10 1'2 Time (min)
14
SB
38
40
Fig. 4. Effect of a-actinin u p o n the s u p e r p r e c i p i t a t i o n of reconstituted a c t o m y o s i n at various ATP concentrations. Final concn. : 5 ° mM KCI; i mM MgC12; 2o mM T r i s - m a l e a t e (total) (pH 6,8); o.S m g / m l a c t o m y o s i n ; o.2 4 m g / m l a-actinin; ATP as indicated.
Variation of pH The effect of p H in a series of experiments in 30 mM KC1 with a total concn. of 60 mM of the components of Tris-maleate buffer, is shown in Fig. 5 a, over the limited range of p H values covered by such buffers and presumably encompassing the values of possible physiological significance. This experiment is at I mM MgC12 and 3 mM ATP. The curve for actomyosin as such follows the familiar pattern, including the first indication of the alkaline activation2L With a-actinin, the moderate activation is seen to be limited mostly to the more neutral regions. The solid lines without individual data points give the difference between the two experimental curves, and thus represent the activation b y c~-actinin as a function of pH. Thus, it is seen that the majority of experiments performed at p H 6.8 are situated toward
i!
....
I'"o ......
h-
6.0
65
Z0
75
8D
8.5
pH
6D
6.5
70
75
80
85
Fi~. 5. Effect of ~-actinin upon the Mg2+-activated ATPase of r e c o n s t i t u t e d a c t o m y o s i n at various pi-I values. Final concn.: I 111~{ MgCI2; 6o mM T r i s - m a l e a t e (total); o.i8 m g / m l actomyosin; o.o54 m g / m l ~-actinin. a, KC1 30 mM, ATP 3 mM; b, KCI 30 mM, A T P z mM; c, KC1 ioo raM, ATP 3 mM; d, KC1 125 raM, ATP 3 raM.
Biochim. Biophys. Acta, 133 (19671 399-411
410
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
the alkaline side but still close to the activation maximum. Under the same conditions but for the I mM ATP concentration used in most experiments, the results are about identical above p H 7, but below this p H the activities are less both with and without a-actinin. This is illustrated in Fig. 5b. Here the optimum p H for the activation by a-actinin is distinctly below the p H 6.8 used in most of this work. At higher concentrations of KC1 the ATPase activity and the activation by a-actinin become less at all p H values, as shown in Fig. 5c for 125 mM KC1. The turbidity responses, not illustrated, have broad optima in the range between about 6. 3 and 7-5, and the choice of p H 6.8 is indeed suitable for the demonstration of the dependence upon the composition of the medium, and of the activation by a-actinin. DISCUSSION
Since this paper is of an introductory nature, surveying the conditions influencing the ATPase activity and the turbidity response of actomyosin as affected by a-actinin, no extensive discussion is required. Two conclusions, however, do emerge with some prominence, and warrant explicit formulation. The finding b y EBASHI AND EBASHI 2, that the turbidity increase induced by ATP in a suspension of actomyosin requires a-actinin, can be confirmed only under very specific conditions of ATP and Mg ~+ concentrations, and at low ionic strength. Under other conditions, the turbidity response requires no a-actinin, but is merely accelerated b y this component; and at higher salt concentrations, approaching or including the physiological range, no activation by cc-actinin is found at all. On the other hand, extending the work of EBASHI b y including ATPase determinations, we find that actomyosin requires no a-actinin in order to split ATP. At low ionic strengths, a-actinin m a y cause a moderate activation of the ATPase activity, at higher ionic strengths including the vital range, this protein turns indifferent and even inhibitory. These orientating investigations, therefore, cast doubt upon the necessity of a-actinin as a required factor and upon its significance as a physiological accelerator. They do confirm, however, that c~-actinin interacts with actomyosin and can modify its responses. In the following papers, we present a detailed analysis of the nature of this modification, and a sharper discussion as to its possible physiological meaning. ACKNOWLEDGEMENTS
We would like to thank Mrs. M. SUH and Mrs. M. MARUSICHfor their invaluable technical assistance in all phases of this work. Appreciation is also expressed to Mrs. M. SLATER for m a n y useful discussions. One of us (E.J.B.) expresses appreciation to A.M.I.F., Chicago, Ii1., for fellowship support. This work was supported in part b y Research Grant HE03o67 of The National Heart Institute, National Institutes of Health, Bethesda, Md. REFERENCES
I S. ]~BASHI AND l~'. EBASHI, J. Biochem., 55 (1964) 6042 S. EBASHI AND F. EBASHI, f . Bioch~m., 58 (196.5) 7. 3 S. V. PERRY AND T. C. GREY, Biochem. J., 64 (1956) 5.
Biochim. Biophys. dcta, 133 (1967) 3 9 9 - 4 I I
ACTOMYO/,qlN AND ~x-ACTININ 4 5 6 7 ,~ 9 lo i[ i2 I3 ]4 15 16 17 18 19 20 2t 22
411
A. WEBER AND S. W1NICUR, J . Biol. Chem., 236 (1961) 3198. A. M. 1-~ATZ, dr. Biol. Chem., 239 (1964) 3304. _~I. MORALES AND J. BOTTS, Arch. Biochem. Biophys., 37 (1952) 283. ~. EIC;;ASHI AND I~. MARUYAMA, .]. Biochem., 58 (I965) 20. \\;. F. t-~. M. MOMMAERTS, J. Biol. Chem., 198 (1952) 445. ~I. CARSTEN AND V~7. F. H. M. MOMMAERTS, Biochemistry, 2 (1963) 28. \\~. 12~. J~[. M. MOMMAERTS, Methods Med. Bes. 7 (1958) 3A SZENT-GY6RG¥I, Chemistry of Muscular Contraction, A c a d e m i c Press, N e w Y o r k , i 9 5 t, p. r46. N. AZI'MA AND S. WATANABE, J. Biol. Chem., 240 (1965) 3847 • V. NESSLER AND S. S. SPICER, Biochim. Biophys. Acta, 8 (1952) 474. W. F. H. M. MOMMAERTS AND R. G. PARRISH, J . Biol. Chem., 188 (1951) .545. R. G. PARRISH AND V~7. F. H. M. MOMMAERTS, J . Biol. Chem., 209 (1954) 9Ol. C. H. FISKE AND Y. SOBBAROW, J. Biol. Chem., 66 (1925 ) 375. S. EBASHI, dr . Biochem., 5 ° (1961) 236. A. YASUI AND S. XVATANABE, J. Biol. Chem, 240 (1965) 98. M. ENDO, .[. Biochem., 55 (1964) 614K. MARUYAMA, J . Biochem., 59 (1966) 422. W . F. H. M. MOlVIMAERTS AND K. SERAYDARIAN, J. Gen. Physiol., 305 (I947) 4Ol. I. GREEN AND W , F. H . ~I~. MOMMAERTS, J. Biol. Chem., 202 (1953) 541.
Biochim. Biophys. Acta, 133 (1967) 3 9 9 - 4 1 1
BIOCHIMI@A ET BIOPHYSICA ACTA
BBA 3 5 o 5 4
T H E MODIFICATION OF ACTOMYOSIN BY a-ACTININ* I. A SURVEY OF E X P E R I M E N T A L CONDITIONS K. S E R A Y D A R I A N , E. J. B R I S K E Y * * AND VVT. F. H. M. MOMMAERTS
The Departments of Medicine (The Los Angeles County Heart Association, Cardiovasczdar Researcli Laboratory), and Physiology, UCLA School of Medicine, Los Angeles, Calif. (U.S.A.) (Received S e p t e m b e r ieth, 1966)
SUMMARY
The finding by EBASHI that the superprecipitation of actomyosin, as measured in a turbidimetric assay, requires a-actinin can be confirmed under specific conditions of ATP concentration and cationic composition. Under other conditions, however, including notably ionic strengths in the physiological range, the presence of a-actinin merely accelerates the turbidity change or is without influence. The ATPase activity of actomyosin is activated only little by a-actinin, only at low ionic strength. This study constitutes a survey of experimental conditions basic to the following publications.
INTRODUCTION
Recent contributions by EBASHI AND EBASHI1,2 on the subject of regulatory proteins affecting the interactions between actin, myosin and ATP, have led to new viewpoints in the interpretation of the control of these interactions as occurring in contraction and relaxation of muscle. Foreshadowed by the observations of PERRY AND GREY3 and WEBER AND WINICUR¢ on the variability of actin (giving rise to 'relaxing' and 'non-relaxing' actomyosins), by KATZ5 on effects of tropomyosin upon the turbidity changes in actomyosins, and indeed b y the early suspicion expressed by MORALES AND BOTTS6 on possible differences between 'natural' and 'synthetic' actomyosin, EBASHI AND EBASHI 1,2 have announced tile discovery of troponin*** conferring a calcium requirement upon actomyosin, and of a-actinin necessary for contraction to occur at all. In our extension of this work, we have confirmed the described properties of troponin, but have found the effects of a-actinin to be much more complex; we conclude that they are not to be interpreted in the indicated sense of identifying a-actinin as a required factor in the contraction process. " This series of three papers is dedicated to Prof. HANS H. ~VEBER at the occasion of his 7oth a n n i v e r s a r y , and in recognition of his continued pioneering investigation in the contractile protein s y s t e m of muscle. ** F o r the period 1965-1966, E. J. B. h a s been a Visiting Scholar from the D e p a r t m e n t of Meat a n d Animal Science, The U n i v e r s i t y of Wisconsin, Madison, Wisc., U.S.A. ** * According to a personal c o m m u n i c a t i o n by S. EBASHI (1965) the fraction of native tropomyosin t which confers calcium sensitivity to a c t o m y o s i n is being t e r m e d ' t r o p o n i n ' .
Biochim. Biophys. Acta, 133 (1967) 399-411
400
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
SCHEME I PREPARATION OF ~-ACTININ-FREE ACTIN W i t h m i n o r m o d i f i c a t i o n s from p a r t i a l d e s c r i p t i o n s in s e v e r a l m a n u s c r i p t s l , 2 , ~. A n i m a l s are gi ve n s o d i u m p e n t o b a r b i t a l (90 rag) a n d ~ - t u b o c u r a r i n e c h l o r i d e (1. 5 rag) pri or to e x s a n g u i n a t i o n . Muscles are excised i m m e d i a t e l y , i m m e r s e d in ice, a n d c o a r s e l y (2 mm) g r o u n d . All e x t r a c t a n t v o l u m e s up to S t e p X V I , for ~-actinin-free a c t i n p r e p a r a t i o n s are b a s e d on w e i g h t of a c e t o n e - d r i e d powder. I MUSCLE TISSUE (IO0 gin) (a) E x t r a c t w i t h 3.3 voh (O.15 M KH~PO4, o. 3 M KC1) (pH 6.5, o°). (b) Stir 15 rain a n d t h e n s l o w l y a d d 13. 3 vol. H 2 0 w hi l e s t i r r i n g . (c) S t r a i n t h r o u g h one l a y e r of g a u z e a n d squeeze.
FILTRATE
n RESIDUE
(use for m y o s i n A preparation)
FILTRATE
(a) W a s h t w i c e (IO m i n each) w i t h 3 vol. (o.o2 M KCI-o.2 mM N a H C O a ). (b) W a s h t w i c e (5 m i n each) w i t h 3 v o h H 2 0 . (c) S t r a i n t h r o u g h one l a y e r of g a u z e a f t e r e a c h w a s h a n d squeeze g e n t l y .
I I I RESIDUE
(discard)
I
(a) A d d I vol. H~O a n d l e t s t a n d 4 h a t 20 °. (b) F i l t e r ( W h a t m a n No. 4 paper), for 4 h, a t 20 °.
FILTRATE I V RESIDUE ( e x t r a c t A for a - a c t i n i n preparation) (a) A d d i voh i mM N a H C O 3 a n d l e t s t a n d 5 h a t 20 ° (b) F i l t e r for 4 h a t 20 °.
FILTRATE
V RESIDUE
( e x t r a c t B for a - a c t i n i n preparation)
FILTRATE (discard)
(a) A d d 2 vol. I mM N a H C O 3 a n d l e t s t a n d 2 h a t 20 °. (b) F i l t e r 2 h a t 20 °.
V I RESIDUE (a) A d d M KC1 to final concn, of o.oi M (I m l / I o o g). (b) L e t s t a n d i h a t 20 ° a n d filter for i h a t 2o ° .
FILTRATE (discard)
vii
RESIDUE
(a) A d d 2 vol. o . o i M N a H C O 3 a n d s t i r I h a t 20 ° . (b) F i l t e r 1-2 h t h r o u g h filter p a p e r .
FILTRATE
viii
RESIDUE
(discard) (a) A d d 4 voh I mM N a H C O 3 a n d s t i r I h a t 20 ° . (b) P a s s t h r o u g h gauze.
Biochim. Biophys..4cta, 333 (1967) 399-411
401
ACTOMYOSIN AND ~-ACTININ SCHEME I
(continued)
I FILTRATE
I X RESIDUE
(discard) (a) Repeat Step V I I I
FILTRATE
X RESIDUE
(discard) (a) Add 2 voh H20 and stir 3 ° rain. at 2o °. (b) Pass through gauze.
FILTRATE
X I RESIDUE
(discard) (a) Repeat Step X.
FILTRATE
X I I RESIDUE
(discard) (a)Add 3 vol. acetone at o °. (b) After 5 min pass through gauze.
FILTRATE
X I I I RESIDUE
(discard) (a) Add I vol. acetone at o °. (b) After 5 min pass through gauze.
FILTRATE
XIV RESIDUE
(discard) (a) Repeat Steps X I I and X I I I .
FILTRATE
XV RESIDUE
(discard) (a) Dry at room temperature.
X V I ACETONE-DRIED POWDER
(a) Extract with 0.2 mM ATP and 0.2 mM ascorbate (pH 7-9) and continue as described in Scheme II Step VI.
t METHODS
A ctin M u c h of t h e e x p e r i m e n t a l f o u n d a t i o n of t h i s w o r k rests u p o n t h e a v a i l a b i l i t y of p u r e a c t i n , n o t a b l y a p r e p a r a t i o n n o t c o n t a m i n a t e d w i t h ~-actinin. A l e n g t h y p r o c e d u r e d e v e l o p e d b y EBASHI AND MARUYAMA7 assures t h i s d e g r e e of p u r i t y , a n d we h a v e used, as s t a n d a r d s of c o m p a r i s o n , a c t i n s p r e p a r e d a c c o r d i n g to t h i s m e t h o d , w h i c h , w i t h s o m e m o d i f i c a t i o n s a n d e x t e n s i o n s is g i v e n in t h e f l o w s h e e t of S c h e m e I. B e s i d e s t h i s e l a b o r a t e p r o c e d u r e w e h a v e also, a n d i n c r e a s i n g l y , u s e d a m o d i f i -
Biochim. Biophys. Acta, 133 (1967) 399-411
402
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
cation of our original methodS, 9 by incorporating a precipitation step with 3-3 M KC1 (ref. 2) to remove ~-actinin. This method is given in the flowsheet of Scheme II. The KC1 precipitation is applied directly to the crude extract, and is followed by the ultracentrifugation step desirable to clear the extract (no polymerization occurs at this high ionic strength). This centrifugation results in the removal of some fatty material by flotation, and of some debris; no observable protein precipitate appears at this point, nor is any precipitate obtained b y 3-o-3.3 M KC1 at later stages in the preparation when the protein concentrations are higher. Since, furthermore, the same results are obtained with our routine actin preparations as with the elaborately purified standard, we conclude that the standard method of this laboratory, when properly executed, yields pure actin also according to this new criterion. However, this conclusion was drawn only after the experience gained in the course of this work; all experiments reported here were done with actin subjected to the additional KC1 step, i.e. with procedures outlined in Schemes I or II. Actin prepared b y any of our methods shows, after polymerization, one distinct hypersharp boundary during ultracentrifugal sedimentation at 20410 rev./min. SCHEME II PREPARATION OF PURE ACTIN Modification of the s t a n d a r d m e t h o d of this laboratory. Animals given s o d i u m p e n t o b a r b i t o l (9o mg) a n d D-tubocurarine chloride (I.5 rag) prior to exsanguination. Muscles excised immediately and coarsely (2 mm) ground. All procedures except drying conducted at o °. All e x t r a c t a n t volumes u p to Step VI are based on the weight of the muscle used for the first extraction. E x t r a c t a n t volumes, after Step V, are based on weight of acetone-dried powder. I MUSCLE TISSUE (IO0 g) (a) E x t r a c t w i t h 3.3 vol. G u b a - S t r a u b soln. (o.15 M K H 2 P O ~, 0. 3 M KC1) (pt-I 6.5). (b) Stir 15 min a n d t h e n slowly add 13. 3 vol. H 2 0 while stirring. (c) Strain t h r o u g h two layers of gauze and squeeze.
FILTRATE (use for m y o s i n A preparation)
Filtrate (discard)
I I RESIDUE (a) Add 4 vol. 0.05 M N a H C O a and e x t r a c t for 20 nlin while stirring. (b) Strain t h r o u g h two layers of gauze.
I I I RESIDUE (a) E x t r a c t with i vol. soln. (0.05 M N a H C O 3 and o.o5 M Na~CO3) tor io min and add io vol. 5" lO-5 M CaCI v (b) After IO rain strain t h r o u g h two layers of gauze and squeeze.
FILTRATE (discard)
IV ~ESIDUE (a) W a s h 3 times (5 min each) with 3 vol. each of cold acetone a n d pass t h r o u g h gauze after each wash.
Biochim. Biophys. Acta, I33 (1967) 399-411
4o3
ACTOMYOSIN AND ~-ACTININ S C H E M E II
(continued)
I V RESIDUE
FILTRATE
(discard) (a) S p r e a d on filter p a p e r a n d d r y at r o o m t e m p e r a t u r e . ~7I ACETONE-DRIED POVCDER
(a) E x t r a c t twice (3 ° rain each) w i t h 0.2 m M A T P a n d 0.2 m M a s c o r b a t e (pH 7.9) a t o (io vol. each). (b) Filter, u s i n g s u c t i o n in cold a n d c o m b i n e filtrates (wash p a p e r w i t h H 2 0 a n d A T P ascorbate)
V i i FILTRATE l (a) Centrifuge a t 7 8 o o o × g at o ° f o r 6 o m i n .
RESIDUE
(discard) i
V I I I SUPERNATANT
SEDIMENT
(discard) (a)Make 3-3 M KC1 u s i n g solid KC1. (b) H o l d IO m i n a t o ° t h e n c e n t r i f u g e at 20000 × g for io rain. (c) Filter at o ° u s i n g s u c t i o n (wash p a p e r w i t h H 2 0 a n d A T P - a s c o r b a t e ) . I X FILTRATE
(a) Dialyze a g a i n s t 33 vol. of 0. 3 m M Na]-ICO 3 for lO h. (b) C e n t r i f u g e at 78000 × g for 3 h.
4 SUPERNATANT
X PELLET
(discard) (a) R i n s e walls of t u b e w i t h H 2 0 of o °. (b) T a k e u p in io vol. of 0.2 m M A T P a n d 0,2 niM a s c o r b a t e (pH 7.9) (glass homogenizer). (c) Centrifuge depolynIerized actin at 78000 × g for 3 ° rain.
SEDIMENT
X I SUPBRNATANT
(discard) (a) B r i n g to o.I M KC1 a n d let polymerize. (b) Centrifuge at 78ooo × g for 3 h.
SUPERNATANT
XlI PELLET
(discard) (a) R i n s e wall of t h e t u b e w i t h H 2 0 of o °. (b) T a k e u p in 2 - 3 vol. of o.i M KC1 (final concn.). (c) Dialyze a g a i n s t large vol. of o.i M KC1 for 6 h. X I I I PURE ACTIN
Myosin Routinely, this protein was prepared by the method of this laboratory 1°. The final solution is in 0.5 M KC1. In the course of this work, a number of variations have also been studied, by preparing myosin according to SZENT-GY6RGYIn, AZUMA AND Biochim. Biophys. dcta, 133 (i967) 399-411
404
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
WATANABE12 and EBASHI AND ~ B A S H I 1. NO differences in myosin behavior were seen as a result of these modifications. A sensitive criterion for the purity of myosin, in terms of the presence or absence of actin or actomyosin, is due to KESSLER AND SPICER as modified by AZUMA AND WATANABE12. At a suitably selected ion composition, while the addition of ATP gives rise to no turbidity change in myosin, marked increases in turbidity are evident in the presence of 5-1o parts of actin to IOOOparts of myosin (w/w). All myosin samples tested were pure by this criterion, as well as by ultracentrifugal inspection according to MOMMAERTS AND PARRISH 14,15.
e-A ctinin Our preparation of this protein is derived from the procedures of EBASHI AND EBAStII 2, with an additional purification step, as shown in the flowsheet of Scheme I I I . The protein so obtained shows a single peak in the ultracentrifuge (Fig. I), but has not otherwise been extensively tested for purity.
Fig. i. U l t r a c e n t r i f u g a l s e d i m e n t a t i o n p a t t e r n s of purified ~-actinin. A p r e p a r a t i o n c o n t a i n i n g 2.25 m g p r o t e i n / m l in I m M N a H C O z w a s p h o t o g r a p h e d (2o°), 4, 26, 34, a n d 42 rain a f t e r r e a c h i n g a speed of 59 780 r e v . / m i n (a-d, respectively).
Actomyosin In order to maintain direct comparison with the experiments of EBASHI AND EBASI-II~, actomyosin was prepared b y mixing polymerized actin and myosin in the proportion 1:2 (w/w). Other proportions, when used, will be so identified.
A denosine triphosphatase For survey purposes, and in certain experiments of long duration requiring continuous observation, the electrotitrimetric method of GREEN AND MOMMAERTS~2 was used, b y means of the Radiometer pH-stat system. At the chosen p H of 6.8 (which was used in the majority of the turbidity and gel contraction experiments), close to 0.5 equivalents of acid are produced per mole of ATP split, and this is little dependent upon variations in the salt composition15; an initial and final phosphate determination serve to establish the equivalence ratio in each experiment. However, all crucial experiments aiming at the measurements of initial rates and at the accurate comparison of enzymatic activities were done b y phosphate analysis according to FISKE AND SUBBARow16. The assay temperature was 27 °.
Turbidimetric test for superprecipitation As practiced by EBASH117, a major test has been the recording of transmittance changes in actomyosin suspensions upon the addition of ATP. This is done in I cm × I cm glass cells of 4 cm height, in the optical path of the Beckman DU spectrophotometer (at 660 mt~ wavelength) in the Gilford arrangement for recording, at 27 °. The electrolyte medium will be specified in the description of individual experiments; Biochim. Biophys, Acta, 133 (1967) 3 9 9 - 4 I I
405
ACTOMYOSIN AND ~ - A C T I N I N SCHEME
22I
PREPARATION OF ~-ACTININ W i t h m i n o r m o d i f i c a t i o n s f r o m d e s c r i p t i o n s of EBASHI AND EBASHI 1 a n d EBASHI AND MARUYAMAT. All p r o c e d u r e s w e r e c o n d u c t e d a t o °. I COMBINED EXTRACTS A AND B (SCHEME I) (a) A d d 22. 5 g r e c r y s t a l l i z e d NH4SO4/IOO m l e x t r a c t . (b) L e t s t a n d a t o ° for 20 m i n . (c) C e n t r i f u g e a t 5000 x g for i o rain.
SUPERNATANT (contains troponin)
I I SEDIMENT (a) D i s s o l v e i n o. 5 vol. (on b a s i s of o r i g i n a l m u s c l e w t . ) i mM NaHCO~. (b) C e n t r i f u g e a t 2 o o o o × g for 3 ° m i n .
SEDIMENT (discard)
I I I SOPERNATANT (a) A d d i o g NH4SO4/IOO m h (b) L e t s t a n d a t o ° for 20 m i n . (c) C e n t r i f u g e a t 5000 × g for i o rain.
SUPERNATANT (discard)
I V SEDIMENT (a) D i s s o l v e s e d i m e n t in o. 4 vol. i m M N a H C O 3. (b) C e n t r i f u g e a t 2 o o o o × g for 3 ° m i n .
SEDIMENT (discard)
V SUPERNATANT (a) D i a l y z e a g a i n s t I m M N a H C O s for i o h. (b) C e n t r i f u g e a t 2 o o o o × g for i h.
SEDIMENT (discard)
SUPERNATANT (discard)
7 2 SUPERNATANT (Partially purified ~-actinin) (a) A d d KC1 t o 3.3 M. (b) L e t s t a n d a t o ° for 20 m i n . (c) C e n t r i f u g e a t 500o × g for IO rain.
V I I SEDIMENT (a) D i s s o l v e i n 0.32 vol. i m M N a H C O a. (b) D i a l y z e a g a i n s t i m M N a H C O a for IO h. (c) C e n t r i f u g e a t 2 o o o o × g for i h.
SEDIMENT (discard)
V I I I SOPERNATA•T ~-actinin (Steps VI and VII may be repeated). Purified ~-actinin.
Biochim. Biophys. Acta, 133 (1967) 3 9 9 - 4 i r
406
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
the protein concn, is 0.80 mg of actomyosin per ml of final suspension. As will be set forth in the subsequent paper, there is much uncertainty about the actual meaning of this measurement. However, with the given optical arrangement, our results appear to be identical with those of EBASH117 and other workers12, is. A number of precautions must be observed. According to EBASH117, the actomyosin must be in a sufficiently fine suspension, before the addition of ATP, to prevent it from settling during the observation time. Following t~NDO 19, the suspension is to be brought to low ionic strength in KC1 first, and the Mg 2+ is added last, just prior to the introduction of the ATP. Constant temperature is maintained in the cell holder and, in view of the work of Y a s u I AND WATANABE is we have examined the effect of stirring the contents of the cuvet by a magnetically rotated disc in the bottom; with fresh actomyosin, this is not advantageous. In our own experience, it is advisable to dilute the original actomyosin solution slowly with water in small portions, lO-15 volume percent at a time, with gentle mixing.
Protein concentration The amounts of actomyosin and other protein are determined b y the biuret method according to Weichselbaum-Kirk as modified by Beisenherz (see ref. i0), after calibration against a nitrogen analysis for the protein in question.
Treatment of protein fractions Unless stated otherwise, and indeed always in the experiments reported in these first three papers, all proteins needed in an experimental series are freshly prepared in the course of 2 successive days, and stored in closed vessels kept in crushed ice. The experiments are then performed within the next 2 days. RESULTS
The findings presented in this paper comprise a description of the basic experiment of EBASHI AND EBASI-II~ concerning the turbidity changes in a suspension of actomyosin with and without a-actinin, performed under a variety of circumstances, and further extended by simultaneous determinations of the ATPase activity. The influence of several variables will be given seriatim; until the effect of p H itself is discussed, all descriptions refer to experiments at p H 6.8 which was selected in order to remain in conformity with the work of EBASHI AND EBASHI2; it represents a favorable choice, being near the optimal range in some, and within a broad optimal plateau in other media. The temperature is 27 ° , but several crucial points were checked at other temperatures, including 18 ° as employed b y EBASHI AND EBASHI 2, without indications of differences other than with respect to reaction rates.
The effect of the KCl concentration Typical results for tile turbidity response are given in Fig. 2 from which it is seen that in the lower range of KC1 concentrations a-actinin always accelerates the increase in turbidity. None of the experiments used in the illustration duplicates EBASHI'S findings but as will be documented in the following paper, some further modifications of the medium do indeed give rise to his finding that a turbidity response m a y be pronounced in the presence, and not occurring in the absence of a-actinin t~iochim. Biophys. Acta, 133 (1967) 399-41i
407
ACTOMYOSIN AND 0~-ACTININ
also. Thus, the presented material shows that his result is not universal but restricted to very specific circumstances, and that otherwise the occurrence of superprecipitation, as far as indicated by the optical method, is merely accelerated by ~-actinin. At higher KC1 concentrations (Fig. 2d) there is no clear effect : ~-actinin actually lengthens the clearing phase, although it increases the turbidity rise once it occurs. 0.6 = - Actinin , ~ ) -
25mM
=oE "66 g
c
~
(c)
0.4 0.3
~ControI v
==-A
05
0.2
KCI
0.1
a - Actinin
~5 :6 23
IOOmM KCI
0.:
Control
0.1
~ ~ ~ Time (min)
ib
z
16
~
20 22 Time ( rain)
24
26
28
3b
Fig. 2. Effect of ~-actinin u p o n superprecipitation of r e c o n s t i t u t e d a c t o m y o s i n at various KC1 concentrations. Final concn.: KC1 as indicated; i inM MgCI2; i mM ATP; 20 mM T r i s - m a l e a t e (total) (pH 6.8); 0.8 m g actomyosin/inl; 0.24 mg ~-actinin/m]. Temp., 27 °.
2
~-
a - Actinin
5
I0
5
I0 15 20 150raM KCI
ft 5
10
15
15
20
5
I0
15
2'0
5
I0
15
20
Controt
Time [ min )
45
Fig. 3. Effect of 0~-actinin u p o n tile Mg2+-activated ATPase of reconstituted a c t o m y o s i n at various KC] concentrations. Final conch. : KC1 as indicated ; I mM MgC12 ; t m M ATP; 2o mM T r i s - m a l e a t e ( p H 6.8); actomyosin o.18 mg/inl; ~-actinin 0.054 mg/ml. Temp., 27 °.
Biochim. J~iophys. Acta, 133 (1967) 399-411
408
I~. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
The physiological ionic strength is above o. I, thus above the region of an acceleration b y a-actinin. The ATPase activities in a parallel series of experiments (Fig. 3) show an influence of a-actinin which is roughly parallel with the effects upon the turbidity change, but of small magnitude. The largest accelerations are found at low ionic strengths, but they amount to less than 50 % increase; at ioo mM KC1, the addition of a-actinin is indifferent (Fig. 3d), at 15o mM (Fig. 3e) this component becomes inhibitory. A similar stimulation of the ATPase activity has just been announced in a note by MARUYAMA 2°. Without further documentation it can be stated that identical results are obtained whether the actin has been prepared by our own procedure of Scheme I I (as in Figs. 2 and 3) or with an actin extensively purified as outlined in Scheme I. It can further be stated that the results are not affected b y the absence or presence of small amounts of CaC12 (e.g. 5"1o-5 M) or of EDTA, neither with respect to the ATPase activity or turbidity response as such, nor with regard to the effect of a-actinin upon these rates. Thus, all proteins are free of troponin, and the effect of c~-actinin is not related to, or influenced by, the effects of Ca 2+.
Tropomyosin We have also examined the reactions of actomyosin and a-actinin in the presence of tropomyosin. Under certain conditions 5, this protein can modify the turbidity response of actomyosin, notably by increasing the clearing phase at KC1 concentrations above o.I M. However, the effects of ~-actinin are not further modified by tropomyosin, present to the amount of one-fourth of the actin, regardless of whether the tropomyosin is added to the actin prior to its polymerization, or afterwards, or added only subsequently to the actomyosin.
Composition of the buffer A considerable amount of experimentation has been devoted to surveying the reactions in different buffers. Phosphate, Tris-maleate, Tris-acetate, and histidine buffers were included. Despite individual differences in the rates and degrees of the turbidity responses, the picture presented with respect to the effect of a-actinin is not modified by this additional material. Reactions in some of these media will be described in the following papers.
Effects of Mg 2+ and of A TP concentration While in the experiments so far both Mg 2+ and ATP were present in I mM concentration, some variations of this condition have been studied. Without Mg 2+ at 50 mM KC1 and I mM ATP, there is a very rapid turbidity response and an inhibition by a-actinin whereas with Mg 2+ at I or 2 mM the turbidity response b y itself becomes suppressed, but is now markedly accelerated by a-actinin. The ATPase is moderately activated by Mg 2+, and its acceleration b y ~-actinin becomes pronounced only in the presence of this ion. Variation of the ATP concentration, illustrated in Fig. 4 for I mM MgC12 and 50 mM KC1 modifies the turbidity response in the sense that increasing ATP concentrations suppress the rise in turbidity. The addition of a-actinin then promotes the turbidity rise; shown in Fig. 4 for I and 3 mM ATP only, this applied equally to the Biochim. Biophys. Acta, 133 (1967) 399-411
409
ACTOMYOSIN AND ~-ACTININ
intermediate concentrations. It will be observed that the experiments at 3 mM ATP in this figure provide an example of the validity of the basic experiment of EBASHI AND EBASHI 2, the same result can be obtained at 3 mM MgC12.
-6
ImM ATP ,'~'--"-~+ a - Actinin ~ _ _ 3 r n M
ATP ÷ (]-Aclinin
:t..5E
o ~0
I mM ATP
g h~
2.SmM ATP
~ll
3mM ATP f
4
6
8 10 1'2 Time (min)
14
SB
38
40
Fig. 4. Effect of a-actinin u p o n the s u p e r p r e c i p i t a t i o n of reconstituted a c t o m y o s i n at various ATP concentrations. Final concn. : 5 ° mM KCI; i mM MgC12; 2o mM T r i s - m a l e a t e (total) (pH 6,8); o.S m g / m l a c t o m y o s i n ; o.2 4 m g / m l a-actinin; ATP as indicated.
Variation of pH The effect of p H in a series of experiments in 30 mM KC1 with a total concn. of 60 mM of the components of Tris-maleate buffer, is shown in Fig. 5 a, over the limited range of p H values covered by such buffers and presumably encompassing the values of possible physiological significance. This experiment is at I mM MgC12 and 3 mM ATP. The curve for actomyosin as such follows the familiar pattern, including the first indication of the alkaline activation2L With a-actinin, the moderate activation is seen to be limited mostly to the more neutral regions. The solid lines without individual data points give the difference between the two experimental curves, and thus represent the activation b y c~-actinin as a function of pH. Thus, it is seen that the majority of experiments performed at p H 6.8 are situated toward
i!
....
I'"o ......
h-
6.0
65
Z0
75
8D
8.5
pH
6D
6.5
70
75
80
85
Fi~. 5. Effect of ~-actinin upon the Mg2+-activated ATPase of r e c o n s t i t u t e d a c t o m y o s i n at various pi-I values. Final concn.: I 111~{ MgCI2; 6o mM T r i s - m a l e a t e (total); o.i8 m g / m l actomyosin; o.o54 m g / m l ~-actinin. a, KC1 30 mM, ATP 3 mM; b, KCI 30 mM, A T P z mM; c, KC1 ioo raM, ATP 3 mM; d, KC1 125 raM, ATP 3 raM.
Biochim. Biophys. Acta, 133 (19671 399-411
410
K. SERAYDARIAN, E. J. BRISKEY, W. F. H. M. MOMMAERTS
the alkaline side but still close to the activation maximum. Under the same conditions but for the I mM ATP concentration used in most experiments, the results are about identical above p H 7, but below this p H the activities are less both with and without a-actinin. This is illustrated in Fig. 5b. Here the optimum p H for the activation by a-actinin is distinctly below the p H 6.8 used in most of this work. At higher concentrations of KC1 the ATPase activity and the activation by a-actinin become less at all p H values, as shown in Fig. 5c for 125 mM KC1. The turbidity responses, not illustrated, have broad optima in the range between about 6. 3 and 7-5, and the choice of p H 6.8 is indeed suitable for the demonstration of the dependence upon the composition of the medium, and of the activation by a-actinin. DISCUSSION
Since this paper is of an introductory nature, surveying the conditions influencing the ATPase activity and the turbidity response of actomyosin as affected by a-actinin, no extensive discussion is required. Two conclusions, however, do emerge with some prominence, and warrant explicit formulation. The finding b y EBASHI AND EBASHI 2, that the turbidity increase induced by ATP in a suspension of actomyosin requires a-actinin, can be confirmed only under very specific conditions of ATP and Mg ~+ concentrations, and at low ionic strength. Under other conditions, the turbidity response requires no a-actinin, but is merely accelerated b y this component; and at higher salt concentrations, approaching or including the physiological range, no activation by cc-actinin is found at all. On the other hand, extending the work of EBASHI b y including ATPase determinations, we find that actomyosin requires no a-actinin in order to split ATP. At low ionic strengths, a-actinin m a y cause a moderate activation of the ATPase activity, at higher ionic strengths including the vital range, this protein turns indifferent and even inhibitory. These orientating investigations, therefore, cast doubt upon the necessity of a-actinin as a required factor and upon its significance as a physiological accelerator. They do confirm, however, that c~-actinin interacts with actomyosin and can modify its responses. In the following papers, we present a detailed analysis of the nature of this modification, and a sharper discussion as to its possible physiological meaning. ACKNOWLEDGEMENTS
We would like to thank Mrs. M. SUH and Mrs. M. MARUSICHfor their invaluable technical assistance in all phases of this work. Appreciation is also expressed to Mrs. M. SLATER for m a n y useful discussions. One of us (E.J.B.) expresses appreciation to A.M.I.F., Chicago, Ii1., for fellowship support. This work was supported in part b y Research Grant HE03o67 of The National Heart Institute, National Institutes of Health, Bethesda, Md. REFERENCES
I S. ]~BASHI AND l~'. EBASHI, J. Biochem., 55 (1964) 6042 S. EBASHI AND F. EBASHI, f . Bioch~m., 58 (196.5) 7. 3 S. V. PERRY AND T. C. GREY, Biochem. J., 64 (1956) 5.
Biochim. Biophys. dcta, 133 (1967) 3 9 9 - 4 I I
ACTOMYO/,qlN AND ~x-ACTININ 4 5 6 7 ,~ 9 lo i[ i2 I3 ]4 15 16 17 18 19 20 2t 22
411
A. WEBER AND S. W1NICUR, J . Biol. Chem., 236 (1961) 3198. A. M. 1-~ATZ, dr. Biol. Chem., 239 (1964) 3304. _~I. MORALES AND J. BOTTS, Arch. Biochem. Biophys., 37 (1952) 283. ~. EIC;;ASHI AND I~. MARUYAMA, .]. Biochem., 58 (I965) 20. \\;. F. t-~. M. MOMMAERTS, J. Biol. Chem., 198 (1952) 445. ~I. CARSTEN AND V~7. F. H. M. MOMMAERTS, Biochemistry, 2 (1963) 28. \\~. 12~. J~[. M. MOMMAERTS, Methods Med. Bes. 7 (1958) 3A SZENT-GY6RG¥I, Chemistry of Muscular Contraction, A c a d e m i c Press, N e w Y o r k , i 9 5 t, p. r46. N. AZI'MA AND S. WATANABE, J. Biol. Chem., 240 (1965) 3847 • V. NESSLER AND S. S. SPICER, Biochim. Biophys. Acta, 8 (1952) 474. W. F. H. M. MOMMAERTS AND R. G. PARRISH, J . Biol. Chem., 188 (1951) .545. R. G. PARRISH AND V~7. F. H. M. MOMMAERTS, J . Biol. Chem., 209 (1954) 9Ol. C. H. FISKE AND Y. SOBBAROW, J. Biol. Chem., 66 (1925 ) 375. S. EBASHI, dr . Biochem., 5 ° (1961) 236. A. YASUI AND S. XVATANABE, J. Biol. Chem, 240 (1965) 98. M. ENDO, .[. Biochem., 55 (1964) 614K. MARUYAMA, J . Biochem., 59 (1966) 422. W . F. H. M. MOlVIMAERTS AND K. SERAYDARIAN, J. Gen. Physiol., 305 (I947) 4Ol. I. GREEN AND W , F. H . ~I~. MOMMAERTS, J. Biol. Chem., 202 (1953) 541.
Biochim. Biophys. Acta, 133 (1967) 3 9 9 - 4 1 1