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Conformational changes of F-actin in myosin-free ghost single fibre induced by either phosphorylated or dephosphorylated heavy meromyosin
Biochirnica et Biophysica Acta 913 (1987) 1-9
1
Elsevier BBA 32820
C o n f o r m a t i o n a l c h a n g e s of F - a c t i n in m y o s i n - f r e e ghost single fibre i n d u c e d by e i t h e r p h o s p h o r y l a t e d or d e p h o s p h o r y l a t e d heavy m e r o m y o s i n I r e n a K ~ k o l a, Yurii S. B o r o v i k o v b, D a n u t a Szcz~sna a V a l e n t i n a P. K i r i l l i n a b a n d D i m i t r i i I. L e v i t s k y c " Department of Cellular Biochemistry, Nencki Institute of E~perimental Btoloev. l+arsaw (Poland). 6 Group of Cell Motilio', Institute of Qvtolo~, A caden 9' of Sciences of USSR, Leningrad and ' A.N. Belozerski Lat~oratorv, Moscow b'ni~'ersit~', Mo.s~ow ( U. S. S. R. t
(Received 20 Janua~' 1987)
Key words: F-actin; Myosin; Heavy meromyosin, (de)phospho~lated: Ghost fibre, m~osin-frce: Fluorescence, polarized
The changes in F-actin conformation of myosin-free single ghost fibre induced by binding of phosphorylated or dephosphorylated heavy meromyosin have been studied by measuring polarized fluorescence of F-actin intrinsic tryptophan and of phalloidin-rhodamin bound to F-actin. The changes of polarization of both fluorescences were found to be dependent on low or high Ca2+ concentration and on the phospho~lated or dephosphorylated form of heavy meromyosin. Computer analysis of polarized fluorescence has shown that binding of phosphorylated heavy meromyosin with divalent ion binding sites saturated with Mg 2+ (in the presence of 1 mM MgCI 2 and 1 mM EGTA) and dephosphorylated heavy meromyosin with divalent ion binding sites saturated with Ca2+ (in the presence of 1 mM MgCI 2 and 0.1 mM Ca 2+) decreases the angles of emission and absorption dipoles and the angle between the F-actin axis and the fibre axis, thus suggesting that F-actin in ghost fibre becomes more flexible. On the other hand, the above-mentioned angles increase when phosphoryalted heavy meromyosin at high and dephosphorylated heavy meromyosin at low Ca2+ concentration were bound to thin filaments, thus showing the decrease of F-actin flexibili~ under these conditions.
Introduction
The explanation of the molecular mechanism of muscle contraction requires detailed information about the conformational changes of contractile proteins in muscle. Considerable attention has been focused on the interaction of myosin heads with F-actin, by which the sliding force of actin and myosin filaments was generated. Recently, spectroscopic techniques have been Correspondence: I. Kgkol, Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland. 0167-4838/87/$03.50
used to study the rotational motion of myosin heads and F-actin filaments [1-10]. By using these techniques it was possible to show that the rate of rotational motion of the actin monomer or its segment is approximately 4-fold reduced by binding of the heavy meromyosin or myosin subfragment 1 [6]. On the other hand, the flexibility of F-actin was increased by the binding of myosin heads to the actin filament [5,8]. Moreover, the conformational changes of F-actin in ghost fibre induced by the binding of myosin subfragment 1 and heavy meromyosin were controlled by Ca 2+ when both myosin fragments contained intact 18 kDa light chains [11-13].
1987 Elsevier Science Publishers B.V. (Biomedical Division/
Additionally, some findings allowed the assumption that phosphorylation of 18 kDa light chains of myosin induces a modulating effect of myosin-actin interaction [14-17]. To obtain more information on this phenomenon, the conformational changes in F-actin of ghost fibre induced by the binding of phosphorylated and dephosphorylated heavy meromyosin having divalent ion binding sites saturated with Mg 2+ or Ca 2+ were studied. The information about these changes was obtained from studies of intrinsic (tryptophan residues of F-actin) and extrinsic (rhodamin-phalloidin bound to F-actin) polarized fluorescence. The four components of polarized fluorescence were registered and the obtained data allowed the calculation of the angles of absorption and emission dipoles both of tryptophan and phalloidin-rhodamin and the angle between the F-actin axis and fibre axis. The latter was used as an indicator of flexibility of F-actin [5]. Preliminary reports of this work have been presented [18-20]. Materials and M e t h o d s
Preparation of heavy meromyosin Phosphorylated and dephosphorylated heavy meromyosin were prepared by brief digestion of phosphorylated and dephosphorylated rabbit skeletal muscle myosin solution with chymotrypsin in the presence of CaC12 [16]. The purity of heavy meromyosin preparations was examined electrophoretically. SDS electrophoresis [21] and electrophoresis in 8 M urea [22] were performed and then the density of the electrophoretic pattern was measured. The ratio of the sum of alkali light chains to the regulatory light chains was approx. 1 : 1 and the amount of phosphorylated regulatory light chains was about 95-100% in phosphorylated and lower than 1% in dephosphoryalted heavy meromyosin. The heavy meromyosin preparations were freshly prepared and kept not longer than 3-5 days at 0°C. The amount of phosphorylated and dephosphorylated light chains (LC 2) was measured immediately before use.
Preparations of muscle ghost fibre Single fibre obtained as previously described [11] was placed in a chamber, then glycerol was
washed out with a solution containing 100 mM KC1/1 mM MgC12/67 mM phosphate buffer (pH 7.0) (standard solution). Myosin, tropomyosin, and troponin were removed by treatment of the single ghost fibre with 800 mM KCI/10 mM MgCI2/10 mM ATP/67 mM phosphate buffer (pH 7.0) for 40 min at 4°C. The removal of myosin, tropomyosin and troponin was controlled electrophoretically in parallel samples of ghost fibre treated as described above. Moreover, each ghost fibre used for the experiments was examined by measuring tryptophan fluorescence anisotropy, and for further studies only the ghost fibres showing typical parameters for pure F-actin filaments were used [23]. Muscle ghost fibre contained about 80% of actin. It was found by densitometrical measurements of sodium dodecyl sulphate electrophoretic pattern of parallelly prepared samples of ghost fibre. Sodium dodecyl sulphate electrophoresis of those samples was run in 6 8% gels. Some single fibres were treated with phalloidin-rhodamin in the following way. The ghost fibres were incubated with 4 mM phalloidinrhodamin diluted in standard solutions during 20 rain, and then the unbound reagent was removed by washing with standard solution.
Binding of heavy meromyosin to F-actin in ghost fibre Ghost fibres both modified and unmodified by phalloidin-rhodamin were incubated with phosphorylated or dephosphorylated heavy meromyosin (2 mg/ml) dissolved in 30 mM phosphate buffer (pH 7.0)/1 mM MgCI 2 and 1 mM EGTA or 0.1 mM CaCI 2, considered in the present paper as low or high Ca 2+ concentration, respectively. The molar ratio of bound heavy meromyosin to actin in decorated ghost fibre was calculated as proposed by Yanagida and Oosawa [5]. The amount of heavy meromyosin bound to F-actin in decorated ghost fibre was estimated by measuring tryptophan fluorescence after removal of unbound heavy meromyosin by washing with buffer containing 20 mM phosphate (pH 7.0) and 1 mM MgC12. Measurement was performed with the same apparatus after removal of polarization filter and wollaston prism.
Measurements of polarized fluorescence of intrinsic tryptophan and phalloidin-rhodamin bound to F-actin in ghost fibre The t r y p t o p h a n fluorescence was excited at 303 _+ 4 n m a n d the e m i t t e d light was r e c o r d e d at 3 2 0 - 3 8 0 nm. T h e fluorescence of p h a l l o i d i n - r h o d a m i n b o u n d to F - a c t i n in ghost fibre was excited at 436 _+ 5 n m a n d registered at 5 0 0 - 6 5 0 nm. M e a s u r e m e n t s were m a d e with a m i c r o s p e c t r o p h o t o m e t e r [24]. T h e four c o m p o n e n t s intensities of p o l a r i z e d fluorescence iiIll, i i I , , ~_I 1 , 1111 i n d i c a t i n g the d i r e c t i o n of incident light (on the left) a n d the d i r e c t i o n of the e m i t t e d light (on the right), relative to the fibre axis were measured. T h e degrees of fluorescence p o l a r i z a t i o n of PII a n d P l a n d the value of QII were defined as;
e,, = (,~6,-,,1. )/(,,1,, +,,1. )
3, ± I l I = F [ ( S - S S ) C I + ( 1 - S ) H 3 + ( 2 - S ) F 3
+ ( 2 S - Q I S S ) R 1 ] + FG1 4. ±I± = F [ ( S - O , 7 5 S S ) C 4 + ( 2 - S ) H 4 +(3SS-(S-2)Q1R4]+ FG2 where F=I-N;
S=sin20;
C2 = 2I sin2Acos2E -cos2A .cos2E + 12sin2Ecos2A +2 cosD sinA sinE cosA cosE; H2 = 1 - sin2A- sin2E
FG1 = i!~(2-cos2y), FG2 = ~tsN(1+2 cos27); Q2 = J~(1+ cos2D). SS = sin40
Pa_ = ( l l ~ - ±III)/(±l± + ±III)
T h e angle between the a b s o r p t i o n a n d emission d i p o l e s 7 was calculated as p r o p o s e d in Refs. 5. 25, 28 from e q u a t i o n
QII = (lllll- • lll)/(Jii 4- ± 111)
PNO = (3 COS27-- 1)/(COS2y +3)
E x p e r i m e n t a l d a t a were a n a l y s e d within the m a t h e m a t i c a l m o d e l s described in Refs. 5, 25 a n d 26. T h e m o d e l s are b a s e d on the a s s u m p t i o n that the a n i s o t r o p y of muscle fibre F - a c t i n fluorop h o r e s fluorescence can be well d e s c r i b e d by the system of f l u o r o p h o r e s aligned along the helix a n d r a n d o m l y a r r a n g e d emitting fluorophores. The four c o m p o n e n t s of p o l a r i z e d fluorescence were expressed as functions of the angles ¢bA, D E, 0 a n d the value N: where ¢bA and ¢bE are the angles b e t w e e n the long axis of F - a c t i n and the a b s o r p tion a n d emission dipoles, respectively; 0 is the angle between the long axis of F - a c t i n and the fibre axis (c.f. Fig. 1); N is the n u m b e r of rand o m l y a r r a n g e d fluorophores. The c o r r e s p o n d i n g expressions derived as d e s c r i b e d in Refs. 5, 25 a n d 26 are: 1.,111 = F[( S - SS)C2 + ( 1 - S)H2 + ( 1 - S + SSQ2)8R1] + FO2
2. iii± = F[( S - SS)CI + ( 1 - S ) U l + ( 2 - S) Vl + (28 - Q1SS)RI] + FG1
where PNo is the degree of fluorescence p o l a r i z a tion of r a n d o m l y o r i e n t e d oscilatores (dipoles) R1 = 'ssin2Asin2E; C1 - 2t cos2A cos2E --4
4I
sin2E cos2A
sinZA cos2E-cos D sin A sin Ecos A cos E:
H1 = 12(1- sin2A-cos2E); F1 = 14sinZAcos2E: Q1 = 0,5 +cos2D: H3 = 2t (1 - sin2E - cos2A) H4 = Q1 sI (1 - cos2A - cos2E ) ; C4 = 4t sin2A cos2E + ~4sin2Ecos2A QI t~sin2Asin2E +cos D sin E cos A cos E. T h e angles ¢bA, q)E, a n d the value of N were chosen by the least-squares fit of e x p e r i m e n t a l ratios of iii±/111,, ± I J , l l l and 11./11111. The elastic m o d u l u s for b e n d i n g or the flexural regidity-E [5] of the thin filament were o b t a i n e d by using the e q u a t i o n sin2~b = 0.8 ( k T / e ) L , where k and T are the Boltzman c o n s t a n t a n d the a b s o l u t e t e m p e r a -
Z
ture, respectively and L is the length of the filament. A TPase assays
The activities of K +- and Ca+-ATPases and actin-activated Mg2+-ATPase of heavy meromyosins were determined in the following media: K +ATPase: 0.5 mM KC1/20 mM Tris-acetate (pH 7.0)/2 mM dithiothreitol/5 mM EDTA/Ca +ATPase: 10 mM CAC12/20 mM Tris-acetate (pH 7.0)/50 mM KCI. Actin-stimulated Mg+-ATPase: 1 mM MgC12/20 mM Tris-acetate (pH 7.0)/50 mM KC1 with 0.08 mg/ml of actin. The final volume was 2 ml, concentration of heavy meromyosin 0.2 mg/ml. The reaction was started by addition of ATP to the final concentration of 1 mM and after 1 min incubation at 25°C was stopped by the addition of 1 ml of 10% trichloroacetic acid. Liberated phosphate was measured by the method of Fiske and SubbaRow [27]. Protein determination
The amount of actin and heavy meromyosin were determined spectrophotometrically [17] and by the biuret method [28]. Results
The enzymatic activities of heavy meromyosin used for the studies of conformational changes in F-actin of muscle ghost fibre and the amount of phosphorylated heavy meromyosin light chains are given in Table I. As mentioned in Materials and Methods the molar ratio of two regulatory light chains to the sum of alkali light chains was, in heavy meromyosin approx. 1:1, and approxi-
?
rl
Fig. 1. Diagram explaining the calculation of the polarized components of fluorescence [51 0 is the angle between the filament axis of F-actin OW and the fiber axis OZ. tbA, q)t:. are angles between the F-actin axis and the absorption dipole (,~) and emission dipole (E) in F-actin, respectively. 3' is the angle between ,~ and I~.
mately all regulatory light chains of the respective heavy meromyosin preparations were either in phosphorylated or dephosphorylated form. The enzymic properties (K +- and Ca+-ATPase activities) of heavy meromyosin used were similar, independent of the form of those regulatory light chains. The values of specific actin-stimulated MgZ+-ATPase activities of phosphorylated and dephosphorylated heavy meromyosin at high (0.1 mM CaCl2) and low (in the presence of 1 mM EGTA) Ca 2+ concentrations were similar (Table I). The binding of heavy meromyosin to F-actin in myosin and troponin-trypomyosin free ghost fibre induced changes in the degree of F-actin intrinsic
TABLE l THE ENZYMIC PROPERTIES OF PHOSPHORYLATED A N D DEPHOSPHORYLATED HEAVY MEROMYOSIN Activities are expressed as tool of ATP hydrolyzed per tool of heavy meromyosin per s. ATPase assays were as detailed in Methods. The ratio of light chains (LC) (LC 1 + LC3)/LC 2 was approx. I : 1. The data represent mean _+S.E. from three determinations for five different preparations. Amount of phosphorylated
ATPase activity (s l)
light chains (LC2) (% of total LC2)
K+
Ca 2+
Mg 2+ plus actin Ca 2 +
EGTA
95_+5% 0-5%
13.8__+0.2 14.2_.+_0.3
12.2_+0.3 12.4_+0.1
4.2±0.1 4.4_+0.2
6.3_+0.2 6.7±0.3
tryptophan fluorescence polarization. A significant increase of Ptt can be observed when dephosphorylated heavy meromyosin at high and phosphorylated heavy meromyosin at low Ca 2+ concentration were bound. The values of P . are increased by binding of both phosphorylated and dephosphorylated heavy meromyosin to actin, but the values of P± differ depending on Ca 2+ concentration (Fig. 2A, B). The value of Qtt for F-actin both in the presence of 1 mM EGTA or 0.1 mM CaC12 was 0.087. However, after binding of phosphorylated heavy meromyosin in the presence of 1 mM EGTA and dephosphorylated heavy meromyosin in the presence of 0.1 mM CaC12, the values are increased to 0.212 and 0.149, respectively. When phosphory-
i
'8
'A
~7 0.20
A
r-
0.18
P ~3
0.1b 0
I t
c ._o
Ic
i
0.48
~D
"6 N c.O
0.40
c
-0.36
0
. . . . . . . . . . . . . . . . . . . .
O
I1
-0.44
I
I
I
0.15
0.30
0.15
I
0,30
Bound heovy rneromyosin ( too[ /tool of octin ) Fig. 2. Changes of polarized fluorescence of F-actin intrinsic tryptophan (A, B) and phalloidin-rhodamin bound to F-actin (C, D) in single ghost fibre induced by the binding of phosphorylated (C) o) or dephosphorylated (vv) heavy meromyosin. Open symbols express the values of Pz and closed symbols those of Pit" The binding studies were performed in a medium containing 30 mM phosphate buffer (pH 7.0), 1 mM MgCI 2 and 1 mM EGTA (A, C) or 0.1 mM CaC1 z (B, D).
lated heavy meromyosin in the presence of Ca 2+ and dephosphorylated heavy meromyosin in the absence of Ca 2+ are bound to F-actin, the values of QII are decreased to 0.07 and 0.047, respectively. The changes of Qtl' PIt and P± reaching their maximum by binding of 0.15 mol heavy meromyosin per mol ofactin, remain practically constant even when the amount of heavy meromyosin bound to actin is increased. The lack of correlation between the changes in polarized fluorescence and the amount of heavy meromyosin bound to F-actin in ghost fibre (see (Fig. 2 A, B) allows the assumption that the amount of tryptophan of heavy meromyosin does not influence the values of Pll and P± under the conditions studied. Moreover, the differences in the values of PtI' QII and P± produced by the binding of both phosphorylated and dephosphorylated heavy meromyosin to F-actin in ghost fibre, estimated at the same molar ratio of bound heavy meromyosin to actin are different, depending on the low or high concentration of Ca 2+. Actin of ghost fibre was also labelled by treatment with phalloidin-rhodamin and the influence of the binding of both phosphorylated and dephosphorylated heavy meromyosin at low and high Ca 2+ concentration on the degree of polarization of phalloidin-rhodamin bound to F-actin was examined. As shown in Fig. 2C, D the binding of phosporylated heavy meromyosin at low and dephosphorylated heavy meromyosin at high Ca 2+ concentration induced a significant increase of Ptl, whereas phosphorylated heavy meromyosin at high Ca 2+ concentration induced a significant increase of Pti' whereas phosphorylated heavy meromyosin at high and dephosphorylated heavy meromyosin at low Ca 2+ concentration decreased the values of PIr The values of P± are only slightly changed by the binding of dephosphorylated heavy meromyosin. Significant changes of P± were observed caused by binding of phosporylated heavy meromyosin, the values of P± increasing at high and decreasing at low Ca 2+ concentration. Thus, the character of the changes of polarization of phalloidin-rhodamin bound to F-actin in ghost fibre, induced by the binding of heavy meromyosin, depends both on the phosphorylated or dephosphorylated form of heavy meromyosin and
low or high Ca 2+ concentration, analogically to the changes of polarized fluorescence of intrinsic tryptophan of F-actin in ghost fibre (cf. Fig. 2A,
B). The three ratios of four components of polarized fluorescence of intrinsic tryptophans of F-actin in ghost fibre, measured in the presence of 0.1 mM CaC1 z or 1 mM E G T A before and after binding of phosphorylated or dephosphorylated heavy meromyosin as well as the calculated angles of absorption ~A and emission q~E dipoles and sin20 (0 is the angle between the F-actin axis and the fibre axis) are given in Table II. The values of q~A and q)E and sin20 were determined on the basis of equations proposed for elaboration of polarized fluorescence data [5], by computer analysis to give the best agreement with the ratios of iii±/111,, ± l J , lll and l lj,/lll, obtained by measuring the four components of polarized fluorescence of intrinsic tryptophan of F-actin in ghost fibre. The angle between the dipoles A and E (Fig. 1) of the tryptophan molecule was adopted as ~ = 38". At the molar ratio of bound heavy meromyosin to actin equal to 0.15 in the presence of 1 mM E G T A the values of q~A and ~E decreased when bound heavy meromyosin was phosphorylated and
increased when it was in dephosphorylated form. The values of both q)A and q~E change in the opposite direction in the presence of 0.1 mM CaCI 2. The most probable value of sin20 for F-actin obtained by computer analysis was 0.054 _+ 0.003 in the presence of 1 mM E G T A or 0.1 mM CaCI 2. This value changed significantly depending on low or high Ca 2+ concentration when phosphorylated or dephosphorylated heavy meromyosin was bound to F-actin in ghost fibre (Table I1). The relation between sin20 and the elastic modulus of the F-actin filament (c), as proposed by Yanagida and Oosawa [5], can be described by the equation sin20 = 0.87. (kT/~). L, where L is the length of the semiflexible filament with one end fixed and the other free, The calculated value of of F-actin both at low and high Ca 2+ concentration, assuming L = 1.0 /,tm, is equal to 5.5 _+ 0.l 10 -26 N e w t o n - m 2 ( N - m 2 ) , The values e of Factin containing bound heavy meromyosin were calculated using the values of sin20 shown in Table II. They were 3.3 _+ 0.2-10 26 N - m 2 and 3.6 _+ 0.2 - 10 26 N • m 2 when phosphorylated heavy meromyosin at low, and dephosphorylated heavy meromyosin at high Ca 2+ concentration
TABLE II P O L A R I Z E D F L U O R E S C E N C E OF T R Y P T O P H A N RESIDUES OF F-ACTIN IN T H E G H O S T FIBRE BEFORE A N D A F T E R B I N D I N G O F P H O S P H O R Y L A T E D OR D E P H O S P H O R Y L A T E D HEAVY M E R O M Y O S I N The molar ratio of bound heavy meromyosin to actim 0.15. The data are averages determined by computer analysis of 30-40 measurements. Calculation was performed on the basis of equations proposed by Treager and Mendelson [25] and by Yanagida and Oosawa [5]. The angle between absorption and emission dipoles in the same tryptophan molecule, y = 38 °. Angles of absorption (q~A) and emission ( ~ E ) dipoles; sin'0 where 0 is the angle between the filament axis and fibre axis, the number of random fluorophores (N), ratios of four components of polarized fluorescence ill±/llllp, • 1!1/11111, • I L/11111, where ilia., • 111"II111' ± 1± are intensities of polarized fluorescence indicating parallel (11) or perpendicular ( ± ) direction of the incidient light (on the left side) and emitted light (on the right side) relative to the fibre axis.
irl • /llllr
± llJilllf
• Ix/lllll
q)A
~E
sin2
N
1 m M EGTA 0.1 m M CaCI z
0.71 _+0.02
0.84_+0.03
1.26_+0.04
62.4_+0.8
57.5_+0.1
0.054_+0.003
0.55_+0.2
F-actin + phosphorylated heavy mermyosin
1 m M EGTA 0.1 m M CaC12
0.68_+0.01 0.71 _+0.02
0.65_+0.02 0.87_+0.02
1.00_+0.02 1.32_+0.05,
54.5_+0.1 63.7_+0.1
56.5-+0.1 57.7_+0.1
0.081 -+0.010 0.044_+0.004
0.66_+0.03 0.54___0.1
F-actin + dephosphorylated heavy meromyosin
1 m M EGTA 0.1 m M CaCI 2
0.71 +0.01 0.67_+0.03
0.91 -+0.03 0,74-+0.03
1.37-+0.03 1.13_+0.03
64.7-+0.5 60.9_+0.5
58.0_+0.1 56.7-+0.2
0.052_+0.004 0.088_+0.002
0.54___0.02 0.64_+0.02
Additions F-actin
were bound to F-actin in ghost fibre, respectively, and 6.2 +_ 0.1 and 6.6 _+ 0.2 • 1 0 - 2 6 N - m 2 for Factin containing bound phosphorylated heavy meromyosin at high, and dephosphorylated heavy meromyosin at low Ca z+ concentration, respectively. The angle between the absorption and emission dipoles of phalloidin-rhodamin bound to F-actin was estimated as ~' = 46 °. The ratios of ,I±/,I,, ± 111/11Iii and ± Ii/NIii obtained by measuring four components of polarized fluorescence of phalloidin-rhodamin bound to F-actin in ghost fibre at low and high Ca 2+ concentration and in the presence or absence of bound phosphorylated or dephosphorylated heavy meromyosin are given in Table III. The values of q~A, q~E and sin20 were calculated by computer analysis by approximation to the values of the above given three ratios of the measured four components of polarized fluorescence of phalloidin-rhodamin bound to F-actin in a ghost fibre. Those values of q~A and q~E and sin20 are presented in Table II1. The binding of phosphorylated and dephosphorylated heavy meromyosin induced significant changes in the values of four components of polarized fluorescence of phalloidin-rhodamin bound to F-actin in a ghost fibre and consequently in the three ratios shown in Table III. The character of those changes depended on Ca 2+ concentration. Therefore, the values of the angles q~A and q~E as well as sin20 differ, depending on the phosphorylated or de-
phosphorylated form of heavy meromyosin bound to F-actin in a ghost fibre as well as on low or high Ca 2+ concentration. A marked increase in the values of sin20 could be observed when dephosphorylated heavy meromyosin was bound at high Ca 2+ concentration. The values of sin20 decreased in binding of dephosphorylated heavy meromyosin at low Ca 2+ concentration. Small changes of the values of sin20 were obtained by approximation of the calculated values to the observed ratios of the four components of polarized fluorescence of phalloidin-rhodamin bound to actin in ghost fibre, when phosphorylated heavy meromyosin at low and high Ca 2+ concentration was bound to F-actin in a ghost fibre (Table III). Discussion
The observed changes in polarization of intrinsic tryptophan fluorescence of F-actin in a ghost fibre induced by binding of heavy meromyosin allows the assumption that the kind of binding or its effect depends strongly on the phosphorylated or dephosphorylated form of myosin heads as well as on low or high Ca 2+ concentration. Since the binding of heavy meromyosin was studied in the presence of 1 mM MgC12 and 1 mM EGTA or 0.1 mM CaC12 non-specific divalent cation binding sites of heavy meromyosin were occupied by Mg 2+ or by Ca 2+ (29-31]. The elastic modulus for bending of F-actin (~)
T A B L E II1 POLARIZED FLUORESCENCE OF PHALLOIDIN-RHODAMIN B O U N D T O F - A C T I N IN T H E G H O S T F I B R E S B E F O R E AND AFTER BINDING OF PHOSPHORYLATED OR DEPHOSPHORYLATED HEAVY MEROMYOSIN The angle between the a b s o r p t i o n and emission dipoles, ), = 46 °. Symbols as in legend to T a b l e II. Additions
i11±
± Iii/11111
_ 1±/11111
~A.
~E
sin20
1 mM EGTA 0.1 m M CaCI 2
0.38 + 0.01
1.22 + 0.03
0.51 _+0.02
59.3 +_0.2
42.6 + 0.2
0.061 + 0.002
F-actin + phosphorylated heavy meromyosin
I mM EGTA 0.1 m M C a C I 2
0.35_+0.01 0.41 - 0.01
1.40+_0.04 1.16 - 0.03
0.57+_0.03 0.53 - 0.02
61.2+-0.9 58.7 - 0.3
41.8_+0.2 43.9 - 0.4
0.061 + 0 . 0 0 2 0.066 - 0.008
F-actin + dephosphorylated heavy meromyosin
1 mM EGTA 0.1 m M CaC12
0.39_+0.01 0.37+-0.01
1.36_+0.03 1.26+-0.03
0.59+-0.03 0.53+-0.03
60.7_+0.4 60.0+-0.4
43.1-+0.3 42.2+-0.2
0.053_+0.02 0.068_+0.002
F-actin
[5] in a ghost fibre both in the presence of 1 mM E G T A or 0.1 mM CaCl 2 was estimated as 5.5 _+ 0.1 • 1 0 2 6 N • m 2. The obtained value of { is comparable with that obtained by Yanagida and Oosawa on the basis of polarized fluorescence of N6-ethenoadenosine 5-diphosphate bound to Factin in ghost fibre measurements followed by mathematical analysis of the data [5]. The influence of the binding of heavy meromyosin was studied by the same method. The authors found the value of { to decrease in binding of heavy meromyosin to the value of 3.9-10 26 N . m2; however, nothing is known about the amount of phosphorylated light chains of heavy meromyosin used for the binding to F-actin in ghost fibre. The above values of { decreased by the binding of dephosphorylated heavy meromyosin at high and phosphorylated heavy meromyosin at low Ca 2+ concentration to the values 3.3 and 3.6.10 26 N . m 2, respectively. Thus, the phosphorylated heavy meromyosin with bound Mg 2+ favours the kind of binding to F-actin which exerts an effect similar to that exerted by the binding of dephosphorylated heavy meromyosin having non-specific metal ions binding sites saturated with Ca 2+. In both cases the flexibility of F-actin increases, in contrast to the slightly decreased flexibility of F-actin in a ghost fibre when phosphorylated heavy meromyosin with bound Ca 2+ and dephosphorylated heavy meromyosin with bound Mg 2+ interacts with F-actin in a ghost fibre. It was manifested by the values of { increased to 6.2 _+ 0.1 and 6.6 + 0.6 • 1 0 _26 N • m 2, respectively. The values of the four components of polarized fluorescence of phalloidin-rhodamin bound to Factin are significantly changed by the binding of heavy meromyosin to F-actin in a ghost fibre and the character of those changes depends both on low or high Ca 2+ concentration and on whether the phosphorylated or dephosphorylated heavy meromyosin was bound to F-actin. Since phalloidin affects actin-actin bonds in the F-actin structure [32] and slightly decreases the flexibility of actin filaments [33], the values of sin20 related to the elastic modulus for bending of F-actin and the changes of those values are smaller than that obtained from the values of four components of polarized fluorescence of intrinsic tryptophan residues of F-actin in a ghost fibre.
Thus, it seems reasonable to assume that phosphorylation of myosin heads modulates the interaction of this protein with actin and the character of this modulation depends on whether the non-specific divalent ion binding sites of myosin heads are occupied by magnesium or calcium ions. When the divalent ion-binding sites of phosphorylated myosin are saturated with Mg 2+ the effect of induced changes on F-actin conformation seems to be similar to that induced by dephosphorylated myosin saturated with Ca 2+. Assuming that the increased flexibility of actin filaments is connected with tension development by a single muscle fibre, the influence of phosphorylation of myosin heads on the parameters of isometric tension could be observed at low Ca 2+ concentration. Therefore, the above described observation seems to be in good agreement with the findings of Persechini et al. [34]. The authors found that at low Ca 2+ concentration, phosphorylation of myosin increases isometric tension. The authors suggested that, in vertebrate skeletal muscle, phosphorylation of myosin heads increases the amplitude of tension at submaximal Ca 2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament. Moreover, the observed differences in the electron micrographs of F-actin filaments decorated by phosphorylated and dephosphorylated heavy meromyosin [16] seem to be connected to the possible difference in the kind of binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin under the same conditions. The difference is most probably related to the orientation of myosin heads.
Acknowledgments The authors thank Professor Theodor Wieland for supplying them with phalloidin-rhodamin and Dr. S.A. Krolenko for a stimulating discussion.
References 1 Mendelson, R.A., Morales, M.F. and Botts, J. (1973) Bioc h e m i s t r y 12, 2250-2255 2 Mendelson, R.A. a n d Cheung, P. (1976) Science 194, 190-192
3 Thomas, D.D., Seidel, J.C., Hyde, J.S. and Gergely, J. (1975) Proc. Natl. Acad. Sci USA 72, 1729-1733 4 Thomas, D.D., Seidel, J.C. and Gergely, J. (1976) in Contractile Systems in Non-Muscle Tissues (Perry, S.V., et al., eds.), pp. 13-21, Elsevier/North Holland, New York 5 Yanagida, T. and Oosawa, F. (1978) J. Mol. Biol. 126, 507-524 6 Thomas, D.D., Seidel, J.C. and Gergely, J. (1979) J. Mol. Biol. 132, 257-273 7 Cooke, R., Crowder, M.S. and Thomas, D.D. (1982) Nature (Lond.) 300, 776-778 8 0 o s a w a , F. (1983) in Actin Structure and Function in Muscle and Non-Muscle Cells (Dos Remidos, C.G. and Bersten, J.A., eds.), Academic Press, New York 9 Pr6chniewicz-Nakajama, E., Yanagida, T. and Oosawa, F. (1983) J. Cell. Biol. 97, 1663-1667 10 Yanagida, T. (1985) J. Muscle Res. Cell. Motility 6, 43-52 11 Borovikov, Yu.S., Levitsky, D.I., Kirillina, V.P. and Poglazov, E.F. (1982) Eur. J. Biochem. 125, 343-347 12 Borovikov, Yu.S. and Karandashov, E.A. (1983) Biochem. Int. 7, 319-328 13 Borovikov, Yu.S. and Levitsky, D.I. (1985) Gen. Physiol. Biophys. 4, 457 464 14 Pemrick, S.M. (1980) J. Biol. Chem. 255, 8836-8841 15 Michnicka, M., Kasman, K. and K~kol, 1. (1982) Biochim. Biophys. Acta 704, 470-475 16 St~pkowski, D., Osihska, A., Szcz~sna, D., Wrotek, M. and K~tkol, I. (1985) Biochim. Biophys. Acta 830, 337-340 17 St~pkowski, D., Szcz~sna, D., Wrotek, M. and K~]kol, I. (1985) Biochim. Biophys. Acta 831, 321-329 18 K~kol, I., Borovikov, Yu.S. and Levitsky, D.I. (1986) J. Muscle Res. Cell. Motility 7 (1), 66
19 Szcz~sna, D. and Borovikov, Yu.S. (1986) J. Muscle Res. Cell. Motility 7 (1), 67 20 Borovikov, Yu.S., K~.kol, I., Szcz~sna, D., Kirillina, V.P. and Levitsky, D.I. (1985) Biochimia 51,691-694 21 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244. 4406-4412 22 Perrie, W.T. and Perry, S.V. (1970) Biochem. J. 119, 31-38 23 Borovikov, Yu.S. and Gusev, N.B. (1983) Eur. J. Biochem. 136, 363-369 24 Joffe, V.A., Borovikov, Yu.S., Barskii, J.Ja. and Rozanov, Yu.M. (1974) Tsitologiya 16, 112-116 25 Tregear, R.T. and Mendelson, R.A. (1975) Biophys. J 15, 455-467 26 Rozanov, Yu.M., Chernogriadskaia, N.A., Barskii, J.Ja., Shudel, M.S. and Borovikov, Yu.S. (1971) Tsitologiya 13, 190 200 27 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375-400 28 Gornall, A.G., Bardawill, C.J. and David, M.N. (1949) J. Biol. Chem. 177, 751-766 29 Morimoto, K. and Harrington, W.F. (1974) J. Mol, Biol. 88, 693-709 30 Holroyed, M.J., Potter, J.D. and Solara, K.J. (1979) J. Biol. Chem. 254, 6478-6482 31 Bagshaw, C.R. and Kendrick-Jones, J. (1979) J. Mol. Biol. 130, 317-336 32 Dancker, P., L~Sw, 1., Hasselbach, W. and Wieland, T.H. (1975) Biochim. Biophys. Acta 400, 407-414 33 Borovikov, Yu.S., Bukatina, A.E. and San'kin B.Ja. (1984) FEBS Lett. 176, 441-443 34 Persechini, A., Stull, J.T. and Cooka, R. (1985) J, Biol. Chem. 260, 7951-7954
1
Elsevier BBA 32820
C o n f o r m a t i o n a l c h a n g e s of F - a c t i n in m y o s i n - f r e e ghost single fibre i n d u c e d by e i t h e r p h o s p h o r y l a t e d or d e p h o s p h o r y l a t e d heavy m e r o m y o s i n I r e n a K ~ k o l a, Yurii S. B o r o v i k o v b, D a n u t a Szcz~sna a V a l e n t i n a P. K i r i l l i n a b a n d D i m i t r i i I. L e v i t s k y c " Department of Cellular Biochemistry, Nencki Institute of E~perimental Btoloev. l+arsaw (Poland). 6 Group of Cell Motilio', Institute of Qvtolo~, A caden 9' of Sciences of USSR, Leningrad and ' A.N. Belozerski Lat~oratorv, Moscow b'ni~'ersit~', Mo.s~ow ( U. S. S. R. t
(Received 20 Janua~' 1987)
Key words: F-actin; Myosin; Heavy meromyosin, (de)phospho~lated: Ghost fibre, m~osin-frce: Fluorescence, polarized
The changes in F-actin conformation of myosin-free single ghost fibre induced by binding of phosphorylated or dephosphorylated heavy meromyosin have been studied by measuring polarized fluorescence of F-actin intrinsic tryptophan and of phalloidin-rhodamin bound to F-actin. The changes of polarization of both fluorescences were found to be dependent on low or high Ca2+ concentration and on the phospho~lated or dephosphorylated form of heavy meromyosin. Computer analysis of polarized fluorescence has shown that binding of phosphorylated heavy meromyosin with divalent ion binding sites saturated with Mg 2+ (in the presence of 1 mM MgCI 2 and 1 mM EGTA) and dephosphorylated heavy meromyosin with divalent ion binding sites saturated with Ca2+ (in the presence of 1 mM MgCI 2 and 0.1 mM Ca 2+) decreases the angles of emission and absorption dipoles and the angle between the F-actin axis and the fibre axis, thus suggesting that F-actin in ghost fibre becomes more flexible. On the other hand, the above-mentioned angles increase when phosphoryalted heavy meromyosin at high and dephosphorylated heavy meromyosin at low Ca2+ concentration were bound to thin filaments, thus showing the decrease of F-actin flexibili~ under these conditions.
Introduction
The explanation of the molecular mechanism of muscle contraction requires detailed information about the conformational changes of contractile proteins in muscle. Considerable attention has been focused on the interaction of myosin heads with F-actin, by which the sliding force of actin and myosin filaments was generated. Recently, spectroscopic techniques have been Correspondence: I. Kgkol, Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland. 0167-4838/87/$03.50
used to study the rotational motion of myosin heads and F-actin filaments [1-10]. By using these techniques it was possible to show that the rate of rotational motion of the actin monomer or its segment is approximately 4-fold reduced by binding of the heavy meromyosin or myosin subfragment 1 [6]. On the other hand, the flexibility of F-actin was increased by the binding of myosin heads to the actin filament [5,8]. Moreover, the conformational changes of F-actin in ghost fibre induced by the binding of myosin subfragment 1 and heavy meromyosin were controlled by Ca 2+ when both myosin fragments contained intact 18 kDa light chains [11-13].
1987 Elsevier Science Publishers B.V. (Biomedical Division/
Additionally, some findings allowed the assumption that phosphorylation of 18 kDa light chains of myosin induces a modulating effect of myosin-actin interaction [14-17]. To obtain more information on this phenomenon, the conformational changes in F-actin of ghost fibre induced by the binding of phosphorylated and dephosphorylated heavy meromyosin having divalent ion binding sites saturated with Mg 2+ or Ca 2+ were studied. The information about these changes was obtained from studies of intrinsic (tryptophan residues of F-actin) and extrinsic (rhodamin-phalloidin bound to F-actin) polarized fluorescence. The four components of polarized fluorescence were registered and the obtained data allowed the calculation of the angles of absorption and emission dipoles both of tryptophan and phalloidin-rhodamin and the angle between the F-actin axis and fibre axis. The latter was used as an indicator of flexibility of F-actin [5]. Preliminary reports of this work have been presented [18-20]. Materials and M e t h o d s
Preparation of heavy meromyosin Phosphorylated and dephosphorylated heavy meromyosin were prepared by brief digestion of phosphorylated and dephosphorylated rabbit skeletal muscle myosin solution with chymotrypsin in the presence of CaC12 [16]. The purity of heavy meromyosin preparations was examined electrophoretically. SDS electrophoresis [21] and electrophoresis in 8 M urea [22] were performed and then the density of the electrophoretic pattern was measured. The ratio of the sum of alkali light chains to the regulatory light chains was approx. 1 : 1 and the amount of phosphorylated regulatory light chains was about 95-100% in phosphorylated and lower than 1% in dephosphoryalted heavy meromyosin. The heavy meromyosin preparations were freshly prepared and kept not longer than 3-5 days at 0°C. The amount of phosphorylated and dephosphorylated light chains (LC 2) was measured immediately before use.
Preparations of muscle ghost fibre Single fibre obtained as previously described [11] was placed in a chamber, then glycerol was
washed out with a solution containing 100 mM KC1/1 mM MgC12/67 mM phosphate buffer (pH 7.0) (standard solution). Myosin, tropomyosin, and troponin were removed by treatment of the single ghost fibre with 800 mM KCI/10 mM MgCI2/10 mM ATP/67 mM phosphate buffer (pH 7.0) for 40 min at 4°C. The removal of myosin, tropomyosin and troponin was controlled electrophoretically in parallel samples of ghost fibre treated as described above. Moreover, each ghost fibre used for the experiments was examined by measuring tryptophan fluorescence anisotropy, and for further studies only the ghost fibres showing typical parameters for pure F-actin filaments were used [23]. Muscle ghost fibre contained about 80% of actin. It was found by densitometrical measurements of sodium dodecyl sulphate electrophoretic pattern of parallelly prepared samples of ghost fibre. Sodium dodecyl sulphate electrophoresis of those samples was run in 6 8% gels. Some single fibres were treated with phalloidin-rhodamin in the following way. The ghost fibres were incubated with 4 mM phalloidinrhodamin diluted in standard solutions during 20 rain, and then the unbound reagent was removed by washing with standard solution.
Binding of heavy meromyosin to F-actin in ghost fibre Ghost fibres both modified and unmodified by phalloidin-rhodamin were incubated with phosphorylated or dephosphorylated heavy meromyosin (2 mg/ml) dissolved in 30 mM phosphate buffer (pH 7.0)/1 mM MgCI 2 and 1 mM EGTA or 0.1 mM CaCI 2, considered in the present paper as low or high Ca 2+ concentration, respectively. The molar ratio of bound heavy meromyosin to actin in decorated ghost fibre was calculated as proposed by Yanagida and Oosawa [5]. The amount of heavy meromyosin bound to F-actin in decorated ghost fibre was estimated by measuring tryptophan fluorescence after removal of unbound heavy meromyosin by washing with buffer containing 20 mM phosphate (pH 7.0) and 1 mM MgC12. Measurement was performed with the same apparatus after removal of polarization filter and wollaston prism.
Measurements of polarized fluorescence of intrinsic tryptophan and phalloidin-rhodamin bound to F-actin in ghost fibre The t r y p t o p h a n fluorescence was excited at 303 _+ 4 n m a n d the e m i t t e d light was r e c o r d e d at 3 2 0 - 3 8 0 nm. T h e fluorescence of p h a l l o i d i n - r h o d a m i n b o u n d to F - a c t i n in ghost fibre was excited at 436 _+ 5 n m a n d registered at 5 0 0 - 6 5 0 nm. M e a s u r e m e n t s were m a d e with a m i c r o s p e c t r o p h o t o m e t e r [24]. T h e four c o m p o n e n t s intensities of p o l a r i z e d fluorescence iiIll, i i I , , ~_I 1 , 1111 i n d i c a t i n g the d i r e c t i o n of incident light (on the left) a n d the d i r e c t i o n of the e m i t t e d light (on the right), relative to the fibre axis were measured. T h e degrees of fluorescence p o l a r i z a t i o n of PII a n d P l a n d the value of QII were defined as;
e,, = (,~6,-,,1. )/(,,1,, +,,1. )
3, ± I l I = F [ ( S - S S ) C I + ( 1 - S ) H 3 + ( 2 - S ) F 3
+ ( 2 S - Q I S S ) R 1 ] + FG1 4. ±I± = F [ ( S - O , 7 5 S S ) C 4 + ( 2 - S ) H 4 +(3SS-(S-2)Q1R4]+ FG2 where F=I-N;
S=sin20;
C2 = 2I sin2Acos2E -cos2A .cos2E + 12sin2Ecos2A +2 cosD sinA sinE cosA cosE; H2 = 1 - sin2A- sin2E
FG1 = i!~(2-cos2y), FG2 = ~tsN(1+2 cos27); Q2 = J~(1+ cos2D). SS = sin40
Pa_ = ( l l ~ - ±III)/(±l± + ±III)
T h e angle between the a b s o r p t i o n a n d emission d i p o l e s 7 was calculated as p r o p o s e d in Refs. 5. 25, 28 from e q u a t i o n
QII = (lllll- • lll)/(Jii 4- ± 111)
PNO = (3 COS27-- 1)/(COS2y +3)
E x p e r i m e n t a l d a t a were a n a l y s e d within the m a t h e m a t i c a l m o d e l s described in Refs. 5, 25 a n d 26. T h e m o d e l s are b a s e d on the a s s u m p t i o n that the a n i s o t r o p y of muscle fibre F - a c t i n fluorop h o r e s fluorescence can be well d e s c r i b e d by the system of f l u o r o p h o r e s aligned along the helix a n d r a n d o m l y a r r a n g e d emitting fluorophores. The four c o m p o n e n t s of p o l a r i z e d fluorescence were expressed as functions of the angles ¢bA, D E, 0 a n d the value N: where ¢bA and ¢bE are the angles b e t w e e n the long axis of F - a c t i n and the a b s o r p tion a n d emission dipoles, respectively; 0 is the angle between the long axis of F - a c t i n and the fibre axis (c.f. Fig. 1); N is the n u m b e r of rand o m l y a r r a n g e d fluorophores. The c o r r e s p o n d i n g expressions derived as d e s c r i b e d in Refs. 5, 25 a n d 26 are: 1.,111 = F[( S - SS)C2 + ( 1 - S)H2 + ( 1 - S + SSQ2)8R1] + FO2
2. iii± = F[( S - SS)CI + ( 1 - S ) U l + ( 2 - S) Vl + (28 - Q1SS)RI] + FG1
where PNo is the degree of fluorescence p o l a r i z a tion of r a n d o m l y o r i e n t e d oscilatores (dipoles) R1 = 'ssin2Asin2E; C1 - 2t cos2A cos2E --4
4I
sin2E cos2A
sinZA cos2E-cos D sin A sin Ecos A cos E:
H1 = 12(1- sin2A-cos2E); F1 = 14sinZAcos2E: Q1 = 0,5 +cos2D: H3 = 2t (1 - sin2E - cos2A) H4 = Q1 sI (1 - cos2A - cos2E ) ; C4 = 4t sin2A cos2E + ~4sin2Ecos2A QI t~sin2Asin2E +cos D sin E cos A cos E. T h e angles ¢bA, q)E, a n d the value of N were chosen by the least-squares fit of e x p e r i m e n t a l ratios of iii±/111,, ± I J , l l l and 11./11111. The elastic m o d u l u s for b e n d i n g or the flexural regidity-E [5] of the thin filament were o b t a i n e d by using the e q u a t i o n sin2~b = 0.8 ( k T / e ) L , where k and T are the Boltzman c o n s t a n t a n d the a b s o l u t e t e m p e r a -
Z
ture, respectively and L is the length of the filament. A TPase assays
The activities of K +- and Ca+-ATPases and actin-activated Mg2+-ATPase of heavy meromyosins were determined in the following media: K +ATPase: 0.5 mM KC1/20 mM Tris-acetate (pH 7.0)/2 mM dithiothreitol/5 mM EDTA/Ca +ATPase: 10 mM CAC12/20 mM Tris-acetate (pH 7.0)/50 mM KCI. Actin-stimulated Mg+-ATPase: 1 mM MgC12/20 mM Tris-acetate (pH 7.0)/50 mM KC1 with 0.08 mg/ml of actin. The final volume was 2 ml, concentration of heavy meromyosin 0.2 mg/ml. The reaction was started by addition of ATP to the final concentration of 1 mM and after 1 min incubation at 25°C was stopped by the addition of 1 ml of 10% trichloroacetic acid. Liberated phosphate was measured by the method of Fiske and SubbaRow [27]. Protein determination
The amount of actin and heavy meromyosin were determined spectrophotometrically [17] and by the biuret method [28]. Results
The enzymatic activities of heavy meromyosin used for the studies of conformational changes in F-actin of muscle ghost fibre and the amount of phosphorylated heavy meromyosin light chains are given in Table I. As mentioned in Materials and Methods the molar ratio of two regulatory light chains to the sum of alkali light chains was, in heavy meromyosin approx. 1:1, and approxi-
?
rl
Fig. 1. Diagram explaining the calculation of the polarized components of fluorescence [51 0 is the angle between the filament axis of F-actin OW and the fiber axis OZ. tbA, q)t:. are angles between the F-actin axis and the absorption dipole (,~) and emission dipole (E) in F-actin, respectively. 3' is the angle between ,~ and I~.
mately all regulatory light chains of the respective heavy meromyosin preparations were either in phosphorylated or dephosphorylated form. The enzymic properties (K +- and Ca+-ATPase activities) of heavy meromyosin used were similar, independent of the form of those regulatory light chains. The values of specific actin-stimulated MgZ+-ATPase activities of phosphorylated and dephosphorylated heavy meromyosin at high (0.1 mM CaCl2) and low (in the presence of 1 mM EGTA) Ca 2+ concentrations were similar (Table I). The binding of heavy meromyosin to F-actin in myosin and troponin-trypomyosin free ghost fibre induced changes in the degree of F-actin intrinsic
TABLE l THE ENZYMIC PROPERTIES OF PHOSPHORYLATED A N D DEPHOSPHORYLATED HEAVY MEROMYOSIN Activities are expressed as tool of ATP hydrolyzed per tool of heavy meromyosin per s. ATPase assays were as detailed in Methods. The ratio of light chains (LC) (LC 1 + LC3)/LC 2 was approx. I : 1. The data represent mean _+S.E. from three determinations for five different preparations. Amount of phosphorylated
ATPase activity (s l)
light chains (LC2) (% of total LC2)
K+
Ca 2+
Mg 2+ plus actin Ca 2 +
EGTA
95_+5% 0-5%
13.8__+0.2 14.2_.+_0.3
12.2_+0.3 12.4_+0.1
4.2±0.1 4.4_+0.2
6.3_+0.2 6.7±0.3
tryptophan fluorescence polarization. A significant increase of Ptt can be observed when dephosphorylated heavy meromyosin at high and phosphorylated heavy meromyosin at low Ca 2+ concentration were bound. The values of P . are increased by binding of both phosphorylated and dephosphorylated heavy meromyosin to actin, but the values of P± differ depending on Ca 2+ concentration (Fig. 2A, B). The value of Qtt for F-actin both in the presence of 1 mM EGTA or 0.1 mM CaC12 was 0.087. However, after binding of phosphorylated heavy meromyosin in the presence of 1 mM EGTA and dephosphorylated heavy meromyosin in the presence of 0.1 mM CaC12, the values are increased to 0.212 and 0.149, respectively. When phosphory-
i
'8
'A
~7 0.20
A
r-
0.18
P ~3
0.1b 0
I t
c ._o
Ic
i
0.48
~D
"6 N c.O
0.40
c
-0.36
0
. . . . . . . . . . . . . . . . . . . .
O
I1
-0.44
I
I
I
0.15
0.30
0.15
I
0,30
Bound heovy rneromyosin ( too[ /tool of octin ) Fig. 2. Changes of polarized fluorescence of F-actin intrinsic tryptophan (A, B) and phalloidin-rhodamin bound to F-actin (C, D) in single ghost fibre induced by the binding of phosphorylated (C) o) or dephosphorylated (vv) heavy meromyosin. Open symbols express the values of Pz and closed symbols those of Pit" The binding studies were performed in a medium containing 30 mM phosphate buffer (pH 7.0), 1 mM MgCI 2 and 1 mM EGTA (A, C) or 0.1 mM CaC1 z (B, D).
lated heavy meromyosin in the presence of Ca 2+ and dephosphorylated heavy meromyosin in the absence of Ca 2+ are bound to F-actin, the values of QII are decreased to 0.07 and 0.047, respectively. The changes of Qtl' PIt and P± reaching their maximum by binding of 0.15 mol heavy meromyosin per mol ofactin, remain practically constant even when the amount of heavy meromyosin bound to actin is increased. The lack of correlation between the changes in polarized fluorescence and the amount of heavy meromyosin bound to F-actin in ghost fibre (see (Fig. 2 A, B) allows the assumption that the amount of tryptophan of heavy meromyosin does not influence the values of Pll and P± under the conditions studied. Moreover, the differences in the values of PtI' QII and P± produced by the binding of both phosphorylated and dephosphorylated heavy meromyosin to F-actin in ghost fibre, estimated at the same molar ratio of bound heavy meromyosin to actin are different, depending on the low or high concentration of Ca 2+. Actin of ghost fibre was also labelled by treatment with phalloidin-rhodamin and the influence of the binding of both phosphorylated and dephosphorylated heavy meromyosin at low and high Ca 2+ concentration on the degree of polarization of phalloidin-rhodamin bound to F-actin was examined. As shown in Fig. 2C, D the binding of phosporylated heavy meromyosin at low and dephosphorylated heavy meromyosin at high Ca 2+ concentration induced a significant increase of Ptl, whereas phosphorylated heavy meromyosin at high Ca 2+ concentration induced a significant increase of Pti' whereas phosphorylated heavy meromyosin at high and dephosphorylated heavy meromyosin at low Ca 2+ concentration decreased the values of PIr The values of P± are only slightly changed by the binding of dephosphorylated heavy meromyosin. Significant changes of P± were observed caused by binding of phosporylated heavy meromyosin, the values of P± increasing at high and decreasing at low Ca 2+ concentration. Thus, the character of the changes of polarization of phalloidin-rhodamin bound to F-actin in ghost fibre, induced by the binding of heavy meromyosin, depends both on the phosphorylated or dephosphorylated form of heavy meromyosin and
low or high Ca 2+ concentration, analogically to the changes of polarized fluorescence of intrinsic tryptophan of F-actin in ghost fibre (cf. Fig. 2A,
B). The three ratios of four components of polarized fluorescence of intrinsic tryptophans of F-actin in ghost fibre, measured in the presence of 0.1 mM CaC1 z or 1 mM E G T A before and after binding of phosphorylated or dephosphorylated heavy meromyosin as well as the calculated angles of absorption ~A and emission q~E dipoles and sin20 (0 is the angle between the F-actin axis and the fibre axis) are given in Table II. The values of q~A and q)E and sin20 were determined on the basis of equations proposed for elaboration of polarized fluorescence data [5], by computer analysis to give the best agreement with the ratios of iii±/111,, ± l J , lll and l lj,/lll, obtained by measuring the four components of polarized fluorescence of intrinsic tryptophan of F-actin in ghost fibre. The angle between the dipoles A and E (Fig. 1) of the tryptophan molecule was adopted as ~ = 38". At the molar ratio of bound heavy meromyosin to actin equal to 0.15 in the presence of 1 mM E G T A the values of q~A and ~E decreased when bound heavy meromyosin was phosphorylated and
increased when it was in dephosphorylated form. The values of both q)A and q~E change in the opposite direction in the presence of 0.1 mM CaCI 2. The most probable value of sin20 for F-actin obtained by computer analysis was 0.054 _+ 0.003 in the presence of 1 mM E G T A or 0.1 mM CaCI 2. This value changed significantly depending on low or high Ca 2+ concentration when phosphorylated or dephosphorylated heavy meromyosin was bound to F-actin in ghost fibre (Table I1). The relation between sin20 and the elastic modulus of the F-actin filament (c), as proposed by Yanagida and Oosawa [5], can be described by the equation sin20 = 0.87. (kT/~). L, where L is the length of the semiflexible filament with one end fixed and the other free, The calculated value of of F-actin both at low and high Ca 2+ concentration, assuming L = 1.0 /,tm, is equal to 5.5 _+ 0.l 10 -26 N e w t o n - m 2 ( N - m 2 ) , The values e of Factin containing bound heavy meromyosin were calculated using the values of sin20 shown in Table II. They were 3.3 _+ 0.2-10 26 N - m 2 and 3.6 _+ 0.2 - 10 26 N • m 2 when phosphorylated heavy meromyosin at low, and dephosphorylated heavy meromyosin at high Ca 2+ concentration
TABLE II P O L A R I Z E D F L U O R E S C E N C E OF T R Y P T O P H A N RESIDUES OF F-ACTIN IN T H E G H O S T FIBRE BEFORE A N D A F T E R B I N D I N G O F P H O S P H O R Y L A T E D OR D E P H O S P H O R Y L A T E D HEAVY M E R O M Y O S I N The molar ratio of bound heavy meromyosin to actim 0.15. The data are averages determined by computer analysis of 30-40 measurements. Calculation was performed on the basis of equations proposed by Treager and Mendelson [25] and by Yanagida and Oosawa [5]. The angle between absorption and emission dipoles in the same tryptophan molecule, y = 38 °. Angles of absorption (q~A) and emission ( ~ E ) dipoles; sin'0 where 0 is the angle between the filament axis and fibre axis, the number of random fluorophores (N), ratios of four components of polarized fluorescence ill±/llllp, • 1!1/11111, • I L/11111, where ilia., • 111"II111' ± 1± are intensities of polarized fluorescence indicating parallel (11) or perpendicular ( ± ) direction of the incidient light (on the left side) and emitted light (on the right side) relative to the fibre axis.
irl • /llllr
± llJilllf
• Ix/lllll
q)A
~E
sin2
N
1 m M EGTA 0.1 m M CaCI z
0.71 _+0.02
0.84_+0.03
1.26_+0.04
62.4_+0.8
57.5_+0.1
0.054_+0.003
0.55_+0.2
F-actin + phosphorylated heavy mermyosin
1 m M EGTA 0.1 m M CaC12
0.68_+0.01 0.71 _+0.02
0.65_+0.02 0.87_+0.02
1.00_+0.02 1.32_+0.05,
54.5_+0.1 63.7_+0.1
56.5-+0.1 57.7_+0.1
0.081 -+0.010 0.044_+0.004
0.66_+0.03 0.54___0.1
F-actin + dephosphorylated heavy meromyosin
1 m M EGTA 0.1 m M CaCI 2
0.71 +0.01 0.67_+0.03
0.91 -+0.03 0,74-+0.03
1.37-+0.03 1.13_+0.03
64.7-+0.5 60.9_+0.5
58.0_+0.1 56.7-+0.2
0.052_+0.004 0.088_+0.002
0.54___0.02 0.64_+0.02
Additions F-actin
were bound to F-actin in ghost fibre, respectively, and 6.2 +_ 0.1 and 6.6 _+ 0.2 • 1 0 - 2 6 N - m 2 for Factin containing bound phosphorylated heavy meromyosin at high, and dephosphorylated heavy meromyosin at low Ca z+ concentration, respectively. The angle between the absorption and emission dipoles of phalloidin-rhodamin bound to F-actin was estimated as ~' = 46 °. The ratios of ,I±/,I,, ± 111/11Iii and ± Ii/NIii obtained by measuring four components of polarized fluorescence of phalloidin-rhodamin bound to F-actin in ghost fibre at low and high Ca 2+ concentration and in the presence or absence of bound phosphorylated or dephosphorylated heavy meromyosin are given in Table III. The values of q~A, q~E and sin20 were calculated by computer analysis by approximation to the values of the above given three ratios of the measured four components of polarized fluorescence of phalloidin-rhodamin bound to F-actin in a ghost fibre. Those values of q~A and q~E and sin20 are presented in Table II1. The binding of phosphorylated and dephosphorylated heavy meromyosin induced significant changes in the values of four components of polarized fluorescence of phalloidin-rhodamin bound to F-actin in a ghost fibre and consequently in the three ratios shown in Table III. The character of those changes depended on Ca 2+ concentration. Therefore, the values of the angles q~A and q~E as well as sin20 differ, depending on the phosphorylated or de-
phosphorylated form of heavy meromyosin bound to F-actin in a ghost fibre as well as on low or high Ca 2+ concentration. A marked increase in the values of sin20 could be observed when dephosphorylated heavy meromyosin was bound at high Ca 2+ concentration. The values of sin20 decreased in binding of dephosphorylated heavy meromyosin at low Ca 2+ concentration. Small changes of the values of sin20 were obtained by approximation of the calculated values to the observed ratios of the four components of polarized fluorescence of phalloidin-rhodamin bound to actin in ghost fibre, when phosphorylated heavy meromyosin at low and high Ca 2+ concentration was bound to F-actin in a ghost fibre (Table III). Discussion
The observed changes in polarization of intrinsic tryptophan fluorescence of F-actin in a ghost fibre induced by binding of heavy meromyosin allows the assumption that the kind of binding or its effect depends strongly on the phosphorylated or dephosphorylated form of myosin heads as well as on low or high Ca 2+ concentration. Since the binding of heavy meromyosin was studied in the presence of 1 mM MgC12 and 1 mM EGTA or 0.1 mM CaC12 non-specific divalent cation binding sites of heavy meromyosin were occupied by Mg 2+ or by Ca 2+ (29-31]. The elastic modulus for bending of F-actin (~)
T A B L E II1 POLARIZED FLUORESCENCE OF PHALLOIDIN-RHODAMIN B O U N D T O F - A C T I N IN T H E G H O S T F I B R E S B E F O R E AND AFTER BINDING OF PHOSPHORYLATED OR DEPHOSPHORYLATED HEAVY MEROMYOSIN The angle between the a b s o r p t i o n and emission dipoles, ), = 46 °. Symbols as in legend to T a b l e II. Additions
i11±
± Iii/11111
_ 1±/11111
~A.
~E
sin20
1 mM EGTA 0.1 m M CaCI 2
0.38 + 0.01
1.22 + 0.03
0.51 _+0.02
59.3 +_0.2
42.6 + 0.2
0.061 + 0.002
F-actin + phosphorylated heavy meromyosin
I mM EGTA 0.1 m M C a C I 2
0.35_+0.01 0.41 - 0.01
1.40+_0.04 1.16 - 0.03
0.57+_0.03 0.53 - 0.02
61.2+-0.9 58.7 - 0.3
41.8_+0.2 43.9 - 0.4
0.061 + 0 . 0 0 2 0.066 - 0.008
F-actin + dephosphorylated heavy meromyosin
1 mM EGTA 0.1 m M CaC12
0.39_+0.01 0.37+-0.01
1.36_+0.03 1.26+-0.03
0.59+-0.03 0.53+-0.03
60.7_+0.4 60.0+-0.4
43.1-+0.3 42.2+-0.2
0.053_+0.02 0.068_+0.002
F-actin
[5] in a ghost fibre both in the presence of 1 mM E G T A or 0.1 mM CaCl 2 was estimated as 5.5 _+ 0.1 • 1 0 2 6 N • m 2. The obtained value of { is comparable with that obtained by Yanagida and Oosawa on the basis of polarized fluorescence of N6-ethenoadenosine 5-diphosphate bound to Factin in ghost fibre measurements followed by mathematical analysis of the data [5]. The influence of the binding of heavy meromyosin was studied by the same method. The authors found the value of { to decrease in binding of heavy meromyosin to the value of 3.9-10 26 N . m2; however, nothing is known about the amount of phosphorylated light chains of heavy meromyosin used for the binding to F-actin in ghost fibre. The above values of { decreased by the binding of dephosphorylated heavy meromyosin at high and phosphorylated heavy meromyosin at low Ca 2+ concentration to the values 3.3 and 3.6.10 26 N . m 2, respectively. Thus, the phosphorylated heavy meromyosin with bound Mg 2+ favours the kind of binding to F-actin which exerts an effect similar to that exerted by the binding of dephosphorylated heavy meromyosin having non-specific metal ions binding sites saturated with Ca 2+. In both cases the flexibility of F-actin increases, in contrast to the slightly decreased flexibility of F-actin in a ghost fibre when phosphorylated heavy meromyosin with bound Ca 2+ and dephosphorylated heavy meromyosin with bound Mg 2+ interacts with F-actin in a ghost fibre. It was manifested by the values of { increased to 6.2 _+ 0.1 and 6.6 + 0.6 • 1 0 _26 N • m 2, respectively. The values of the four components of polarized fluorescence of phalloidin-rhodamin bound to Factin are significantly changed by the binding of heavy meromyosin to F-actin in a ghost fibre and the character of those changes depends both on low or high Ca 2+ concentration and on whether the phosphorylated or dephosphorylated heavy meromyosin was bound to F-actin. Since phalloidin affects actin-actin bonds in the F-actin structure [32] and slightly decreases the flexibility of actin filaments [33], the values of sin20 related to the elastic modulus for bending of F-actin and the changes of those values are smaller than that obtained from the values of four components of polarized fluorescence of intrinsic tryptophan residues of F-actin in a ghost fibre.
Thus, it seems reasonable to assume that phosphorylation of myosin heads modulates the interaction of this protein with actin and the character of this modulation depends on whether the non-specific divalent ion binding sites of myosin heads are occupied by magnesium or calcium ions. When the divalent ion-binding sites of phosphorylated myosin are saturated with Mg 2+ the effect of induced changes on F-actin conformation seems to be similar to that induced by dephosphorylated myosin saturated with Ca 2+. Assuming that the increased flexibility of actin filaments is connected with tension development by a single muscle fibre, the influence of phosphorylation of myosin heads on the parameters of isometric tension could be observed at low Ca 2+ concentration. Therefore, the above described observation seems to be in good agreement with the findings of Persechini et al. [34]. The authors found that at low Ca 2+ concentration, phosphorylation of myosin increases isometric tension. The authors suggested that, in vertebrate skeletal muscle, phosphorylation of myosin heads increases the amplitude of tension at submaximal Ca 2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament. Moreover, the observed differences in the electron micrographs of F-actin filaments decorated by phosphorylated and dephosphorylated heavy meromyosin [16] seem to be connected to the possible difference in the kind of binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin under the same conditions. The difference is most probably related to the orientation of myosin heads.
Acknowledgments The authors thank Professor Theodor Wieland for supplying them with phalloidin-rhodamin and Dr. S.A. Krolenko for a stimulating discussion.
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