Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase

.I. Mol. Biol. (1985) 186, 185-195

Effects of Phosphorylated and Unphosphorylated C-protein on Cardiac Actomyosin ATPase H. Criss Hartzelly Department of Anatomy Emory University School of Medicine Atlanta, GA 30322, IJ.S.A. (Received 20 March

1985, and in revised form 24 June 1985)

C-protein, a component of the thick filaments of striated muscles, is reversibly phosphorylated and dephosphorylated in heart. It has been hypothesized that C-protein may be involved in regulating contraction, because the extent of C-protein phosphorylation correlates with the rate of cardiac relaxation. To test this hypothesis, the effects of phosphorylated and unphosphorylated C-protein on the actin-activated ATPase activity of myosin filaments prepared from DEAE-Sephadex-purified myosin were examined. Unphosphorylated C-protein (0.1 pM to 1.5 PM) stimulated actin-activated myosin ATPase activity in a dose-dependent manner. With a myosin : C-protein molar ratio of N 1, actinactivated myosin ATPase activity was elevated up to 3.2 times that of the control. Phosphorylated C-protein (2.5 mol PO,/mol C-protein) stimulated the activity somewhat less (2.5 times that of control). The stimulation of ATPase activity by C-protein was due to an increase in the V,,, value (from 0.25/second to 0.62/second) and a decrease in the K, value (from 11.9 PM to 6.7 /AM). The addition of C-protein to actomyosin solutions produced an increase in the light-scattering of the actomyosin solution and a distinct precipitation of the actomyosin with time. Phosphorylated C-protein had a smaller effect on light-scattering than dephosphorylated C-protein. C-protein had a negligible effect on Ca-ATPase, EDTAK-ATPase, or Mg-ATPase activities in the absence of actin. C-protein had only small effects on the actin-activated ATPase of heavy meromyosin. These results suggest that C-protein at,imulates actin-activated myosin ATPase activity by enhancing the formation of stable aggregat,es between actin and myosin filaments.

1. Introduction

Winegrad: 1984). For example, there is good evidence that Ca influx through the sarcolemmal calcium channel (Isi) is regulated by CAMP and CAMP-dependent protein kinase (Tsien et al., 1972;

The frequency and strength of contra&ion of the vertebrate heart are regulated by the autonomic nervous system: norepinephrine stimulates the heartbeart, whereas acetylcholine inhibits it. The effects of these transmitters are thought to be mediated, at least in part, by the CAMPS-dependent phosphorvlation and dephosphorylation of several prot’eins involved in regulating contraction (Tsien, 1977; St,ull, 1980; England, 1983; Perry, 1983;

Tsien,

t Present address until 1 July 1986: Unite de Recherches de Physiologie Cellulaire Cardiaque, Centre Universitaire Paris-Sud (Bat. 443), F-91405 Orsay Cedex. France. $ Abbreviations used: cAMI’, cyclic AMP; DTT. dithiothreitol: HMM. heavy meromyosin; Mg-HMM, heavy mrromyosin digested in the presence of Mg; EDTA-HMM. HMM digested in the presence of EDTA: S-l, myosin subfragment-1: 5-1, myosin subfragment-2: IL-2. myosin light, chain-2 (phosphorylat,able. DTXB light chain); s.11.. standard deviation. 0022-2836/85/210185-11

$03.00/O

1973;

Reuter,

1974;

Tsien

& Weingart,,

1976;

Josephson & Sperelakis, 1978; Vogel & Sperelakis, 1981; Rinaldi et aZ., 1981, 1982; Trautwein et al., 1982; Osterrieder et al., 1982; Reuter et al., 1982; Xargeot et al., 1983). Phospholamban, which is phosphorylated in viva in response to fi-adrenergic et stimulation (Kranias & Solaro, 1982; Lindemann al., 1983; Lindemann & Watanabe, 1985) and in U&O hy CAMP-dependent protein kinase (Kirchberger et al., 1972; Wray et al., 1973; LaRaia & Morkin, 1974; Tada et al., 1975)? regulates Ca uptake by the sarcoplasmic reticulum (Kirchberger et al.. 1974; Katz et al., 1975; Schwartz et al., 1976; Wray & Gray, 1977; Katz, 1979). Troponin-I is also phosphorylated in viva in response to fi-adrenergic stimulation (England, 1975, 1976, 1977: Solar0 et al., 1976; Stull, 1980) and in vitro by CAMPdependent protein kinase (Reddy et al., 1973; Cole 186

0

1985 Academic

Press Inc.

(London)

Ltd

H. C. HartzeEl

186

& Perry, 1975; Ray & England, 1976; Stull & Buss, 1977; Blumenthal et al.. 1978). Troponin-T phosphorylation is likelv to be involved in regulating the calcium sensitivity of the contractile apparatus (Ray & England, 1976; Reddy & Wyborny: 1976; Bailin, 1979; Holroyde et al., 1979; McClellan & Winegrad, 1980; Mope et al.. 1980: Herzig et al., 1981; Stull, 1980; Robertson et al.. 1982). It has been found that C-protein, which is a component of the myosin thick filament (Offer, 1972: Offer ef al., 1973), is phosphorylated in a CAMP-dependent manner in cardiac muscle exposed to j-adrenergic agonists (Jeacocke & England, 1980; Hartzell &, Titus, 1982; England, 1983; Hartzell, 1984). C-protein purified from cardiac muscle is phosphorylated at three to five sites by purified CAMP-dependent’ protein kinase (Hartzell & Glass, 1984; Lim & Walsh, 1985). Although the function of C-protein remains obscure, we have shown that the level of C-protein phosphorylation correlates with the rate at which cardiac muscle relaxes after a twitch (Hartzell, 1984). We have proposed that C-protein is involved in regulating twitch relaxation in cardiac muscle by modulating actin-myosin interaction. In this paper, I have tested the hypothesis that (‘-protein is involved in regulating actin-myosin interaction by examining the effect of purified (‘-protein on the ATPase activity of purified cardiac myosin. Other investigators have also studied the effect of C-prot,ein on myosin ATPase activity (Offer et al.: 1973; Moos et al.. 1978; Moos & Feng. 1980: Yamamoto & Moos, 1983). These studies have shown that, at low ionic strength, (T-protein inhibits a,ctin-activated skeletal muscle myosin ATPase but stimulates actin-activated cardiac muscle tnyosin ATPase. Our results confirm and ext’end the observations (‘-protein

of Yamamoto stimulates cardiac

& hloos (1983) that actomyosin ATPase

and support the hypothesis that C-protein regulatory role in the myofibril.

plays a

2. Materials and Methods (a) Solutions Citrate/Tris buffer was composed of 10 rnM-citric acid titrated with Tris base to give a final pH of 8.0 at 4°C. The final Tris concentration was approximately 35 mM. Buffer M contained 0.5 M-KCI. 10 m,n-KH,PO,. 1 tnMEDTA. 1 mM-NaPu’,, and 1 mM-DTT (pH 7.0). (b) Protein (i) Protein

purijkation

concentrations

Protein concentrations were determined at, A,,,. assuming the following extinction coefficients: myosin. 0.53 mg ml-’ cm- ‘; HMM, 0.65 mg ml- ’ cm- ‘; actin. 1.15 mg ml-’ cm-‘; C-protein, 1.09 mg ml-’ cm-‘. (ii) C-protein C-protein was purified from frozen muscle exactly as described (Hartzell

chicken cardiac & Glass. 1984).

except that 1O-4 M-phenylmethylsulfonyl-fluoridr kvas added to all solut,ions and the number of Triton ?1100 washes used in preparing mgofibrils was increased from 2 t’o 5. Unphosphorylated C-protein contained. on the average, 0.2 mol PO,/mol C-protein. The PO, content of each batch of C-protein, however. was not routin+ measured because. from past experience. we found that there was lit’tle variability in the PO, cont’ent (Hartzrll & Glass, 1984). C-protein was phosphorylated with purified catalytic. subunit of CAMP-dependent prot,ein kinase as described (Hartzell 8r Glass, 1984). Usually 5 to 10 mp of purified C-protein were incubated in a 1 ml reaction containing 3 pg catalytic subunit, of cAMP-dependent prot,ein kinase/ml (bovine heart), 48 mM-?u‘d?. 3 mm-i&So,. 1.6 m>f-Na?u’,. I In.\l3 mM-DTT. 0.08 mh~-EI)TL4. [y-“P]ATP (- 1 ct/min per pmol). and 20 mmTris H(‘I (pH 8). The reaction was incubated for 30 to 45 min at 30°C. The reaction was terminated by addition of cold, saturated (NH,)&), to makr a final w’,, saturat,ed solution. The mixture was centrifuged at 15,000 g for 20 min. The pellet was dissolved in ;thr~t 0.5 ml of citrate/Tris buffer. C-protein was separated from 011 il the other react)ants chromatograph1 by 1.2 cm x 25 cm column of Biogel PlOO rquil;bratrd ill 10 mu-cit,rate/Tris buffer. The C-prot’ein peak v as concentrated by precipitation with ammonium sulfate. dissolved in cit,rate/Tris buffer to give a protein concentration of 10 mg/mi. and dialyzed against citratcx, Tris buffer overnight. The phosphate c&t.cant of ttrtl phosphorglated C-protein was determined by scintillaticm counting of a known amount of the final dialyet product. I’nphosphorylat,rd (‘-protein received itlenti<~al treatment, except that protein kinase and .4TP ww omitted from t’he reaction. After purification. C-protein was divided into saml)lt~s containing 5 mg of protein and Iyophilized. The I,v,Iphilized protein was stored at - 2OY’ for up to 3 tnont 11s.

Mysin was purifird from fresh chicken hearts I)\- :( method similar t’o that described by Sirtnankowski B White (1984). Myosin was extracted from Tritolt-washed myofibrils. which were prepared by a modifica,tion of thtl met,hod of Ha,rtzell 8 Glass (1984). Myofibrils \I c’rt’ prepared b>- repeated homogenization and crntrifugaticm (13 repetitions) of fresh chicken ventricles in 50 m*r-K(‘l. 2 miv-EDTA. 1 nimXaR’,, 1 mrv-DTT and 2Om~ Tris.HCl (pH 7.9). In t,he washes 5 through 9. the buffer contained I”, (v/v) Triton X100. Myosin was taxtractrd from the myofibrils by adding 3 rols 0.3 M-K(‘l. 0.15 >IKH,PO,. 2 mM-Sa,P,O,. 5 mM-ATP. IO msl-Mg(‘l,. 0.1 tnnl-EDTA, and 1 m&f-DTT (pH 7.0) and stirring for at IO.000 g for 20 min. the* 30 min. After centrifugation supernate was rapidly diluted ISfold with cold I mu DTT. The myosin was allowed to precipit,atr overnight at 4°C’. About’ half of the supernate was aspira,trd and thr remainder was centrifuged at 8OOOg for 20 min. Tht, myosin pellet was dissolved in an equal volume of I >IK(‘1. 20 mM-KH,PO,. 1 rnM-EDTA. 1 IllM-xxx,. tltltl I mnf-TITT (pH 7.0). A solution of 0.2 M-;ITP and 0.2 *IMgSO, (pH 7.0) was added to the dissolved tnyosin to make a final concrntration of 20 m.wATP and 20 mw MgSO,. The viscous solution was mixed gently in a Dounce homogenizer and immediately centrifuged iit .?O,OOOrevs/min in a Beckman type .50.2 Ti rotor for 3 h. The pellet from this spin. which wa,s largely actomyotiin. was saved for t’he preparation of actin (SW below). Myosin was salted out from the superna,te at 36 to 4S0,

C-protein EJfects on Actomyosin saturated ammonium sulfate. This crude myosin, dialyzed into buffer M was used for several experiments. Usually, however, the myosin was dialyzed into 10 mMEDTA. 135 mM-K,HPO,, 15.3 KH,PO,, and 1 mM-DTT. The crude myosin ( -0.5 g) was purified on a 2.5 cm x35 cm column of DEAE-Sephadex A50, as described by Richards et al. (1967) and Offer et al. (1973). Purified myosin was dialyzed and stored in buffer M at 4°C. (iv) Mini$Zaments Minifilaments were prepared as described by Reisler et al. (1980). Myosin in buffer-M was diluted to 5 mg/ml and dialyzed into 5 mM-Na,P,07 (pH 8). After 24 h of dialysis. the myosin was centrifuged at 24,000 g for 20 min. Only fresh myosin (< 1 week old) was used for preparing minifilaments, because aged myosin often precipitated at this stage. The clarified myosin was then dialyzed extensively into citrate/Tris buffer (pH 8). The final minifilaments were clarified by centrifugation at 24,000 g for 20 min. The minifilaments were stored for up to several weeks at, 4°C. Immediately before use. minifilaments were clarified by centrifugation. (v) Heawy meromyosin DEAE-purified myosin at 10 mg/ml in buffer M was digested in buffer M (EDTA-HMM) or in buffer M containing 3 mM-MgSO, (Mg-HMM) with 50 pg chymotrypsin/ml (Sigma type I-S) for 10 min at 25°C. The digestion was terminated by addition of phenylmethylsulfonylfluoride to make 5 x 10e4 M. The mixture was dialyzed overnight against 1 1 of 40 mM-NaCl, 10 mMKH,PO, buffer (pH 6.5), 1 mM-DTT, 1 mM-NaN,. The precipitated protein was removed by centrifugation at 10,OOOg for 20 min. The soluble HMM was then dialyzed against citrate/Tris buffer. Under these conditions - 25% (w/w) of the myosin was converted to soluble material. (vi) Actin Actin was purified from the actomyosin pellet prepared during t,he myosin purification described above. The very viscous actomyosin pellet was resuspended using a Dounce homogenizer in 0.2 mM-Na,ATP, 0.2 miv-CaCl,, 0.2 mM-NaN,, 0.5 mM-DTT, and 2 mM-Tris. HCI (pH 8). This suspension was dialyzed for 72 h with 3 to 4 changes against 2 1 of this same buffer. The suspension was then centrifuged at 50.000 revs/min in the Beckman type 50.2 Ti rotor for 3 h. The supernate was filtered through a 0.22 pm pore size nitrocellulose filter and a solution of 10 mw-sodium ATP (pH 7.5) 0.5 M-KCl, 20 mM-Mgso,, was added to make the solution up to 50 mM-KCl, 2 mMMgS04, 1 mm-ATP. The actin solution was incubated at 37°C for 1 h. cooled on ice to 4”C, made 0.6 M-KC1 by addition of solid KCl, and then centrifuged in the type 50.2 Ti rotor at 50,000 revs/min for 3 h. The crude actin pellet was furt,her purified by at least 3 cycles of depolymerizat’ion and polymerization (Spudich & Watt, 1971; Pardee & Spudich, 1982). In a few experiments, rabbit skeletal muscle actin, prepared by the method of Spudich & Watt (1971) was used and the results obtained were identical with those for cardiac actin. Actin was stored in 50 mmKC1, 1 mM-MgSO,, 1 rnMNa,ATP, 0.2 mM-NaN,, 10 mm-Tris. HCl (pH 8) at 4°C. Before use, fresh Na,ATP (pH 7) was added to make 1 UlM. The actin was incubated for 1 h and centrifuged at 50,000 revs/min, 3 h in the Beckman type 50.2 Ti rotor. The pellet was resuspended in 50 mmKCl, 1 mM-MgSO,, 10 mw-Tris . HCl 1 mw-sodium ATP, 0.2 mM-NaNv’,, (pH 8) by brief sonication and used within 48 h.

187

(c) ATPase

assays

ATPase activity was measured by the release of 32P from [Y-~‘P]ATP. [y3*P]ATP was synthesized by the method of Walseth & Johnson (1979) and purified by the method of Palmer & Avruch (1981). ATPase activities were measured at 25°C in triplicate in a 50-~1 assay containing - 1 PM-myosin and the following buffers: MgATPase: 0.6 M-KCl, 2 mM-MgCl,, 1 mM-DTT. 50 mMTris . HCl (pH 7.5); Ca-ATPase: 0.6 M-KCI. 5 mM-CaCl,, 50 mw-Tris . HCl (pH 7.5); EDTA-KCI1 mM-DTT. ATPase: 0.6 M-KCl, 1 mM-EDTA, 1 mM-DTT, 50 mm Tris.HCl (pH 7.5). Assays were initiated by the addition of [Y-~~P]ATP (50 cts/min per pmol) to a final concentration of 0.4 to 1.2 mM. Unless noted otherwise, actin-activated ATPase was measured in a buffer composed of citrate/Tris buffer (pH 8), 10 mM-KCl, 1.9 mM-MgSO,, 0.4 mM-(y-32P]ATP ( - 50 cts/min per pmol), - 1 PM-myosin, 2 to 50 PM-actin, and, if appropriate, C-protein dissolved in citrate/Tris buffer. The myosin and C-protein were mixed and preincubated for >30 min at 4°C before the initiation of the assay by addition of a mixture of actin and [y3*P]ATP. The length of time the minifilaments were preincubated with C-protein did not seem to affect the results significantly. The time-dependence of the effect, however, was not investigated systematically. Typically, the length of preincubation was between 30 min and 2 h. The assay was incubated at 25°C for 2 to 3 min. Actin-activated ATPase was expressed as the activity in the presence of actin minus the activity in the absence of actin measured in the same buffer. This “minus-actin” activit’y (<0.02/s) was less than the Mg-ATPase activity measured under more standard conditions (above). The release of 32P from [Y-~*P]ATP was measured as described by Chock 6 Eisenberg (1979). The assay was terminated by the addition of 10 ~1 of 6 M-HCl followed by 25 ~1 of 2.5 M-sulfuric acid, 6% (v/v) silicotungstic acid. Ammonium molybdate (25 ~1 at 10% (w/v)) was added, the sample was vortexed for 10 s, and the phosphomolybdate complex was extracted with 100 ~1 of isobutanol/benzene (1: 1, v/v). The extraction of the phosphomolybdate complex was completed within 1 min after the termination of the assay. The tubes were then centrifuged at 3000 revs/min for 5 min, and 25 ~1 of the organic phase were counted by Cherenkov counting in 3 ml of water. ATPase activities were expressed as mol ATP hydrolyzed/mol myosin per s (l/s). Each point was run in triplicate. Variation between replicate tubes was less than + 10%. Under all the conditions used here, the assays were linear with time. (d) Light-scattering Light-scattering was measured in a Beckman spectrophotometer by the absorbance at 350 nm at room temperature (22 to 25°C).

3. Results (a) P,rotein preparations The purpose of these experiments was to evaluate the effect of C-protein upon the actin-activated ATPase activity of myosin filaments and HMM. Thus, it was important to know the purity of the proteins used. Figure 1 is a sodium dodecyl sulfate/ polyacrylamide gel stained with Coomassie blue to

188

H. C. Hartzell

PC

C

A

EDTA HMM

M

Mg

HMM

f?

iC

‘Li

Figure 1. Sodium dodecyl sulfate/polyacrylamide gel of proteins used for these experiments. Proteins \vert~ electrophoresed as described (Hartzell & Glass, 1984). PC. phosphorylated C-protein (5 pg): C. unphosphorylatrd C-protein (5 pg): A. actin (12 pg); EDTA-HMM: heavy meromyosin (12 pg) prepared by digest,ion of DEAE-purified myosin in buffer M; M, DEAE-purified myosin (12 /*g); Mg-HMM: heavy meromyosin (50 pg) prepared by digestiotl of DEAE-purified myosin in buffer M containing 3 mM-MgSO,. The Mg-HMM channel in this Figure was taken from a gel different from those used for the other channels in this Figure. For this reason, t,here is not an exact, alignment of the bands with the channels on the left. However. the LC-2 band in the Mg-HMM exactly co-migrat’ed with L(‘-2 of undigested myosin run on the same gel. HC marks the position of the myosin heavy chain @I, 200,000), LC2 marks t’he position of the light chain-2 of myosin (J& 18.000).

show the purity of our protein preparations. Most of the Figures in this paper show results from experiments performed using the same protein preparations shown in this gel, although the experiments were replicated with at least two different preparations. The DEAE-purified myosin was free of contaminating C-protein or actin and was composed largely of myosin heavy chains and light chains.

The average ATPase activities of the myosin preparations were: Mg-ATPase, 0.0 1 ( kO.01) s- ‘: Ca-ATPase, 0.63 (kO.14) s-l: EDTA-ATPase, 1W N = 5). These values are (f0.49) s-l (mean+s.n.. comparable to those reported by Siemankowski bz Dreizen (1978) but are lower than the values reported by ,Margossian &. Slayter (1985a) for canine myosin. Heavy meromyosin prepared by digestlion in

C-protein

189

Effects on Actomyosin

two distinct bands in their gel system (Guilian et al., 1983). These two bands may represent different isoforms of C-protein. The actin contained a minor contaminant migrating near M, 115,000.

(b) Effect of C-protein

0

O-15 03

0.6

I.2

25

5.0

C-protein (/al) (b)

Figure 2. The effect of C-protein upon actin-activated myosin ATPase activity of myosin minifilament,s. Minifilaments were pre-incubated with unphosphorylated (c‘. circles, continuous line) or phosphorylated (PC, squares. broken line) C-prot,ein at 4°C for - 1 h. The ATPase reaction was initiated by addition of actin and ATP. Note t’hat the x-axis is a logarithmic scale. (a) [Actin] = 5 PM; [myosin] = 0.85 FM. The ordinate is ATPase activity in mol ATP hydrolyzed/s per mol myosin. In this and other Figures, t’he ATPase activity in the absence of actin was subtracted from the activity in the presence of actin to give the values shown. (b) [Actin] = iP ELM: [myosin] = 1.3 PM. In (b). rabbit skeletal muscle actin was used. buffer containing EDTA (EDTA-HMM) was composed predominately of a heavy chain of molecular weight approximately 150,000 and one light chain. LC-2 was degraded in EDTA-HMM. In addition, there were a variety of proteolytic fragments of the heavy chain. LC-2 in t,he Mg-HMM was c*onsiderably less degraded than that in the EDTA-HMM preparation. Quantitative densitometry showed that approximately 5O”/b of the LC-2 in the Mg-HMM was undegraded. The other 50% of LC-2 was degraded to a fragment of && 13,000 to 14,000. We did not analyze the homogeneity of the HMM by sedimentation velocity. As described previously, C-protein preparations were almost. homogeneous. The main contaminant was a. prot’eolytic fragment of C-protein (J& 130,000). which in this particular preparation may have amounted to 5% of the total protein. The C-protein preparation was free of protein phosphatase and protein kinase activities in the absence of exogenous calcium and calmodulin. Although C-prot)ein migrated as a single band in this gel system. G. CT. Guilian & R. L. Moss (personal communication) have recently found that this preparation of C-prot’ein can be separated into

on ATPase

We examined the effects of C-protein on the actin-activated ATPase activity of myosin filaments produced in two different’ ways. In the first set of experiments, we made myosin minifilaments as described by Reisler et al. (1980). These minifilaments were quite homogeneous in their size distribution. On sucrose gradients in citrate/Tris buffer, minifilaments (1 mg/ml) sedimented as a sharp peak at 17 S (data not shown). Figure 2 shows two typical ATPase experiments using minifilaments. In these experiments, minifilaments were mixed with C-protein and incubated for about one hour at 4°C. The ATPase assay was initiated by addition of actin and [y-32P]ATP and incubated at 25°C for two minutes. In Figure 2(a), the actin-activated ATPase activit’y in the absence of added C-protein was 0.06 per second. C-protein between 0.08 pM and 2.5 pM stimulated the ATPase activity. The maximum ATPase activity was 0.24 per second at a C-protein concentration of 0.6 PM. With high C-protein concentrations, the ATPase activities were often, but not invariably. inhibited to levels below those of the control. There was considerable variability from experiment’ to experiment in the percentage stimulation. Part of the variability seemed to be related to the age of the myosin: the ATPase activities of older myosin preparations were usually stimulated less than more fresh preparations. Phosphorylated C-protein (2.4 mol PO,/mol C-protein) had an effect qualitatively similar to that of unphosphorylated C-protein. However, the maximum actin-activated ATPase activity in t’he presence of phosphorylated C-protein was less than t.hat in the presence of unphosphorylat,ed C-protein. In eight experiments, unphosphorvlated C-protein always stimulated the ATPase act&ties more t’han phosphorylated C-protein did. although the difference bet,ween phosphorylated and unphosphorylated activities was sometimes small. The maximum ATPase activity in the presence of 0.6 ,LiM to 1.2 PM-unphosphorylated C-protein was 3.2OkO.79 (meanhs.n., N = 8) times control, whereas the maximum ATPase activit,v in the presence of same concentration of the phsophorylated C-protein was 2.56 k 0.57 (mean & S.D.) times control. The difference between these was significant at t,he 59b level, as determined by the Wilcoxon paired sample test (Zar, 1984). C-protein had no effect upon the Mg-ATPase activity of myosin measured in the absence of actin. Furthermore. C-protein had no ATPase activity by itself or in the presence of act’in under the conditions of these assays. This confirms previous

190

H. C. Hartzell

0.10

r

0.6

0.08 c

0,621s

;

0.06

0 E a

0.04

(kO.04)

A’,,. =6,7p~

I

0

0.04

008

0.16

0 30

0.60

+ (1.1)

I

I25

2-5

5.0

C-protem Q.LM)

Figure 3. The effect of C-protein on ATPase activit? of myosin filaments prepared by dilution of monomeric> myosin into citrate/Tris buffer. Myosin in 0.25 M-KC? was dilut,ed to 12.5 mM-KC1 in citrate/Tris buffer containing either unphosphorylated (C, circles, continuous line) or phosphorylated (PC, squares, broken line) C-protein. The ATPase reaction was initiated by addition of actin and ATP. The final assay mixture contained O&5 PM-myosin. 17.5 mM-KCl, 5 PM-actin, 1.9 mM-MgSO,, 0.4 rnM[y-32P]ATP, and 10 mM-citrate/Tris buffer (pH 8).

performed under different conditions (Offer et al., 1973; Yamamoto & Moos, 1983: Hartzell & Glass, 1984). In order to determine whether the effect of C-protein on actin-activated ATPase activity was a peculiarity of the minifilament preparation, the effect, of C-protein on the actin-activated ATPase activity of myosin filaments produced by dilution of myosin into low ionic strength was examined. Dilution of solutions of myosin in high ionic strength to low ionic strength produces large filaments with a heterogeneous size distribution & Harrington, 1966). In these (Josephs experiments, myosin in 0.25 M-KC1 was diluted to 12.5 mM-KC1 into citrate/Tris buffer with or without C-protein. The mixture was incubated for about one hour at 4°C and the ATPase assay was initiated by addition of actin and [y-32P]ATP. The actin-activated ATPase act.ivity of these larger filaments in the absence of C-protein was about five to tenfold lower than that of the minifilaments. Nevertheless, the effect of C-protein on the actinactivated ATPase activity of these filaments and on minifilaments was qualitatively the same (Fig. 3). Unphosphorylated C-prot.ein stimulated the actinactivated ATPase activit,y about sixfold and phosphorylated C-protein was less effective in stimulating the actin-activated ATPase activit’y than unphosphorylated C-protein (Fig. 3).

20

IO

0

40

30

Actm CPM) (al

25-e

measurements

(c) Kinetic mechanism of the stimulation

of ATPase

In order to gain insight into the mechanism b) which C-protein stimulated the actin-activated ATPase activities of myosin filaments, ATPase activity was measured as a function of actin concentration in the presence and absence of 1 PM-

, -0.2

0

0.2

0.4

I/actm

0.6

0.8

(,i&‘)

(b)

Figure 4. Kinetic parameters of actin-activated ATPase. Actin-activated minifilament ATI’ase was measured in t,he absence (squares) or presence (circles) of 1 PM-unphosphorvlated V-protein under standard assa? conditions. [Myosm] = 1.Ii pM. {a) Michaelis plot. Points are observed data points: continuous lines are best-tit non-linear regresssions to the Michaelis-Menten equation. (b) Double reciprocal plot of the same dat,a as in (a).

C-protein (Fig. 4). Figure 4(a) shows he data plotted on linear co-ordinat,es. The continuous lines are the best fit lines to the data using the MichaelisMenten equation. The effect of Cprotein was to increase the V,,, value to 25096 of the control and to decrease the K, value to 44”/b of the cont’rol. The differences between the kinetic paramet.ers in the presence and absence of C-protein were statisticalIS significant at ly/, The double reciprocal plot of the same data (Fig. 4(h)) illustrat,ed an additional feature. This plot showed that, in t,he absence of C-protein, the data were not well fit by a single line> but were tit by two lines. This suggested that I he affinity of myosin for actin at low concentrations of actin was higher than the affinity at high concentrations of actin. A similar result has been reported for both cardiac and skelet,al tnuscle myosins (Pope et al.. 1980. 1981). It, seems likely that the larger K,,, value at high actin caoncentrat’ion

C-protein ESfects on Actomyosin Table 1 Kinetic constants of lines in Fig. 4

A

o.25r

Km (*SE.)

~nl:,,,(fS.E.) A. Xon-linear

191

jitt

So C-protein 1 PM-C-protein

0.246/s (kO.016) 0.617/s (kO.036)

IS. Linear jit to double reciprocal$ No Vprotein Low affinity 0.26/s High affinity 0.1 l/s I PM-(‘-protein

(k1.63) 6.7 /LM ( + 1.04)

ll.g/LM

( < 5 PM-aCtin) 14.89 /IM ( > 5 PM-aCtin) 2.47 PM 7.0 /lM

0.615/s

s.E., standard error. t The data in Fig. 4(a) were fit by the Marquart method of iterative least-squares to the Michaelis-Menten equation. $ The data in Fig. 4(b) were fitted by a non-weighted iterative least-squares method.

was caused by steric restrictsions on the accessibility of the actin to the myosin heads. The best-fit parameters for t,he lines in Figure 4 are shown in Table 1. The kinetic parameters derived from the best fit lines in the double-reciprocal plot at artin concentrations greater than 5 PM were in very close agreement with the paramet’ers derived from the non-linear regressions. It was interesting that, in t)he presence of (I-protein, there was no evidence for a biphasic double reciprocal plot, even when the data were replot’ted on expanded axes t,o amplify any inflections in the line. (d) Effect

of (‘-proteinon

HMM

ATPCLSP

The kinetic data show that’ C-protein increased the velocity of the actin-act’ivat’ed ATPase and the affinity of actin for myosin. Since the myosin was in a filament’ous form. I hypothesized that’ C-protein altered the structure of the myosin filaments and increased t,he number of myosin heads available for interaction with actin. In order to test this possibility, the effect of (J-protein on acto-HMM ATPase activity was examined. I reasoned that if C-protein produced its effects on actin-activated myosin ATPase activity by producing changes in filament structure, C-protein should not affect t’he actin-activated ATPase activity of soluble HMM. In t,hese experiments, the ATPase assays were performed in buffer of exactly the same composition as that used for the minifilament ATPase assays. The a&in-activated HMM ATPase activity in the absence of (‘-protein was only slightly higher than that, of the same concentration of minifilaments in the absence of C-protein in the same experiment’. C-protein. whether in the phosphorylated 01 dephosphorylatrd forms. however, had no effect on ATPase activity. except at high concentrations of C-protein, whercs an inhibition was noted (Fig. 5). These data were consistent with the hypothesis that the stimulatory effect’ of C-protein required either myosin in a filamentous form or a portion of the light, meromyosin segment of the myosin molecule in order to exert its effect.

L.+ 0

’ 0 16

1

0.30 I.2 060 C-protein (FM)

I

I

2.5

50

Figure 5. Effect of’ c-protein on actin-activated ATPase of HMM. ATPase was measured as a function of unphosphorylated (C. circles, continuous line) and phosphorylated

(PC. circles.

broken

line) (I-prot’ein

under

standard assav conditions. [Actin] = 5 /lM; [HMM] = l.ldp~.“In the same experiment. the rlTPase activity of minifilaments ([myosin] = 0.78 ~NI) was also measured (triangles). The HMM used for the experiment shown in Figure 5 was prepared in the presence of EDTA. As shown in Figure 1, LC-2 of this preparation was largely degraded. Thus, an alternative explanation for the lack of effect of C-protein on acto-HMM ATPase was that the effect of C-protein required LC-2. This possibility was suggest’ed by work of Margossian & Slayter (1985a,b). To t’est this possibility, we prepared HMM in t’he presenre of Mg. The LC-2 in this preparation was only partly degraded (Fig. 1). Experiments with this HMM preparation, however, yielded exactly the same results as those shown in Figure 5: Cprotein had no st’imulatory effect on the acto-HMM ATPase act’ivities. Sodium dodecyl sulfate/polyacrylamide gels of the acto-HMM reaction ATPase mixtures demonstrated that C-protein was not degraded during the assay, which might occur if the chymotrypsin used for preparing the HMM were not completely inactivated.

(e) Light-scattering To examine the effect’ of
192

H. C. Hartzell

0.06

-.

k

:q 0

,

,

0.4

O-0

,

,

I.2

1.6

,

(

20

24

,

C-protein (PM)

Figure 6. The effect of C-protein on light-scattering of minifilaments. Minifilaments (500 ~1; 2 PM) in 10 mMcitrate/Tris buffer containing 3.2 m&I-MgSO, and 1 mMATP were placed in a cuvette. A sample of 2 ~1 of unphosphorylated continuous line) or (circles. phosphorylat,ed (squares, broken line) C-protein was added at -2-min intervals with stirring. The triangles show the light-scattering of the sum of the separate minifilament and C-protein solutions. Light-scattering was measured by absorbance at 350 nm. By itself, 1 PM-C-protein had an AjSO of
When actin was added to minifilamenk that’ had presence been pre-incubated in the of unphosphorylated C-protein, however, a different result was obtained. Even in the presence of approximately 1 mM-ATP, light-scattering increased with time after addition of actin (Fig. The increase in light-scat’tering was 76)). accompanied by visible turbidity in the cuvette and. after ten minutes, formation of an act,omyosin precipitate (superprecipitation). The rate of increase in light-scattering was dependent upon the time of pre-incubation of the minifilaments with C-protein. The rate of lightscattering increase was distinctly slower if C-protein was incubated wit.h minifilaments for only four minutes, rather than eight minutes, before addition of actin (Fig. 7(a) and (b): see also Fig. S(a) and (c)).

M Cc)

1

A ! 1

(d)

F

(e)

)

II Figure 7. The eflect of C-prot,ein on actin and myosin light-scattering. Light-scattering was measured 1,~ absorbance (,4) at 350 nm. The following stock solutions were mixed together in the ruvette as described below. M. minifilaments (1.4 PM) in 10 mrv-citrate/Tris buffer cont.aining 0.77 mM-ATP. 3.6 mM-MgSO,. and 9-34 MelKCl. C. 10 PM-unphosphorylated C-protein in IO mMcitrate/Tris buffer. A. 20 PM-a&in in 50 rn\f-KCII. 1 m&l-

MgSO,, 1 mM-KaK,, 10 miv-Tris.H(:l

(pH 8). In each of

the traces shown, the solutions were mixed t,ogether at) the downward deflert.ion. (a,) and (b) 400 ~1 of M +50 ~1 of C + 50 ~1 of A. (c) Before the start of the trace. 400 ~1 of M and 50 ~1 of citrate/Tris buffer were mixed. At the downward deflection, 50~1 of .4 were added. (d) Before the start of t,he trace. 400 ~1 of citrat,e/Tris buffer containing 0.77 mM-ATP, 3.6 mM-MgS(>,, and !%37 mMKC1 were mixed with 50 ~1 of CT.At the deflection, 50 pi of A were added. (e) Before the start of the trace. 400~1 of M were mixed with 504 of citrate/Tris buffer. At

the deflection, 50~1 of c’ were added. Final concentrations in all traces were: 9 mM citrate/Tris

ATP,

A-88mr+MgSO,.

present, [actin]

[C-protein] =

= 1 PM.

buffer. 0.62

12-5 mM-KC’I.

and.

[myosin] = 1.l

phi.

IIIM-

when md

1 PM.

The rate of increase in light-scattering produced by unphosphorylated C-protein was great,er than the rate produced by phosphorylated C-protein (Fig. 8). Regardless of whether the minifilaments were pre-incubated with (:-protein for two minutes

C-protein Effects on Actomyosin

M c-Cc)

1

Figure 8. The effect of unphosphorylated and C-protein on light-scattering of phosphorylated minifilaments. Conditions were as described for Fig. 7. (a) and (c) 400 ~1 of M +50 ~1 of unphosphorylated C+50 ~1 of A. (b) and (d) 400 ~1 of M+50 ~1 of phosphorylated (‘+50/d of A. (Fig. 8(a) and (b)) or less than eight minutes and (Fig. S(c) and (d)), the increase in light-scattering was less with phosphorylated C-protein than with C-protein. These results unphosphorylated suggested that C-protein was stimulating the formation of aggregates of actin and myosin and that unphosphorylated C-protein was more effective than phosphorylated C-protein in producing this aggregation. 4. Discussion (a) iMechanism of C-protein, action demonstrate that low These experiments concentrations of C-protein stimulate the actinactivat’ed ATPase activitity of cardiac myosin filaments. The increase in ATPase activity is related to an increase in V,,, value and a decrease in the for a&in. The C-probein-induced Kn value st’imulatjion of ATPase activity is associated with an increase in the turbidit,y of actomyosin solutions and an eventual superprecipitat’ion of the actomyosin. The effect of phosphorylated C-protein is less than effect of quant’itatively the unphosphorylated C-protein on both ATPase a&iv&y and on turbidity. These results suggest

193

that C-protein either directly or indirectly results in more stable attachments between actin and myosin filaments and a higher ATPase activity as a result of higher ordering of the filaments in solution. Studies by other workers have demonstrated a number of complexities in the effects of C-protein on ATPase activity (Offer et al., 1973; Moos et al, 1978; Moos & Feng, 1980; Yamamoto & Moos, 1983). Moos & Feng (1980) have reported that, at ionic strengths below 0.1, skeletal muscle C-protein was a potent inhibitor of skeletal muscle actomyosin ATPase. At higher ionic strengt’hs, however. C-protein was a mild activator of the ATPase. In contrast, C-protein from both cardiac and skeletal muscle caused activation of cardiac muscle actomyosin at all KC1 concentrations test,ed above 25 mM (Yamamoto & Moos, 1983). Recently, Margossian & Slayter (1985a,b) have presented data showing that C-protein stimulates canine cardiac actomyosin, but that this stimulation is abolished by removal of the LC-2 from the myosin. Although our results and those of other investigators show clearly that C-protein can stimulate cardiac actomyosin ATPase in solution, the question remains as to how these in vitro results relate to events occurring in wivo. It seems unlikely that C-protein is interacting with these synthetic myosin filaments in the same way it does in the native thick filament, for two reasons. (1) The arrangement of C-protein in the native thick filament is quite regular and peculiar. C-protein is localized in seven distinct stripes, separated by about 43 nm, in the middle third of each half of the A-band (Offer, 1972; Rome et al., 1973; Pepe & Drucker, 1975; Craig & Offer, 1976; Dennis et al., 1984; see Discussion by Squire, 1981). Although we have not yet examined in detail how C-protein interacts with the synthetic myosin filaments used here, it seems unlikely that the arrangement is similar to that occurring in native thick filaments. Indeed, Koretz (1979) has shown that C-protein added to pre-formed myosin filaments coats their surface. (2) The maximum stimulatory effect of that’ we occurred at C-protein observed approximately equimolar ratios of added C-protein t’o myosin. We do not’ know what fraction of the added C-protein bound to the filaments, but’ this ratio of added C-protein to myosin is higher than the naturally occurring ratio in muscle. In the native thick filament of skeletal muscle, the molar ratio of C-protein t’o myosin is about 1 : 8 (Offer et al., 1973; Morimoto & Harrington, 1974: Squire, 1981). At t,he naturally occurring ratio of C-prot’ein to myosin, the effect of C-protein on ATPase activit’y is rather small (Fig. 2). For these reasons. it’ will be important to design experiments to examine the effect of C-protein in native thick filaments or in synt’hetic filaments t,hat are assembled in the presence of the proper amount of C-protein. From t)he work of Moos et al. (1975) and Starr 62 Offer (1978), it is known that C-protein binds to LMM and S-2 subfragments of skeletal muscle

194

H. C. Hartz11

myosin. It is not known whether C-protein binds to the same regions of cardiac myosin. The effect of C-protein on actin-activated ATPase activity that we observed could theoretically be effected by the binding of C-protein to either LMM or S-2. The absence of an effect of C-protein on actin-activated ATPase activities of HMM can be explained in three ways. (1) C-protein binds to LMM. which is absent in HMM. (2) C-protein binds to S-2 in intact myosin but fails to bind to S-2 in HMM because of LC-2 degradation. (3) The effect of C-protein requires the myosin be in a filamentous form. The present data do not permit us to distinguish between these possibilities. The finding that C-protein stimulates the formation of actomyosin aggregates suggests that C-protein facilitates the association or cross-linking of actin and myosin filaments. This could occur either by C-protein directly cross-linking actin and myosin filaments or by altering thick filament structure. so that more myosin heads are available for interaction with actin. The ATPase and lightscattering data presented here are consistent with both of these possibilities. If C-protein were to cross-link filaments directly in viva, C-protein would need to be sufficiently large to span t’he distance between thick and thm filaments. We (Hartzell & Sale, 1985) have shown that, C-protein purified from cardiac muscle is a V-shaped molecule, with each arm approximately 20 nm long. This is sufficiently large to span the distance between thick and thin filaments at all physiological sarcomere lengths. (b) Relationship

to cardiac

relaxativn

C-protein becomes phosphorylated in heart in response to /3-adrenergic agonists and dephosphorylated in response to cholinergic agonists (Jeacocke & England, 1980; Hartzell & Titus, 1982: Hartzell, 1984). Perfusion of hearts with p-agonists causes both an increase in peak contractile force and a speeding of relaxation. whereas acet’ylcholine has opposite effects. The effects on peak force a,nd on relaxation occur with different’ time-courses and. so. probably occur by different mechanisms. \V;e have suggested that C-prot,ein phosphorglation correlates with t’he relaxation: (l-protein is phosphorylated when relaxation is rapid (Hartzell, 1984). Although this remains no more than a correlation, it is tempting to speculate how our results ile vitro might relate to relaxation in I~~GO. The data described here show that unphosphorylated C-protein has a greater effect in stimulating ATPase and increasing light-scattering of actomyosin than does phosphorylated C-protein. The suggestion that, C-protein either directly or indirectly increases actomyosin interaction would crudely explain the slower relaxatjion in t,he presence of unphosphorylated C-protein. For example, peak contractile force could be determined by the number of act,ive, cycling cross-bridges determined by thin filament activation. whereas the rat,e of relaxation might be affected by t,hick

filament st,ructure and the number of cross-bridges available for intera,ction with actin. I thank Mr Edward King for excellent and dedicated t’echnical assist’ance. Dr David Glass for the gift of catalytic subunit of CAMP-dependent protein kinase. I)r Sarkis Margossian for helpful suggestions and for providing unpublished data, and ,Joe and Win for running with me. Supported by grant HI,-21195 from the P;ational Heart, Lung and Blood Tnstitutr.

References Bailin. (:. (1979). Amer. ./. Physiol. 236, C41--C46. Blumenthal, D. K., Stull. ,I. T. & Gill. (:. PI’. (1978). J. Biol.

Chem. 253. 334-336.

Chock. 8. I’. & Eisenberg. E. (1979). J. Riol. (‘hem. 254, 3229-3235.

Cole. H. A. & Perry. S. \T. (1975). Biochwvn. J. 149. 525 533. Craig, R. & Offer. (:. (1976). Proc. Hay. Sot. ser. H, 192. 451461. Dennis. ,J. E.. Shimizu. T.. Reinach, F. (‘. & Fischman. I). A. (1984). J. C’rll Biol. 98. 1514-1522. England, P. ,J. (1975). FEBS Letters, 50. 57-60. England. P. J. (1976). Biochem. J. 160, 295304. England. P. J. (1977). Biochem. J. 168, 307-310. England, P. *J. (1983). Phil. Trans. Roy. Sot. ser. B. 302. 83-N). Giulian, cf. G.. Moss. R. L. $ Greaser. M. (1983). An.al. Biochem. 129. 277-287. HartzelI, H. (1. (1984). J. Uen. Physiol. 83, 563-588. Hartzell. H. C. & Glass. D. B. (1984). J. Riol. Chem. 259, 15587-15596. Hartzell, H. C. 8r. Sale, W. 8. (1985). .J. (‘ell Biol. 100. 208-215. Hartzell. H. (‘. & Tit,us. 1,. (1982). ./. Biol. C’herrr. 257. 211 l--2120. Herzig, .J. W.. Kohler, G.. Pfizer. (i.. Kuegg. ,J. C’. Pr Wolfe. G. (1981). Z”JlCgers Arch. 391. 208-212. Holroyde. M. ,J.. Howe, E. & Solaro. R. J. (1979). Biochim. Biophys. Acta, 586, 63-69. ,Jeacocke. S. A. & England. P. ,J. (1980). FEBS Letters. 122. 129.-132. Josephs, R. & Harrington. W. F. (1966). Biochemistr,y, 5. 2834-2847. Josephson, I. & Bperelakis, h’. (1978). J. No!. (‘rll. (‘ardiol. 10. 1157-l 166. Katz. A. M. (1979). Advan. C’,yclic A’ucl. Krx. 11. 30Ym343. Katz. A. M., Tada. M. 8z Kirchbergrr. 31. A. (19i5). Advan.

(.‘yclic

,Vucl. Res. 5, 453-471.

Kirchberger, 11. ~1.. Tada, M., Repkr. I). I. & Katz, A. M. (1972). J. Mol. Pell. C’ardiol. 4. 673-680. Kirchberger. M. A., Tada. M. & Katz. A. M. (1974). ./. Biol. C%em. 249, 616G-8173.

Koret,z. .I. F. (1979). Biophys. J. 27. 433.-446. Kranias, E. G. dt Solaro. R. J. (1982). LVatu?r (London). 298, 182-184. LaRaia, P. .J. & Morkin, E. (1974). C’irc. Iles. 35. 29% 306. Lim. M. S. & Walsh. M. 1’. (1985). Biophys. J. 47. 47la. Lindemann, ,J. P. & Watanabe. A. M. (1985). ,/. Biol. (‘hem. 260. 451ci-4525. Lindemann, *J. P.. *Jones, L. R., Hathaway. I). K.. Henry, B. (:. & Wat,anabe, A. M. (l983). ./. Hid. (‘hem. 258. 464-47 1.

Maargossian. S. S. & Rlayter. H. S. (1985a). Uiop/~y,s. J. 47. !309ii.

C-protein Effects on Actomyosin Margossian, S. S. & Slayter, H. S. (19856). J. Biol. Chem. In the press. McClellan, G. B. & Winegrad, S. (1980). J. Gen. Physiol. 75. 283-295. Moos. C. & Feng, I. M. (1980). Biochim. Biophys. Acta, 632, 141-149. Moos, C., Offer. G., Starr, R. & Bennett, P. (1975). J. Mol. Biol. 97, l-9. Moos, C., Mason, C. M.. Besterman, J. M., Feng, I. M. 8: Dubin, J. H. (1978). J. Mol. Biol. 124, 571-586. Mope, I,., McClellan, G. B. & Winegrad, S. (1980). J. Gen. Physiol. 75, U-282. Morimoto, K. & Harrington, W. F. (1974). J. Mol. Biol. 83, 8:%-97. Sargeot, J., Serbonne, J. M., Engels, J. & Lester, H. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 23952399. Offer. G. (1972). Cold Spring Harbor. Symp. Quant. Biol. 37, 87-95. Offer, G., Moos. C. & Starr, R. (1973). J. Mol. Biol. 6. 653-675. Osterrieder. W. G.. Brum, G.. Hesheler. J., Trautwein. W.. Flockerzi, V. & Hoffmann. F. (1982). Nature (London),

298, 576-578.

Palmer, J. L. & Alrruch, J. (1981). Anal. Biochem. 116. 372-373. Pardee, J. D. & Spudich. tJ. 4. (1982). Methods Cell Biol. 24 271-289. Pepe. i. A. & Drucker. B. (1975). J. &foZ. Biol. 99, 609617. Perry, S. V. (1983). Phil. Trans. Roy. Sot. ser. B, 302. 5971. Pope, B., Hoh. J. F. Y. & Weeds, A. (1980). FEBS Letters, 118, 205-208. Pope. B.. Wa,gner, P. D. & Weeds, A. G. (1981). J. Biochem. 117. 201-206. Ray. K. P. & England. P. J. (1976). FEBS Letters, 70. 11-16. L. E. (1976). Biochem. Reddy. Y. S. & Wyborny, Biophys.

Res. Commun.

73, 703-709.

Reddy. Y. S.. Ballard. D., Giri, X. Y. 8r Schwart’z. A. (1973). J. &fol. Cell. Cardiol. 5. 461-471. Reisler. E., Smith. C. B Seegan. G. (1980). J. Mol. Biol. 143. 129-145. Reuter. H. (1974). ,I. Physiol. 242, 429-451. Reuter. H., Stevens. C. F., Tsien, R. W. & Yellen. G. (1982). Yaturu (London), 297, 501-504. Richards, E. G., Chung, C. S.. Menzel. D. B. & Olcott. H. S. (1967). Biochemistry, 6, 528-540. Rinaldi, M., LePeuch. C. J. & Demaille. J. G. (1981). FEBS Letters. 129. 277-281.

195

Rinaldi,

M., Capony, J. P. & Demaille, J. G. (1982). J. Cell. Cardiol. 14, 279-289. Robertson, S. P., Johnson. J. D., Holroyde. M. J., Kranias, E. G., Potter, J. D. & Solaro. R. J. (1982). Mol.

J. Biol.

Chem. 257. 260-263.

Rome, E.. Offer, G. & Pepe. F. (1973). Xafure Ivew Biol. 244, 152-154. Schwartz, A., Entman, M. L.. Kaniike. K., Lane, L. K., VanWinkle. W. B. & Bornet. E. P. (1976). Biochim. Biophys.

Acta, 426, 57-72.

Siemankowski, R. F. & Dreizen, P. (1978). J. Biol. Chem. 253, 8648-8658. Siemankowski. R. F. & White, H. D. (1984). J. Biol. Chem

259, 50455053.

Solaro, R. J.. Moir, A. J. G. & Perry. S. V. (1976). *Vatare (London), 262. 615-616. Spudich. J. A. 8: Watt. 8. (1971). J. Bioi. (‘hem. 246. 4866487 I. Basis oj Muscular $L yuire, J. (1981). The Structural Contraction. Plenum Press, New Stork. 171. 813-816. Starr, R. & Offer, G. (1978). Biochemistry, Stull, J. T. (1980). $dvan. Cyclic ,%cl. Res. 13. 39-93. Stull, J. T. 8: Buss. J. E. (1977). J. Biol. Chem. 252, 851857. Tada, M.. Kirchberger. M. A. & Katz, A. M. (1975). J. Biol.

Chem. 250, 2640-2647.

Trautwein.

W., Taniguchi, J. & ?u’oma. A. (1982). Pjliigers 392, 307-314. Tsien, R. W. (1973). Nature New Biol. 245. 120-1%2. Tsien, R. W. (1977). Advan. Cyclic &cl. Res. 8, 363-420. Tsien, R. 1%‘.& Weingart. R. (1976). J. Physiol. 260. 117141. Tsien, R. IV.. Giles, W. R. & Greengard. P. (1972). Yature LVew Biol. 240. 181-183. Vogel. S. & Sperelakis. N. (1981). .I. Nol. Cell (‘ardiol. 13, 51-64. Walseth; T. F. & Johnson, R. A. (1979). rld~~~. Cyclic LVucl. Res. 10. 135-167. Winegrad. S. (1984). Circ. Res. 55, 565-574. Wray, H. L. & Gray, R. R. (1977). Biochim. Biophys. Acta, 461. 441-459. Wray, H. L.. Gray, R. R. 8r Olsson. R. A. (1973). J. Biol. Chem. 248, 149e-1498. Yamamoto, K. & Moos. C. (1983). J. Biol. (‘hem. 258. Archiv.

8395-8401.

Zar. J. H. (1984). Riostatistical An,aZysis. Prentice-Hall. Englewood Cliffs. X.J.

2nd edit.,