- Email: [email protected]
Myosin-linked calcium regulation in vertebrate smooth muscle
J. Mol. Biol.
(1976)
Myosin-linked
101, 75-92
Calcium Regulation in Vertebrate Smooth Muscle APOLIKARY SOBIESZEK AND ,J. V. SMALL
(Rewived
1 Augwt
19’i.i.
and in revisal
fhw~.
20
October
1975)
may be extracted ill Iligh yield and 13). ttw ,lSR of a new pr0ccd11rc:. actomyosin purity from fowl gizzard which exhibits a calcillm-dependent, actin-activated ATPase activity comparable to that of the pawtlt myofibril-like preparation. Studies of this vertebrate smooth muscle act,omyclsin show that t,hcx regulation of the actin-myosin interaction is effected, as in molluscan muscles. by the myosirr mc~lrc~& it,wlf and not b>- arl actin-link4 regulatory systtlm. as found in verteI)rat,cl skeletal muscle. ‘IIllls, calcium-sensitive smooth muscle actomyosin is composed of only- myosirr. any troponin-like compotrcntx bring absent. Myosin is acatln and tropomyosin. thr only component that binds significant amounts of calcium and shows a calcillrn-deprrldent a&in-activated ATPase acti\Aty iti tltrk pxwnce of IT-actill from (lither gizzard or rabbit skrlat~al musclr. The cross-reaction of gizzard thin filaments \vitJt skeletal muscle myosin pro(111ces an actomyosin whose actin-act,ivated .ATPaso is calcium-insensitive, showing that smooth muscle Olin filaments do not serve a regulatory function. of one of the The effect of Mg2+ and pH, and evidence for the involvement lnyosin light chains in calcinm regulation are dcwrilwtl and discnswd.
1. Introduction Recent, studies have demonstrated that the regulation of bhe actin-myosin interaction in muscles is not a unique property of t’hc thin actin-containing filaments. Thus. while a troponin-tropomyosin complex linked to actin serves to mediate t’hr triggering of contraction by calcium in vertebrate skeletal muscle (see reveiws by Ebashi & Endo, 1968; Weber & Murray, 1973; and Cold ~Spring Harbor Symp., vol. 37, 1973). a rather different system. associated with Dhe myosin molecule has been shown t’o operate in molluscan muscles (Kendrick-Jones of al.. 1970,1973 ; SzentGyorgyi et al., 1973). Further, related studies (Lehman et al., 1973) have indicated that such a myosin-linked system exists in cert’ain other invertebrate muscles and have fllrnished evidence, in some cases, for the presence of a dual system involving regulation on the actin as well as on the myosin component. We have been focussing our attention on identifying the component(s) responsible for calcium regulation in vertebrate smooth muscle. Studies of this muscle type have previously been hampered by the difficulties encountered (see review by Needham & Shoenberg, 1968; Sparrow ef al.. 1970; Murphy. 1!$71; Driska & Hartshorne. 1975) in obtaining an actomyosin with a demonstrated activity and calcium-sensitivity showing some comparison to that of skeletal muscle (Weber & Herz, 1963; Weber et aZ., 1969; Murphy, 1971) or to glycerinated smooth muscle preparations (Filo et al., 1965). In a recent study (Sobieszek & Bremel, 1975) we described a new procedure for obtaining actomyosin from fowl gizza,rd which is highl,v calcium-sensitive and 7.5
i(i
.I. SOHlES%Eli
.\SI)
.I. \.. Skl.-\l,l,
cbxhihits an activity several times greater than that reported for other smooth mus& actomyosins. It was further shown that this calcium-scnsit’ivc actomyosin exhibit,td the presence of only three prot,eins. namely myosin. actin and t ropomyosi n. arid components resembling troponin of skeletal muscle being absent. On this I)asis it was suggested that in vertebrate smooth muscle, as in molluscan muscles. myosin may perform the role of calcium regulat,ion. From the recombination and cross-reaction of purified contract,ile proteins and actomyosins from both smooth and skeletal muscle we have now obtained direct evidence for a myosin-linked control mechanism in vertebrate smooth muscle. Specifically, we show that smooth muscle myosin binds calcium and interacts with F-acbin, from either smooth or skeletal muscle, to produce actomyosins that exhibit a calcium-dependent actin-activated ATPase. Mixtures of smooth muscle thin filaments with skeletal muscle myosin are? in contrast. calcium-insensitive. The results of competition tests of skeletal muscle F-actin with smooth muscle actomyosin are further consistent with a myosin-linked system (see also Bremel. 1974). Evidenctl is further presented which indicates t)hat. in vertebrate smooth muscle at least, one of the mgosin light chains is involved in the calcium regulation process.
2. Materials and Methods As described in a previous paper (Sobieszek $ Bremel, 1975) we have used a myofibrillike preparation of chicken or turkey gizzard as source material for the extraction of smooth muscle actomyosin. Briefly, the myofibrils are produced by thorough llomogenization of finely minced fresh gizzard followed by extraction with Triton Xl00 and extensive washing to produce a white residue. Figure 1 summarizes the procedures used for processing the myofibrils to produce t,he various preparations of contractile proteins. Unless otherwise stated, all procedures were carried out at 4°C and all solutions contained 100 mg streptomycin/l and freshly added 1 mno-cyst&e. For the extraction of actomyosin, myofibrils were resuspended in an ice-cold medium of the following composition : 10 mM-ATP(Na), 1 mm-EDTA, 2 mM-EGTA, 1 mM-CySteinP.
60 mM-KCl,
40 mM-imidazole
(pH 7.1). Undissolved
material
was removed
by centrifu-
gation at’ 14,500 revs/min (15,000 g) for 1 II and the extracted actomyosin filtered through glass wool. For a ratio, by volume, of extraction medium to myofibrils (originally sedimented at 2500 revs/min, 2000 g for 1 h) of between 3 and 4 to 1 the protein concentration of this crude actomyosin (Fig. 1) is greater than 10 mg/ml. To this extract 1 >r-MgCl, solution was slowly added, while stirring, to give a final concentration of 25 to 30 m&l. causing the actomyosin to become opaque. The extract was then left at, 4°C for about 10 to 15 h to form a compact gel. This gel was then centrifuged at 14,500 revs/min (15,000 g) for 30 min and the pellet so obtained resuspended, by gentle homogenization, in a “wash 20 mM-imidazole (pH 6.8). solution” containing 60 mM-KCl, 1 Inn%-MgCl,, 1 rnfiI-cysteine, The actomyosin was pelleted and resuspended and stored in a buffer solution of 60 mMKCl, 1 mar-cysteine, 40 mM-imidazole (pH 7.0 at 25’C) corresponding to that. used for ATPase activity measurements. exhibits the highest calcium-sensil ivit’> Actomyosin purified by Mg2 + precipitation and we refer to this actomyosin as Mg-actomyosin (MgAM in Fig. 1). Precipitation may equally well be effected by using CaCl, (1 to 2 m&l) or CaCl, plus MgCl,; in both cases the actomyosin precipitates without the formation of a gel. We refer to these actomyosins respectively. In contrast to as Ca-actomyosin (CaAM in Fig. 1) and CaMg-actomyosin, Mg-actomyosin, these actomyosins exhibit little if any calcium-sensitivity (see lttesults). Tropomgosin was removed from divalent cation-precipitated actomyosin by ammonium sulphate precipitation (25% saturation) as described previously (Sobieszek & Hremel. 1975), with at least 2 subsequent washes to remove ammonium sulphatr.
,/
_
CAM extraciion
220,000 mol wt
F - octm
I
(crude octomyosin) Can be used for TM extraction 0 6 lo I 0 M-KCL
*I--Discard
MgCl2 - 35 ‘h; 14.500 revs/mm
Myos
I
3’10. 1. Schematic diagram illust,rating t,he processing tractilr proteins. Upper and lower parts of rectangles respectively. Numbers used in composition of media essential components are given. Unless otherwise stated TiCI. 20 t,o 40 mM-imidazole, 1 mwcgst,nine and 100 TN&ho& for furthrr d&ails).
&eps in the preparation of gizzard concorrespond to supernatants and pellets, correspond to ITIM concentrations; only all solutions contain, in addition, 60 mMrng st,repbomycin/ml (see Materials anrl
Hr centrifuging freshly extracted crude actomyosin for about 18 h at 14,500 revs/mill ( 15,WO g) a myosin fraction is ohtained which contains very little actin and tropomyosin impurities; we refer to this as a “crude myosin fraction” (CMF in Fig. 1). This nyosin may be selectively pracipitated by the slow addition of 1 M-MgCl,, with stirring, to a final concentration of about 35 rnM and is then collected by centrifug-ation for 2 h at 14,500 revs.’ rnin (15.000 g). CaCl, is effective in precipitating rnyosin but was not used, since the preeipit&ion of actomyosin wit.h calcium caused a. va.riablc loss of calcium-sensitivity (see section (a), above). Thp pellet formed by t)he centrifugation of crude actomyosin comprises an actomynsin (P-AM, see Fig. 1) enriched in actin and tropomyosin and was preserved and used for rcaombina,tion c>>cperiment’s with rabbit, skelet,al mnscltl myosin (SW Results).
From rabbit skeletal muscle myofibrils extracted with Triton Xl00 and washed in the presence of 2 rnM-pyrophosphatr (Etlinger & Fischman, 1973), myosin was extract,ed in H medium conbaining O-3 M-KCl? 1 mM-MgCl,, 5 rnM-ATP(Mg), 2 mM-pyrophosphste. % HIM-EGTA, 1 mM-cysteina and 40 mm-imidazole (pR 7.0). The extract was centrifuged at 151000 revs/rnin (20,000 g) f or about 15 h and t.he myosin in the supernatant precipitated by dilution with about .5 vol. ice-cold water with added 1 mnr-cysteine. The precipit,ai.tb was collected by eentrifugat,ion at 2.500 revsjrnin (2000 g) for 1 h and resuspended. using a Teflon/glass homogenizer, in buffered 0.06 IVI-KCl. 3 ~-Kc1 was then added with stirring until the myosin dissolved (at about 0.4 M-KCl) and the solution clarified b) ccnt’rifugatian in the presence of P mM-EGTA and 2 mM-ATP(Mg) at 40,000 revs/tin (100,000 g) for 3 h. The precipitation and clarification steps were then repeated bnt wit8hout t’he addition of ATY(Mg) rind EGTA.
mirlimisr myosin filament disaggrcpat~iotl, it11 tbxtract ricll in ttlill filamrrlts nlay bcl obt~ainc~cl. For this purposn bile same basic cxLractior1 mfdil~m \vas used brat) cwrrtaining 2 tn~ wit11 tllrs pH ad,justed to 6.5 at 1 ‘C’. ;-\ftclr wtltrifll,ZTP(My). 1 mwMgC11,. 2 ml%-EGTAantl gation for 20 min at 19.000 rrvs/min (40.000 g) to r(w1ovc tissurl fragm~~tLts. ttrcb pH \+.a~ raised to 7.0 and the thin filarnwlts cwllwtc~tl 1)). c~c,lltrifrl~:i~tiotl for 2 II at 40,000 W\~S,:lllill (100,000 g). The pellets wcro tllcll dissol\~c~tl irl \vastI solrltion Itsing R Il;rrltl-l)~)c~r’at,(~(l, Tc+lon/glass homogrnizw. (t!)
F-a&n
‘I’tre residue remaining aftor actornyositi csxtraction coIlt,ains a large amount. of arti,, rclat,ive to myosin and serves as a ronvc~nirnt scjurce of F-actin. Residual myosin :ultl by rr-rxtractiorL at, Iii@ ionic strengt,h tropomyosin wew wmovfd (0.6 to 1.0 ar-K(‘1. 1 mM-cysteine. 1 mu-EDTA, 20 ml\r-imidazole, pH 7.0) \vith a substqncntj waslr at low ionic strength to removcx KU. The rosultinp residw was resuspended wit11 a Teflon/glass homogenizer in a small volume of u-ash sohltion (not mow than t\+%o that of the residue) and sonicated for 5 t,o 8 min in an MSE sonicator at, an amplitude of 16 pm. The suspension was homogenized again and then scdirnt~ntc~tl folx 1 tr at t 4.500 revs/rnin (15.000 g) to give> a supcrnatant cont,aining puro I?-actin at, :I conccwtration of 1.5 to 2 mg/ml. Gizzard F-actin was also prepared by A second mctllod directly frown t,hin filaments (sot section (d) above) hy soloct,ively removing t~ropomyosin at high ionic strength. Tl~ns, the pH of the thin filament extract was adjusted to 7.0. t)lle KC1 concentratiotl inclrLascXd to 0.56 Y by tile addition of 3 >I-KC1 and F-actin subseqllrntly pelleted by ccntrifugat~iorr for 2 h at 40,000 revs/min (100,000 g). TI>c prllct, was dissolved in \vastI solntiorr using a hand-operated T&on/glass homogenizer. F-a&in from rabbit skeletal muscle IVNS prrlparcd wwntially accwrtlinp to Splidic,ll & ITatt (1971) from the residue rernainirlg after myosirr extraction. (f)
Ijodecyl
sctlphnte
ye1 electrophoresis
gcLs it1 the prescnco of sodium dodecyl sulphate Elcctrophoresis on polyacrylamide (Weber & &born, 1969) was carried out according to Fairhanks et al., (1971) \vith modifications as described by Sohieszek & Rwmcl (1975). (g)
Protein
determination.
and
A TE’ase
activity
measurements
Protein concentrations were drt~ermincd by the Binret method using a protein standard from Sigma for calibration. ATPase activity measurements were carried otlt at 25 C in a medium containing 60 rn>lKCI, 1 mM-ATP(Mg), 1 m>T-cysteine, 40 mnr-imidazolt: (pH 7.0 at 25°C) and with either: 2 m&r-EGTA plus 1 mM-Mgcl,; 0.1 mnr-CnCl, plus 1 mM-MgCt,; or 2 mM-EDTA. The latter measurements. Undw these assay provides an important refcrenw point for activity conditions actin and myosin are dissociated (Sobieszck 8~ Rrc-rnel, 1975) and the act)ivitJ cat,ions. For measured corresponds to that of myosin alone in the absence of divalent buffer system was usotl, assays at different, concentrations of free (‘a2 + , a CaEGTA/EGTA taking the apparent dissociation constant for C’aEGTA at pH 7.0 as 1 Y: 10m6 &I (Ogawa., 1968). The 3-ml assays were pre-rquilibrat,ed at 25°C’ and t)he reaction initiated by adding was torminat,ed aft.w 30 to ATP while mixing gently on a vortex mixer. Tllc waction filternd and 60 s by the addition of 2.0 ml 10% trichloroawt~ic acid, the preparations inorganic phosphate measured according to thr mrtllod of Fiske & SubbaRow (1925). For expressing enzyme activit,y relative to myosin. the myosin content of the preparations of sodium dod~cyl was determined by quantitati\-e gel electrophnresis in t,llr pwsencr sulphate (see Sobieszek & Rremel, 1975). (II) Ca2+ -binding
measurements using the double label method
Ca2 + -bindiny
mere carried out according to Kendrick-Jones et al. (1970) with [3H]glncose as a second label. Assays were performed
3. Results (a) .-Ic/ott(/psi,r. atrri c~nlcil~~rl.-.YPtIS~li)‘i~~/ Depending on t,hr nn:t~hod of precipitation of freshly extracted. crude actoniyosin (CAM. see Fig. 1). actomyosins are ol)ta.incd wlrieh exhibit, different’ characterist~icn. In an earlier study (Sohirszek & Hremel. 197.5) precipitjat8ion \\as effected by the use of’ magnesium followed 1)s ammonium sulphate. Suhseyuently, in t’he present study. it was found that divalent c&ons alone ww sufficient to cause precipitation overnight. The actomyosin so obtained is four to tive times more act’ive than that processed through ammonium sulphatc and it also lacks the cont8aminating component migrating at, a molecular weight of ahout ~10.000 on sodium dodecyl sulphate polyacrpla~mide gels (compare (a) and (c). Fig. 6). The most calcium-sensitive actomyosin is obtained by the use of magnesium precipit~ation and \ve refer to this as Mg-actomyosin (MgAM Fig. 1). The properties of this ac:tomyosin are described hekm~ : a later section will deal with the eharacterist~ics of actonly-osins obtained 1)). precipitation with calcium or calcium plus magnesium. In freshly prepared Mg-actomyosin tOhe ATPasc activity is inhibited 90 t,o 95”;, I)y calcium removal (Table 1) and this degrrc of c~~lciu~n-st~~~sit~i~~it~~~ is only maintained over a period of two t,o three tla;vs. ,Is shown in Figure 2. the activation by calcium occurs Vera’ sharply in the concentration range I to 10 cc”. A significant feature of t,his calcium activation curve is the range of free C:t2 + concentration over which full actjivation occurs. As Carl be seen from Figure 2. therc~ is a ilistinct platea,u between I.5 +V and 200 ~BI free calcium and a marked decrease in act,ivitjy a,l)ove about 500 PM. The same type of inhibition at high Ca2+ concentrat’ions also occurs with myofibrils f’~m skeletal muscle (Portzehl it ab.. 1969) indicating tlliat ATP(Ca) is not as good a substrate as ATP(Mg) for the actomyosin system. While the most active and calcium-sensitive actomyosin is obtained by magnesium precipitation the high concentration of magnesium used commonly acts t,o shift, the wlrium
ac+iv;ltion
curve
relative
to
that
ol&tirnd
for
tin
parent
myofibrils.
As
TABLE
A TPase activityt
and C’a’ + -sensitivity
Preparation
EUTA
Myofibrils Sctomyosins f MgAM MgAM at 37’Y’ P-AM CaAM Ca, MgAM Tropomyosin-free actomyosim MgAM - TM CaAM - TM Ca, MgAM - TM
1
of various yizzard preparation.~ EGTA
(:a2 + -scnsitivity$
Ca2 +
IO.4
Il.6
105
0.89
“2.4
13.9 46~9 22.6
0.94 0.90 0.89
171
""4 472 204 193
1%
176
0.23
66~1 18.3 36.7 32.7 164 48.8
61.0
9.1
106 99.5
0.11
11.5 118
0.78
0.10 0.31
145
t Expressed in nmol.mg protein-‘.min-‘. $ 1 - (EGTA activity/&x2 + activity). § For abbreviations see text and Fig. 1. TM, tropomyosin.
-7
I 6
I 4
I 5
- log ka”
I 3
1
FIG. 2. Dependence of ATPase activity of different gizzard actomyosin preparations on the free Ca’J+ concentration. Open and closed circles, MgAM; triangles, P-AM. Activation commonly occurs at around 1 x lOWE M-Ca2+ (closed circles) although sometimes takes place (open circles) at the lower Ca2+ concentrations characteristic for myofibrils (see Fig. 3).
shown in Figure 3, gizzard myofibrils are normally activated at lower Ca2+ concenand further, their activation curve trations (about 6 x 10d7 M) than actomyosin shifts to the position characteristic of actomyosin when they are assayed in the (10 mlcl Fig. 3) or after pretreatment with presence of high Mg2+ concentrations Mg2+ (25 mM). In some cases the activation curve of actomysin was not shifted but occurred at the same Ca2+ concentration as myofibrils (Fig. 2). The steepness of activation of actomyosin, however, was always the same as for myofibrils. For actomyosin (and also myofibrils) changes in the free Mg2+ concentration (in excess of ATP) between O-1 mM and 10 mM act to modify the magnitude of activalying in the range tion by calcium (Fig. 4), the optimum free Mg2+ concentration
0.2 mM
t0
2 mM.
REGULATION
I 0
I 7
IN
SMOOTH
I 5
I - loo6w+
MUSCLE
I 4
J
I
Fx. 3. Calcium-activation of gizzard actomyosin (&&AM, ---e---a--) and myofibrils of 1 mx. Note similarity between the actina-) at a free Mg2+ concentration (MYF, ---Icurve for myofibrils is shifted aotivated ATPase activities in the presence of Ca 2 + . The activation towards that obtained for actomyosin when these are assayyod in t,he prssencp of high concentrasee Discussion). tions of Mg2+ (10 rnq -n---Q--;
-log luci* : YIG. 4. The effect of changes in Mg2+ concentration on the calcium-activation of gizzard actomyosin (MgAM). In all experiments the ATP(Na) concentration was 1 maI while the total Mg2 + concentration was varied from O-2 rnM to 11 mM. Optimum activation takes place between 0.2 atid 2 rnM free Mg2+ concentration, while almost complete inhibition occurs at 0.2 mM total magnesium resulting from the relaxing effect of the ATP anion (Sobieszek & Bremel, 1976). The tota. magnesium concentrations were, in IIIM :~~~~~~~~2;-x-x-~1~O;~~~-----~-~
1.2; --.‘.----A--,
1.5; ---m-o--,
2.0; -n----o-,
3-o; --m-m---,
11.0.
Figure 5 shows the effect of changes in pH at the normal assay temperature of 35°C and demonstrates an optimum around neutrality. Increasing t’he temperature to 37°C at pH 7-O acts to elevate the ATPase activity to approximately double t’hat at 25°C (Table 1). From gel electrophoresis in the presence of sodium dodecyl sulphate and sedimentat’ion experiments (Sobieszek & Bremel: 1975) purified vertebrate smoot8h muscle 6
PH
FIQ. 5. Inlluence of pH on the ATPase activity (----a-) andabsence (EGTA, -O---O-;
of gizzard actomyosin EDTA, ----A---A-)
(MgXM) in the prewnw of (‘aZ+.
actomyosin shows only the three primary contractile proteins, actin, myosin (with its two light chains) and tropomyosin (Fig. 6 (a) and (b)). Densitometry of photographic negatives of stained gels with corrections applied for the relative stainability of each protein component (Sobieszek & Bremel. 1975) yields proportions for m.yosin : actin : tropomyosin of 2.5 : 1 : 0.3. (b) Loss of calcium-sensitivity For Mg-actomyosin, both the calcium-sensitivity and the ATPase activity in the presence of calcium decay, after 7 to 10 days at 4”C, to about 50% of their original value and calcium-sensitivity is completely lost after a few weeks. Actomyosin produced by precipitation overnight with Ca2 + (1 mM), CaAM, or with Ca2 + plus Mg 2 + , CaMgAM, exhibits rather different properties. In bot,h cases, calciumsensitivity was considerably reduced (Table l), although the absolute activity was comparable to that for Mg-actomyosin. However, while calcium-sensitivity is rapidly lost in these actomyosins during the preparation, they retain, in contrast to Mgactomyosin, almost full MgATPase activity for at least one week. Calcium-sensitivity is also irreversibly lost on brief treatment of Mg-actom,vosin with trypsin or papain : this effect is discussed again in a later section.
(c) Evidence for rnyosin-linked cuEciunl-.sen~s~t~T:jt?/ (i) Recombination of gizzard proteins Using a new and relatively simple procedure (Sobieszek t Bremel, 1975; and Fig. 1) myosin may be readily obtained in high yield and purity from gizzard muscle (Fig. 6(d)). We further describe here (see Materials and Methods) techniques for actin and tropomyosin obtaining native thin filaments from gizzard, containing (Fig. 6 (i)) and a,lso gizzard F-actin. The latter F-actin shows the characteristic thin filaments in the electron microscope and runs as a single band on sodium dodecyl sulphate gels (Fig. 6 (g) and (h)). (j) Rabbit F-actin. (k) Complex of gizzard S-l and gizzard F-a&in. S-1 and Y-aotin were mixed together at low ionio strength and pelleted by low-speed centrifugation. (1) Hybrid aotomyosin from rabbit myosin and gizzard thin filaments. The preparation8 were mixed at about 0.3 M-KCl, dialysed against 0.06 M-KC1 and pelleted by low-speed centrifugation.
84
A. SOBIESZEK
AND TABLE
AT Pase activity
and Ca’ + -sensitivity
J. V. SMALL 2
of recombined gizzard ~re~arf~~i~)~~~-~-
Preparation
Ratio
EGTA
ca= ’
Myosin + F-actin Myosin + thin filam. S-1 + F-e&in
2.5:1 2.6: 1.3 2.5:1
20.6 21.5 180
62.3 137 194
t Expressed
Ca* + -sensit,ivity 0.67 0.84 0.07
in nmol . mg myosin - 1 . min - I.
Table 2 shows the ATPase activities for mixtures of gizzard myosin with gizzard F-actin and with native thin filaments, In each case: the combination shows 70 to 80% calcium-sensitivity. By comparison, gizzard myosin subfragment. 1 produced by papain digestion and which lacks the L,, li g ht, chain (see further below) shows an actin-activated ATPase activity that is insensibive to calcium. (ii) Cross-reactiojzs
between gizzard and skeletal muscle proteins
The actin competition test devised by Lehman et al. (1973) provides a simple test for the presence of a myosin-linked regulatory system. This test relies on the assumpt’ion t#hat unregulated actin (for example ra,bbit F‘-actin) added to a myosin-regulated actomyosin will not affect calcium-sensitivity, since in the absence of calcium it is the myosin that is inhibited and actin cannot activate its ATPase activity. Conrerselv. for an a&in-linked system where there is no myosin control, unregulated P-actin will act,ivat)e the myosin ATPase and act t.o decrease or abolish the calciumsensitivity. ilpplied to gizzard actomyosin this t’est indicates the existence of a myosin-linked control system; thus, the addition of F-actin to MgAM has litt,lrl effect on either the ATPase activity or the calcium-sensitivity (Table 3). The same result, was formerly obtained using acbomyosin prepared with ammonium sulphattb (Bremel, 1974). Table 3 also shows the effect of mixing gizzard myosin and subfragment 1 with rabbit F-a&in. The result is essentially the same as with gizzard F-actin (cf. Table 2). Calcium-sensitivity is exhibited by the intact myosin molecule wit,h F-actin hut, is ;tbsent in t,he mixbure of rabbit F-a&in with gizzard S-l.
TABLES
ATPase
of hybrid uctomyosins formed activity and Ca” -ser&tivity from rabbit skeletal P-act& and gizzard preparation.st Protein-toF-actin mass ratio
Prepamtion
MgAM Myosin 8-l
control + F-ectin + F-actin + F-a&in
7 Expressed
in nmol.mgS1
3.8:O 3.8:1 2:l 2:l (protein
EGTA per mg of protein 11.3 11.2 30.2 140
or myosin).miIl-‘.
myosin 17.5 21.5 45.3 209
ca2 + per mg of protein 181 143 67.6 146
myosin 279 274 101 219
Ce2 + -sensitivity
0.94 0.92 0.45 0.04
REGULATION
IN
SMOOTH
x5
MUSCLE
TABLE 4
ATPase activity and Ca’+ -sensitivity of hybrid actomyosina formed from rabbit skeletal myosin and gizzard preparationst
P-AM
control + myosin Thin fil. + myosin i l&pressed
EGTA per mg of
Protein-to myosin mass ratio
Preparation
protein 36.2 81.1 308
3.8:O 3.8: 2.5 2:l
in mnol’mg-’
(protein
CL32+ per mg of
actin 01.7 341
protein 152 153 305
actin 385 642 -
Caa+ -sensitivity
0.76 0.47 0
or aotin).min-‘.
The absence of any regulatory system associated with the thin filaments of gizzard was indicated by experiments in which skeletal muscle myosin was mixed with gizzard thin filaments. It can be seen in Table 4 that the gizzard thin filaments activate rabbit myosin equally in the presence or absence of calcium. In contrast, and as already indicated (Table 2), the same thin filaments give a fully calciumsensitive actomyosin when combined with smooth muscle myosin. Further, consistent with the absence of any thin filament regulation was the observation that skeletal muscle myosin added to actin-rich smoot,h muscle actomyosin (P-AM, see Fig. 1 and Fig. 6 (c)) resulted in an increase in actin-activated ATPase activity in the a,bsencr of calcium t’o the level of act’ivity of the control actomyosin with calcium present (Table 4). (iii) (Yalcium-binding Gizzard myosin and actomyosin bind calcium with high affinity over the range of concentration where the activation of the ATPase activity occurs and for a Mg2f concentration of 2 InM (Fig. 7). The amounts bound (see Table 6) at 60 to 90 PM free Ca2+ concentration, correspond t’o 3.4 nmol Car2+ per mg myosin and 2.4 nmol Ca2+
7 Pro. 7. Dependence (,MgAM, - -m--m--)
6
5
of calcium-binding of gizzard myosin (--C---C-) on the free Crt2+ concentration at 2 mM.MgCl,.
4
and actomyosin
86
A.
SoHItcsS%15li
.\SI)
.I.
\‘.
SM,\i.I,
per mg actomyosin. Since :tc:t,om~wsin is composed of about N(i”,, (iv/\V) of’ 1r1y0si1,. the value for actomyosin may by accounted for I)y the binding of’ calcium by. myosin (Fig. 7). This is consistent with t’he correspondingly insigniticant, amounts of calcium bound by gizzard thin filaments and gizzard IT-actin (l’ahlc 6). Associated with th(s lower pCa requiwd for their nctivat.ion. myofibrils IY(W found t.o I)intl calcium irt, correspondingly lower Ca2 + concentrations than for actomyosin. It may be not’ed that optimum calcium-binding oc :lrs at’ an apparently higher Ca2 + concentration than required for t’he full actjivation of actin-activated STPaw activity. However. when ATPase activity mca,surement,s were made at the 14-fold lower CaEGTA buffer concentration used for t,hc calcium-binding experiments, full activabion was achieved only at 60 to 90 PM free Ca2+ concentration as compared to 20 pnf for the same preparabion under standard conditions (Fig. 3). The optimum calcium-binding at 60 to 90 PM (Fig. 7) thus correlates directly with the ATPaw activity measurements made under the same conditions. We conclude that a,t lo\\calcium-buffer concentrations the CaEGTA stahiliby constant’ is somewhat dependent on the total Ca2+ concentration. The figure obtained for myosin is approximately t~he same as that obtained for scallop (Kendrick-Jones et al.. 1970;1973 : Szent-Gyorgyi st al., 1973) and corresponds to nearly two calcium ions bound per molecule. A IOO-fold change in the free Mg2’ concentration from 0.1 mM to 10 mM caused only A t’uofold change in the amount of calcium bound by myosin demonstrating a high specificity of thcb binding site for calciuni .
As indicated above (see Table 3), gizzard myosin 8-l produced by papain digestion forms a calcium-insensitive actomyosin when combined with rabbit F-actin. In agreement with the data of Kendrick-dories (1973). we find that this S-l lacks tho L,,, light chain (Fig. 6 (e)). To establish if there was a more specific correlation between the degradation of this light chain and the loss of calcium-sensitivity, we assayed the digestion by papain of gizzard actomyosin for different enzyme-ho-prohein ratios. The results are shown in Tahlt~ 5 and Figure 8 (a) t’o (d). As shown in Figure 8(d): papain causes ext’rnsivt:
EDTA EGTA Calcium Calcium-sensitivity a Expressed in b After 25 min c After 30 min fl Expressed in e Subjected to
TrypsinC
Papainb
Enzyme Enzyme to MgAM ratioa
Conhole
16
80
400
31.5 45.9 134 0.66
31.3 62.6 150 0.58
29.6 63.7 157 0.59
37.4 95.4 195 0.51
nmol . mg protein - 1 . min -- I. digestion. digestion. ng mg - I. iodoacetic acid as for papain digests.
( ‘ontrol
8
21.1 20.0 16.6 19.0 122 160 0.86 0.88
40
?o(J
21.6 14.6 40.7 0.36
33.4 22.3 23,3 0.04
REGULATIOX
IN
SMOOTH
MUSC’LE
ti;
degradation of the myosin heavy chain without noticeably affecting the light chaitl components. Further, as long as the L,, myosin light chain is present! the preparations st,ill exhibit calcium-sensitivity. Unfortunately, for the concentration of papain required to split myosin fully to S-l this light chain is degraded and. as alread! indic;ltrd. t,he calcium-regulatory property is lost. While not conclusive. these finding+ arc consistent’ \vith the L,, light chain being necessary for calcium-sensitivity. Rit)h trypsin the digestion of actomyosin follow-s a mnrked1.v different courw (Fig. 8(r) to (h)): the light chains being affected much more rapidl- than the heav>chain components. The degradation of the L,, light chain in t’his case is associatctl I\ ith t,hr complete loss of actin-activated ATPase activity (Table 5). AMorc direct evidence for the involvement of the L,, light chain in the calciumregulat’or- process has come from the demonstration (Sobieszek. unpublished results) that R phosphorylation of this light chain is triggered by Dhe same Ca2 + concentrat8ioll wquiwd to activat,e the BTPast activity of actomyosin (see Discussion).
4. Discussion (a) General Whiltb the numerous physiological and pharmacological investigat,ions of vertebrate smooth muscle (see reviews by Somlyo & Somlyo. 1968,197O; Biilbring et al., 1970; Phil. Traru. Roy. Xoc. ser. B. (1973). vol. 265, pp. 1-231) attest to its importance in the performance and control of diverse physiological funct’ions rat,her little has been established, in comparison to skeletal muscle, either about the contractile process or its wyulation in this muscle type. In addition to furthering our understanding of IION smooth muscle operates, biochemical studies of this muscle type will clearly complement dat’a from other muscle systems about the fundamental process of cont,raction. Furthermore, the!- promise bo furnish significant information relating to the, operation of non-muscle a&omyosin-containing systems which have been shown to possess actomyosins and myosins t’hat exhibit’ similar properties t#o those of smoot’h muscle (Adelstein & Conti. 1974). (b) I~ertrhrute smooth muscle actomyosinz \Vts describe here a method! as modified from an earlier study (Sobieszek & Bremel, 19i5). for obtaining actomyosin from a’ vertebrat,e smooth muscle which exhibits a calcium-sensitivity and acbivit’y comparable to tha’t shown by the washed, parent myofihrils. The actin-activat,ed ATPase activities that we have obtained for gizzard actomvosin (0.25 prnol Pi/mg protein at’ 25°C and 0.47 PmoI Pi/mg protein at 37°C) now compare realistically wit’h those obtained for striated muscle myofibrils (Weber. 1969: Port,zehl rt al.. 1969) and a’ctom,vosin (Kenttrick-Jones it al., 1970; Murphy, 19i 1) and at physiological temperatures exceed t’llilt obtained for a molluscan smoot,h muscle I Hendrick-Jones et (1.1..1970). Wr have noted that the Ca2+ concentration required for activation is normall>, low,er for myofibrils than for actomyosin and we have attributed this difference bo the modifying effect of the high concentrations of Mg’ + used for actomyosin precipitation. However, while high Mg2 + concentrations clearly shift t.he activation curve for myofibrils the activation of P-&4M (Fig. 2). obtained wit,hout magnesimn precipitat,ion, also occurs in th(l same range of Ca2+ concent,rations as for MgAM. On t’his basis the shift in activation of a.ct’omyosin with respect to myofibrils ma?.
.
FIG. 8. Mild proteolytic digestion of turkey gizzard act,omyosin (MgAM) by papain ((a) ttr. (d)) and trypsin ((0) to (h)). The actomyosin-to-enzyme (papain and trypsin) ratios and the calcmmsensitivity of each actomyosin preparation are given in Table 5. Note the complete removal of the L,, light chain of myosin dhring trypsin digwtion (h) and its presence in the gels of the [‘apain digests.
REGULATION
IN
SMOOTH
TABLE
Calcium
binding
MUSCLE
6
by gizzard preparation-st M&L
b’M) 2.0
Preparation
0.1
Myosin MgAM Xyofibrils C&AM CaAM - TM Thin filaments F-actin Myosin rod:
5.1
3.1 ‘P.4 2.2
2.0 1.3
1.0
2.5 2.7 w07 0.06 II.6
1.3 1.3
10.0
04
t Expressed in nmol of calcium per mg of protein at HO to 90 pM of CaZ+ and at 3 different Mg= + concentrations of 0.1, 2.0 and IO.0 mM. For other condit’ions see Materials and Methods. $ At, pH about 6.
be simply due to the more compact state of actomyosin, produced either by precipitation or centrifugation, which gives rise to some limitation of diffusion during the ATPase assays. In all cases, however, the steepness of activation, reflecting the degree of co-operativity, is the same for both myofibrils and actomyosin, and we may presume from t,his that the fundamental properties of activation are retained by the actomyosin. The reduced actin-activated ATPase activity of actomyosin at high Mg2+ concentrations (Fig. 4) is probably due to the associated higher rate of precipitation of actomyosin during the assays, which could result in a steric limitation of the number of active sites. At high Mg2 + concentrations there is, in addition to a decrease in activation, a shift in the calcium-activation curve (Figs 3 and 4) and also a decrease in calcium-binding (Table 6). This would suggest that at high Mg2+-to-Ca2+ ratios. magnesium successfully competes with calcium for the calcium-binding site on myosin. This is compatible with the findings that in fresh Mg-actomyosin the ATPase activity is inhibited up to 5Oo/o by magnesium (EGTA, Table 1) relative to t,he activity in the absence of both magnesium and calcium (EDTA, Table l), and for Ca-actomyosin, which is calcium-insensitive at 1 mM free Mg2 + concentration, some calcium-sensitivity map he restored by reducing the free Mg2+ concenDration to 0.1 mM. (c) Calcium
regulatiolL
in smooth ~muscle
9s demonstrated in the case of molluscan muscles, the actin-activated ATPase activity of vertebrate smooth muscle myosin is sensitive to Ca2+ with F-actin alone, that is in the absence of any a.ctin-linked regulatory components. Since the combination of thin filaments or F-actin from smooth muscle with rabbit skeletal muscle myosin produces a calcium-insensit’ive actomyosin. we may conclude that, in vert’ebrate smooth muscle, regulation by calcium is a specific property of the myosin molecule. Consi&ent with this conclusion is the absence of any troponin-like proteins either in smooth muscle actomyosin or myofibrils (Sobieszek $ Bremel, 1975) or on t’he thin filaments (Fig. 6(i)). The finding of myosin-linked calcium-regulation in vrrt’ehrate smooth muscle indicates t’hat this type of system is more widespread than
!I0
x. SOHIERZEI< .-\SI) ,I. \‘. S~lAI,I,
f)rrviously su~~~osed and, in particular. not, restricted to invertebra&. At preserlt 21 correlation between muscle t,ypr. for example smooth or striated. and the type of‘ regulation system is not obvious and indeed. for molluscs, bot,h smooth and striat,ed muscles are regulated by- myosin. One inttrest,ing possibility raised bp these results on vertebrate smooth muscle is that the various non-muscle actornyosin-containitl~ syst’ems (see review by Huxley, 1973a; Adelstein & C’onti, 1974) including, for exa,mplc. slime mold. amoeba. blood plat’elets and fibroblasts may also be regulated by nryositr. This suggestion derives from the noted similarit,y between the myosins of thest systems and that of vertebrate smooth muscle. in light chain composition and ill solubility and assembly properties (Adelman & Taylor, 1969 : Sbramolvitz et (11.. 1973 ; Hinssen 8r D’Haese, 1974: Bdelst’ein $ Comi. 1974). Indeed. we might expect that in systems where the contract’ilc elements are continually assembled and disassembled according to need. a myosin system composed of fewer probein components would be a dist,inct’ advantage. In molluscan muscles it has been shown that one of the myosin light chains. specifically that released by EDTA, is intimately involved in the calcium-regulat.ory process. Thus, t.he removal of this light, chain result’ed in a desensitizat,ion of myosin. \\.hile its recombination with desensitised myosin caused a restoration of calciumsensitivity. The light chains of vertebrate smooth muscle myosin are not so readil! released and we have, as yet, been unable to define conditions under which any of the light chains can be reversibly removed. However, some other recent experiments (Sobieszek, unpublished results) have provided strong evidence for the involvement of at least one of the smooth muscle myosin light chains in the regulation process. Thus. it has been shown that the triggering by calcium of the actin-act,ivated ATPase of smoot,h muscle actomyosin is associated with a parallel phosphorylation. at the same Ca2 + concentration, of the 20,000 molecular weight (L2,,) light chain of myosin. From this and a demonstrated correla,tion between the calcium-sensitivity of act,omposin and the degree of phosphoryla,tion. it is clear that the phosphorylation of the L,, light chain is int’imately associated xvith calcium regulation. The concomitant loss of calcium-sensitivity with the digestion of the L,, light chain by papain or trypsin is further consistent with t,his conclusion. A phosphorylat8ion of a myosin light chain has been shown to occur in other systems. including striated muscle (Pires et al., 1974: Adelstein et al.. 1973) but., although not, overlooked (Adelst,ein & Conti, 1974), a link ljetween this and calcium-sensit,ivity was not’ demonstrated. How the L,, light chain and whether the L,, li g ht, chain of smooth muscle myosin operate in the regula.tion process has yet bo be defined. In scallop myosin t#here appea’m to be only one regulatory “EDT,A” light, chain per molecule and, on this basis. son~t’ with the same light chain. co-operativitp between the two heads. each interacting smooth muscle the has been postulated (Szenb-Gyorgyi et al.. 1973). .I n vertebrate available data, suggests that there a,rr at, least, two L,, light chains (Sobieszek & Bremel, 1975) and that two light chains per molecule become phosphorylated in t’he presence of calcium (Sobieszek, unpublished results). Since: in addition, t,he amount of calcium bound by myosin corresponds to about two calcium ions per molecule, it is not unlikely that the tn-o heads (S-l) of myosin are independently activated 1)) calcium. Presumably. this may occur via conformat’ional changes resulting from the itself being dependent phosphorylation of the L,, light, chain, the phosphorylation on calcium. As far as the L,, light chain is concerned, it would appear that this light, chain may be necessary for the hydrolytic activit!, of mvosin. An indication of this
REGULATION
IS
SMOOTH
MUSC!LE
9I
comes from the parallel loss of the L,, light chain (in addition to the L,, light chain) and of actin-activated ATPase act,ivity with trypsin digestion (Fig. 8 and Table 5). The role that tropomyosin plays in the operation of myosin-linked systems is unclear. In striated muscle it serves to propagate the calcium-induced changes on troponin in a co-operative manner along the thin fila.ment, (Bremel & Weber. 1972 : Bremel et u.Z., 1973). In the absence of troponin the same changes clearly cannot occur. I)utj nevertheless from X-ray diffraction evidence (Vibert et nl.. 1972 : Huxley. 1973h : Haselgrove. 1973) a similar conformational change. taken as a shift in the position of tropomyosin. occurs on activation of smooth muscle as it does in striat,ed muscle. That, tropomyosin is bound to nctin in smooth muscle is indicated 1)~ its parallel cbxt,raction and co-sedimentation with actin in the thin filament,s. As we hare shown (see also Sobieszek & Bremel, 1975). t,ropomyosin can be selectively removed from smooth muscle actomyosin by ammonium sulphate precipitation (2524, saturation) and, depending on the type of actomq-osin. A variable amount of’ actin-activated ATPase is lost (see Table 1). Since ammonium sulphatc may act. in general. to decrease the activit’y of total act’omyosin (i.t,. whc,n precipitating all components a.t 6001;~saturation: Sobieszek & Bremel. 1975) it would appear that t,his variability result)s more from differing susceptibilities of actomyosin (e.g. MgAM or C!aAM) to ammonium sulphate t’han t)o the loss of tropomyosin. This is more dircctl). indicat#ed hy the effect of tropomyosin removal on ci~lcium-insc,nsit’ive Ca-actomyosin (&AM). whirh rcxxults in a relat’ivclly minor loss of actin-act,ivnttLd .4TPase activit!, (Table I ). While these lat,ter results would t’end to suggest t’hat t’ropomyosin plays a, minor role in t,hr activation of ATPasr activity, the result’s from the rrcomhination experimentb rTahle 2) may be more rrlwant. These cxpcriments showed that the comhination of’ gizzard myosin with gizzard thin filnment,s. composed of actin and tropomyohill. was twice as active as that with P-actin prepared directly from the thin filaments. A1 similar r+l%ct~ \\.as found in the case of stria,ttld muscle from Lirrrulus (Lehman & Szent-(:yorgyi. 1972). The possihilitg thus exists. that tl'(JpOmyOSiti acts. as in striated muscle. t 0 “turn on” the t*hin filament in a co-operativca manner when act,ivatiotl t:l kes plwre. Indeed. the steepness of the activation curves that L\‘e hare obtained fol snioot8h muscle (Pigs 2 and 3) suggests a co-operative a&n-myosin intera,ction in smooth muscle similar to that’ in striat’ed musclr (Bremel et ccl., 1973) and. on this I,asir. I\ t’ might, ttxpect this co-operat’ivity to he due t,o the presence of tropomyosin on thus thin filamt~nts. We should. however. not exclude the possibility that, myosin alone II~;I~ he thcl component responsible for the c.o-opcrativity seen in the actinmyosiu interaction. This possibility is suggesttad 1,~ the observation that the steepness of the ~.aIciutii-act’ivat’ion curve’ for ni.yosin phosphorylation is the same as for a&omyosin ;\TPase activity and. furthermorr. calcium is needed only for myosin phosphorylation and not for its lrydrolyt’ic actiritj(Sobicszek, unpublished results). Studieh art in progress to cla.rify the specific roles of’ calcium and myosin phosphory. lation in the regula,tion of the ac*tirit’- of rcrtebratc, smooth musclt). Thlc work represents an outgrowt,h of investigations I)egun in collaboration w4th Dr 12. D. Hremel at the Department of Anatomy, l)uke Universit,y Medical Center. \Ve thank Dr Brelnel for stimulating discussions and are indebt,ed to Dr M. K. Reedy for I& support during the early stages of this work (National Institutes of Health grant 1 ROlAM14317). We also thank Professor K. Marcker for his positive at,titude in making worn n\~ailahlo ill t Ile Mnlecular Bio1og.y Inntitlltcx. This \wrk \~as drnle (luring the tenure
92
A. SOHIESZEK
dND
J. V. SMALL
of a research fellowship to one of the authors (A.S.) from the Muscular Dy&rophy .Aewciu. tion Inc. and was supported by grants from the Volkswagen Foundation and the Muscular Dystrophy Association Inc. We thank MS Tove Wiegers for excellent, technical assistanve and Miss Aase Sorensen for t,ypinp. REFERENCES Abramowitz, J., Malik, M. N., Stracher, A. & Detwiler, T. C. (1973). Gold &w&q Harbor Symp. Quant. Biol. 37, 595-598. Adelman, M. R. $ Taylor, E. W. (1969). Biochemistry, 8, 4976-4988. Adelstein, R. S. & Conti, M. A. (1974). In Exploratory Concepts in Muscular lly&oph,?y (Milhorat, A. T., ed.), vol. 2, pp. 70-78, Excerpta Medica, Amsterdam. Adelstein, R. S., Conti, M. A. & Anderson, W. (1973). Proc. Xat. A4carl. Sci., U.S.A. 79, 3115-3119. Bremel, R’. D. (1974). Nature (Londo?~), 252, 405. 407. Bremel, R. D. & Weber, A. (1972). Nature New Biol. 238, 97-101. Bremel, R. D., Murray, .l. M. & Weber, A. (1973). Cold Spring Harbor Symp. Qua&. Bill. 37, 267-275. Biilbring, E., Brading. A. F., Jones, A. VV. & Tomita, T. (1970). Editors ofSmooth. -1fu,scle, Arnold, London. Driska,, S. & Hartshorne, D. J. (1975). Arch. Biochem. Biophya. 167, 203-212. Ebashi, S. & Endo, M. (1968). Prog. Biophys. Mol. Biol. 18, 123-183. Etlinger, J. D. & Fischman, D. A. (1973). Cold Spring Harbor Symp. Quant. Biol. 37, 51 l-522. Fairbanks, G., Stock, T. L. & Wallach, D. F.-H. (1971). Biochemistry, 10, 2606-2617. File, R,. S., Bohr, D. F. & Riiegg, J. c’. (1965). Science, 147, 1581-1583. Fiske, C. H. & SubbaRow, Y. (1926). J. Biol. Chem. 66, 375-400. Haselgrove, J. C. (1973). Cold Spring Harbor Symp. Quunt. Biol. 37, 341-352. Hinssen, H. & D’Haese, J. (1974). J. Cell Sci. 15, 113-129. Huxley, H. E. (1973a). Nature (London), 243, 445-449. Huxley, H. E. (1973b). Cold Spring Harbor Symp. Quant. Biol 37. 361-376. Kendrick-Jones, J. (1973). Phil. Trans. Roy. Sot. ser. B. 265, 183-189. Kendrick-Jones, J., Lehman, W. & Szent-GyGrgyi, A. G. (1970). J. MoZ. Biol. 54,313-326. Kendrick-Jones, ,J., SzentkirBlyi, E. M. & Szent-GyBrgyi, A. G. (1973). Cold Spring Harbor Symp. Quark Biob. 37, 47-53. Lehman, W. & Szent-Gyijrgyi, A. G. (1972). J. G’erc. Physiol. 59, 375-387. A. G. (1973). Cold h’pring Harbor Lehman, W., Kendrick-Jones, J. & Hzmt-Gyargyi, Symp. Qua&. Biol. 37, 319-330. Margossian, S. S. & Lowoy, S. (1973). J. ,IloZ. Biol. 74, 313-330. Murphy, R. A. (1971). Amer. J. Physiol. 220, 1494&1500. Needham, D. M. & Shoenberg, C. F. (1968). In Handbook of Physiology (Code, Ch. I”‘., ed.), vol. 4, sect. 6, pp. 1793-1810, Amer. Physiol. Sot., Washington. Ogawa, Y. (1968). J. Biochem. (Tokyo), 64, 255-257. Pires, E., Perry, S. V. & Thomas, M. A. W. (1974). FEBS I,etters, 41, 292-296. Portzehl, H.? Zaoralek, P. & Gaudin, J. (1969). Biochim. Biophys. Acta, 189, 440-448. Sobieszek, A. & Bremel, R. D. (1975). Eur. ,7. Biochem. 55, 49-60. Sparrow, M. P., Maxwell, I,. C.. Riiegp. .J. C. dz Bohr, D. F. (1970). Amer. J. Phyviol. 219, 1366-1372. Spudi& J. A. & Watts, S. (1971). J. Biol. Chem. 246, 4866--4871. Somlyo, A. P. & Somlyo, A. V. (1968). Pharmacol. Rev. 20, 197-272. Romlyo, A. P. & Somlyo. A. V. (1970). Ph,armacoZ. Rev. 22, 249-353. E. M. B Kenrlrick-Jones, .J. (1973). J. Mol. Biol. 74, Rzent-Gyijrgyi, A. G., SzentkirBlyi, 179-203. Vibert, P. J., Haselgrove, J. C., Lowy, J. d Poulsen, F. Ii. (1972). J. Mol. HioZ. 71, 757-767. Weber, A. (1969). J. Gen. Physiol. 53, 781--791. Weber, A. & Herz, R. (1963). .7. BioZ. Chem. 238, 599-605. Weber, A. & Murray, J. M. (1973). Physiol. Rev. 53, 612-673. Weber, .4., Herz, R. 8: Reiss, I. (1969). Biochemistry, 8, 2266-2270. Wnher, K. & Oshorn, M. (1969). .I. Biol. Chem. 244, 4406-4412.
(1976)
Myosin-linked
101, 75-92
Calcium Regulation in Vertebrate Smooth Muscle APOLIKARY SOBIESZEK AND ,J. V. SMALL
(Rewived
1 Augwt
19’i.i.
and in revisal
fhw~.
20
October
1975)
may be extracted ill Iligh yield and 13). ttw ,lSR of a new pr0ccd11rc:. actomyosin purity from fowl gizzard which exhibits a calcillm-dependent, actin-activated ATPase activity comparable to that of the pawtlt myofibril-like preparation. Studies of this vertebrate smooth muscle act,omyclsin show that t,hcx regulation of the actin-myosin interaction is effected, as in molluscan muscles. by the myosirr mc~lrc~& it,wlf and not b>- arl actin-link4 regulatory systtlm. as found in verteI)rat,cl skeletal muscle. ‘IIllls, calcium-sensitive smooth muscle actomyosin is composed of only- myosirr. any troponin-like compotrcntx bring absent. Myosin is acatln and tropomyosin. thr only component that binds significant amounts of calcium and shows a calcillrn-deprrldent a&in-activated ATPase acti\Aty iti tltrk pxwnce of IT-actill from (lither gizzard or rabbit skrlat~al musclr. The cross-reaction of gizzard thin filaments \vitJt skeletal muscle myosin pro(111ces an actomyosin whose actin-act,ivated .ATPaso is calcium-insensitive, showing that smooth muscle Olin filaments do not serve a regulatory function. of one of the The effect of Mg2+ and pH, and evidence for the involvement lnyosin light chains in calcinm regulation are dcwrilwtl and discnswd.
1. Introduction Recent, studies have demonstrated that the regulation of bhe actin-myosin interaction in muscles is not a unique property of t’hc thin actin-containing filaments. Thus. while a troponin-tropomyosin complex linked to actin serves to mediate t’hr triggering of contraction by calcium in vertebrate skeletal muscle (see reveiws by Ebashi & Endo, 1968; Weber & Murray, 1973; and Cold ~Spring Harbor Symp., vol. 37, 1973). a rather different system. associated with Dhe myosin molecule has been shown t’o operate in molluscan muscles (Kendrick-Jones of al.. 1970,1973 ; SzentGyorgyi et al., 1973). Further, related studies (Lehman et al., 1973) have indicated that such a myosin-linked system exists in cert’ain other invertebrate muscles and have fllrnished evidence, in some cases, for the presence of a dual system involving regulation on the actin as well as on the myosin component. We have been focussing our attention on identifying the component(s) responsible for calcium regulation in vertebrate smooth muscle. Studies of this muscle type have previously been hampered by the difficulties encountered (see review by Needham & Shoenberg, 1968; Sparrow ef al.. 1970; Murphy. 1!$71; Driska & Hartshorne. 1975) in obtaining an actomyosin with a demonstrated activity and calcium-sensitivity showing some comparison to that of skeletal muscle (Weber & Herz, 1963; Weber et aZ., 1969; Murphy, 1971) or to glycerinated smooth muscle preparations (Filo et al., 1965). In a recent study (Sobieszek & Bremel, 1975) we described a new procedure for obtaining actomyosin from fowl gizza,rd which is highl,v calcium-sensitive and 7.5
i(i
.I. SOHlES%Eli
.\SI)
.I. \.. Skl.-\l,l,
cbxhihits an activity several times greater than that reported for other smooth mus& actomyosins. It was further shown that this calcium-scnsit’ivc actomyosin exhibit,td the presence of only three prot,eins. namely myosin. actin and t ropomyosi n. arid components resembling troponin of skeletal muscle being absent. On this I)asis it was suggested that in vertebrate smooth muscle, as in molluscan muscles. myosin may perform the role of calcium regulat,ion. From the recombination and cross-reaction of purified contract,ile proteins and actomyosins from both smooth and skeletal muscle we have now obtained direct evidence for a myosin-linked control mechanism in vertebrate smooth muscle. Specifically, we show that smooth muscle myosin binds calcium and interacts with F-acbin, from either smooth or skeletal muscle, to produce actomyosins that exhibit a calcium-dependent actin-activated ATPase. Mixtures of smooth muscle thin filaments with skeletal muscle myosin are? in contrast. calcium-insensitive. The results of competition tests of skeletal muscle F-actin with smooth muscle actomyosin are further consistent with a myosin-linked system (see also Bremel. 1974). Evidenctl is further presented which indicates t)hat. in vertebrate smooth muscle at least, one of the mgosin light chains is involved in the calcium regulation process.
2. Materials and Methods As described in a previous paper (Sobieszek $ Bremel, 1975) we have used a myofibrillike preparation of chicken or turkey gizzard as source material for the extraction of smooth muscle actomyosin. Briefly, the myofibrils are produced by thorough llomogenization of finely minced fresh gizzard followed by extraction with Triton Xl00 and extensive washing to produce a white residue. Figure 1 summarizes the procedures used for processing the myofibrils to produce t,he various preparations of contractile proteins. Unless otherwise stated, all procedures were carried out at 4°C and all solutions contained 100 mg streptomycin/l and freshly added 1 mno-cyst&e. For the extraction of actomyosin, myofibrils were resuspended in an ice-cold medium of the following composition : 10 mM-ATP(Na), 1 mm-EDTA, 2 mM-EGTA, 1 mM-CySteinP.
60 mM-KCl,
40 mM-imidazole
(pH 7.1). Undissolved
material
was removed
by centrifu-
gation at’ 14,500 revs/min (15,000 g) for 1 II and the extracted actomyosin filtered through glass wool. For a ratio, by volume, of extraction medium to myofibrils (originally sedimented at 2500 revs/min, 2000 g for 1 h) of between 3 and 4 to 1 the protein concentration of this crude actomyosin (Fig. 1) is greater than 10 mg/ml. To this extract 1 >r-MgCl, solution was slowly added, while stirring, to give a final concentration of 25 to 30 m&l. causing the actomyosin to become opaque. The extract was then left at, 4°C for about 10 to 15 h to form a compact gel. This gel was then centrifuged at 14,500 revs/min (15,000 g) for 30 min and the pellet so obtained resuspended, by gentle homogenization, in a “wash 20 mM-imidazole (pH 6.8). solution” containing 60 mM-KCl, 1 Inn%-MgCl,, 1 rnfiI-cysteine, The actomyosin was pelleted and resuspended and stored in a buffer solution of 60 mMKCl, 1 mar-cysteine, 40 mM-imidazole (pH 7.0 at 25’C) corresponding to that. used for ATPase activity measurements. exhibits the highest calcium-sensil ivit’> Actomyosin purified by Mg2 + precipitation and we refer to this actomyosin as Mg-actomyosin (MgAM in Fig. 1). Precipitation may equally well be effected by using CaCl, (1 to 2 m&l) or CaCl, plus MgCl,; in both cases the actomyosin precipitates without the formation of a gel. We refer to these actomyosins respectively. In contrast to as Ca-actomyosin (CaAM in Fig. 1) and CaMg-actomyosin, Mg-actomyosin, these actomyosins exhibit little if any calcium-sensitivity (see lttesults). Tropomgosin was removed from divalent cation-precipitated actomyosin by ammonium sulphate precipitation (25% saturation) as described previously (Sobieszek & Hremel. 1975), with at least 2 subsequent washes to remove ammonium sulphatr.
,/
_
CAM extraciion
220,000 mol wt
F - octm
I
(crude octomyosin) Can be used for TM extraction 0 6 lo I 0 M-KCL
*I--Discard
MgCl2 - 35 ‘h; 14.500 revs/mm
Myos
I
3’10. 1. Schematic diagram illust,rating t,he processing tractilr proteins. Upper and lower parts of rectangles respectively. Numbers used in composition of media essential components are given. Unless otherwise stated TiCI. 20 t,o 40 mM-imidazole, 1 mwcgst,nine and 100 TN&ho& for furthrr d&ails).
&eps in the preparation of gizzard concorrespond to supernatants and pellets, correspond to ITIM concentrations; only all solutions contain, in addition, 60 mMrng st,repbomycin/ml (see Materials anrl
Hr centrifuging freshly extracted crude actomyosin for about 18 h at 14,500 revs/mill ( 15,WO g) a myosin fraction is ohtained which contains very little actin and tropomyosin impurities; we refer to this as a “crude myosin fraction” (CMF in Fig. 1). This nyosin may be selectively pracipitated by the slow addition of 1 M-MgCl,, with stirring, to a final concentration of about 35 rnM and is then collected by centrifug-ation for 2 h at 14,500 revs.’ rnin (15.000 g). CaCl, is effective in precipitating rnyosin but was not used, since the preeipit&ion of actomyosin wit.h calcium caused a. va.riablc loss of calcium-sensitivity (see section (a), above). Thp pellet formed by t)he centrifugation of crude actomyosin comprises an actomynsin (P-AM, see Fig. 1) enriched in actin and tropomyosin and was preserved and used for rcaombina,tion c>>cperiment’s with rabbit, skelet,al mnscltl myosin (SW Results).
From rabbit skeletal muscle myofibrils extracted with Triton Xl00 and washed in the presence of 2 rnM-pyrophosphatr (Etlinger & Fischman, 1973), myosin was extract,ed in H medium conbaining O-3 M-KCl? 1 mM-MgCl,, 5 rnM-ATP(Mg), 2 mM-pyrophosphste. % HIM-EGTA, 1 mM-cysteina and 40 mm-imidazole (pR 7.0). The extract was centrifuged at 151000 revs/rnin (20,000 g) f or about 15 h and t.he myosin in the supernatant precipitated by dilution with about .5 vol. ice-cold water with added 1 mnr-cysteine. The precipit,ai.tb was collected by eentrifugat,ion at 2.500 revsjrnin (2000 g) for 1 h and resuspended. using a Teflon/glass homogenizer, in buffered 0.06 IVI-KCl. 3 ~-Kc1 was then added with stirring until the myosin dissolved (at about 0.4 M-KCl) and the solution clarified b) ccnt’rifugatian in the presence of P mM-EGTA and 2 mM-ATP(Mg) at 40,000 revs/tin (100,000 g) for 3 h. The precipitation and clarification steps were then repeated bnt wit8hout t’he addition of ATY(Mg) rind EGTA.
mirlimisr myosin filament disaggrcpat~iotl, it11 tbxtract ricll in ttlill filamrrlts nlay bcl obt~ainc~cl. For this purposn bile same basic cxLractior1 mfdil~m \vas used brat) cwrrtaining 2 tn~ wit11 tllrs pH ad,justed to 6.5 at 1 ‘C’. ;-\ftclr wtltrifll,ZTP(My). 1 mwMgC11,. 2 ml%-EGTAantl gation for 20 min at 19.000 rrvs/min (40.000 g) to r(w1ovc tissurl fragm~~tLts. ttrcb pH \+.a~ raised to 7.0 and the thin filarnwlts cwllwtc~tl 1)). c~c,lltrifrl~:i~tiotl for 2 II at 40,000 W\~S,:lllill (100,000 g). The pellets wcro tllcll dissol\~c~tl irl \vastI solrltion Itsing R Il;rrltl-l)~)c~r’at,(~(l, Tc+lon/glass homogrnizw. (t!)
F-a&n
‘I’tre residue remaining aftor actornyositi csxtraction coIlt,ains a large amount. of arti,, rclat,ive to myosin and serves as a ronvc~nirnt scjurce of F-actin. Residual myosin :ultl by rr-rxtractiorL at, Iii@ ionic strengt,h tropomyosin wew wmovfd (0.6 to 1.0 ar-K(‘1. 1 mM-cysteine. 1 mu-EDTA, 20 ml\r-imidazole, pH 7.0) \vith a substqncntj waslr at low ionic strength to removcx KU. The rosultinp residw was resuspended wit11 a Teflon/glass homogenizer in a small volume of u-ash sohltion (not mow than t\+%o that of the residue) and sonicated for 5 t,o 8 min in an MSE sonicator at, an amplitude of 16 pm. The suspension was homogenized again and then scdirnt~ntc~tl folx 1 tr at t 4.500 revs/rnin (15.000 g) to give> a supcrnatant cont,aining puro I?-actin at, :I conccwtration of 1.5 to 2 mg/ml. Gizzard F-actin was also prepared by A second mctllod directly frown t,hin filaments (sot section (d) above) hy soloct,ively removing t~ropomyosin at high ionic strength. Tl~ns, the pH of the thin filament extract was adjusted to 7.0. t)lle KC1 concentratiotl inclrLascXd to 0.56 Y by tile addition of 3 >I-KC1 and F-actin subseqllrntly pelleted by ccntrifugat~iorr for 2 h at 40,000 revs/min (100,000 g). TI>c prllct, was dissolved in \vastI solntiorr using a hand-operated T&on/glass homogenizer. F-a&in from rabbit skeletal muscle IVNS prrlparcd wwntially accwrtlinp to Splidic,ll & ITatt (1971) from the residue rernainirlg after myosirr extraction. (f)
Ijodecyl
sctlphnte
ye1 electrophoresis
gcLs it1 the prescnco of sodium dodecyl sulphate Elcctrophoresis on polyacrylamide (Weber & &born, 1969) was carried out according to Fairhanks et al., (1971) \vith modifications as described by Sohieszek & Rwmcl (1975). (g)
Protein
determination.
and
A TE’ase
activity
measurements
Protein concentrations were drt~ermincd by the Binret method using a protein standard from Sigma for calibration. ATPase activity measurements were carried otlt at 25 C in a medium containing 60 rn>lKCI, 1 mM-ATP(Mg), 1 m>T-cysteine, 40 mnr-imidazolt: (pH 7.0 at 25°C) and with either: 2 m&r-EGTA plus 1 mM-Mgcl,; 0.1 mnr-CnCl, plus 1 mM-MgCt,; or 2 mM-EDTA. The latter measurements. Undw these assay provides an important refcrenw point for activity conditions actin and myosin are dissociated (Sobieszck 8~ Rrc-rnel, 1975) and the act)ivitJ cat,ions. For measured corresponds to that of myosin alone in the absence of divalent buffer system was usotl, assays at different, concentrations of free (‘a2 + , a CaEGTA/EGTA taking the apparent dissociation constant for C’aEGTA at pH 7.0 as 1 Y: 10m6 &I (Ogawa., 1968). The 3-ml assays were pre-rquilibrat,ed at 25°C’ and t)he reaction initiated by adding was torminat,ed aft.w 30 to ATP while mixing gently on a vortex mixer. Tllc waction filternd and 60 s by the addition of 2.0 ml 10% trichloroawt~ic acid, the preparations inorganic phosphate measured according to thr mrtllod of Fiske & SubbaRow (1925). For expressing enzyme activit,y relative to myosin. the myosin content of the preparations of sodium dod~cyl was determined by quantitati\-e gel electrophnresis in t,llr pwsencr sulphate (see Sobieszek & Rremel, 1975). (II) Ca2+ -binding
measurements using the double label method
Ca2 + -bindiny
mere carried out according to Kendrick-Jones et al. (1970) with [3H]glncose as a second label. Assays were performed
3. Results (a) .-Ic/ott(/psi,r. atrri c~nlcil~~rl.-.YPtIS~li)‘i~~/ Depending on t,hr nn:t~hod of precipitation of freshly extracted. crude actoniyosin (CAM. see Fig. 1). actomyosins are ol)ta.incd wlrieh exhibit, different’ characterist~icn. In an earlier study (Sohirszek & Hremel. 197.5) precipitjat8ion \\as effected by the use of’ magnesium followed 1)s ammonium sulphate. Suhseyuently, in t’he present study. it was found that divalent c&ons alone ww sufficient to cause precipitation overnight. The actomyosin so obtained is four to tive times more act’ive than that processed through ammonium sulphatc and it also lacks the cont8aminating component migrating at, a molecular weight of ahout ~10.000 on sodium dodecyl sulphate polyacrpla~mide gels (compare (a) and (c). Fig. 6). The most calcium-sensitive actomyosin is obtained by the use of magnesium precipit~ation and \ve refer to this as Mg-actomyosin (MgAM Fig. 1). The properties of this ac:tomyosin are described hekm~ : a later section will deal with the eharacterist~ics of actonly-osins obtained 1)). precipitation with calcium or calcium plus magnesium. In freshly prepared Mg-actomyosin tOhe ATPasc activity is inhibited 90 t,o 95”;, I)y calcium removal (Table 1) and this degrrc of c~~lciu~n-st~~~sit~i~~it~~~ is only maintained over a period of two t,o three tla;vs. ,Is shown in Figure 2. the activation by calcium occurs Vera’ sharply in the concentration range I to 10 cc”. A significant feature of t,his calcium activation curve is the range of free C:t2 + concentration over which full actjivation occurs. As Carl be seen from Figure 2. therc~ is a ilistinct platea,u between I.5 +V and 200 ~BI free calcium and a marked decrease in act,ivitjy a,l)ove about 500 PM. The same type of inhibition at high Ca2+ concentrat’ions also occurs with myofibrils f’~m skeletal muscle (Portzehl it ab.. 1969) indicating tlliat ATP(Ca) is not as good a substrate as ATP(Mg) for the actomyosin system. While the most active and calcium-sensitive actomyosin is obtained by magnesium precipitation the high concentration of magnesium used commonly acts t,o shift, the wlrium
ac+iv;ltion
curve
relative
to
that
ol&tirnd
for
tin
parent
myofibrils.
As
TABLE
A TPase activityt
and C’a’ + -sensitivity
Preparation
EUTA
Myofibrils Sctomyosins f MgAM MgAM at 37’Y’ P-AM CaAM Ca, MgAM Tropomyosin-free actomyosim MgAM - TM CaAM - TM Ca, MgAM - TM
1
of various yizzard preparation.~ EGTA
(:a2 + -scnsitivity$
Ca2 +
IO.4
Il.6
105
0.89
“2.4
13.9 46~9 22.6
0.94 0.90 0.89
171
""4 472 204 193
1%
176
0.23
66~1 18.3 36.7 32.7 164 48.8
61.0
9.1
106 99.5
0.11
11.5 118
0.78
0.10 0.31
145
t Expressed in nmol.mg protein-‘.min-‘. $ 1 - (EGTA activity/&x2 + activity). § For abbreviations see text and Fig. 1. TM, tropomyosin.
-7
I 6
I 4
I 5
- log ka”
I 3
1
FIG. 2. Dependence of ATPase activity of different gizzard actomyosin preparations on the free Ca’J+ concentration. Open and closed circles, MgAM; triangles, P-AM. Activation commonly occurs at around 1 x lOWE M-Ca2+ (closed circles) although sometimes takes place (open circles) at the lower Ca2+ concentrations characteristic for myofibrils (see Fig. 3).
shown in Figure 3, gizzard myofibrils are normally activated at lower Ca2+ concenand further, their activation curve trations (about 6 x 10d7 M) than actomyosin shifts to the position characteristic of actomyosin when they are assayed in the (10 mlcl Fig. 3) or after pretreatment with presence of high Mg2+ concentrations Mg2+ (25 mM). In some cases the activation curve of actomysin was not shifted but occurred at the same Ca2+ concentration as myofibrils (Fig. 2). The steepness of activation of actomyosin, however, was always the same as for myofibrils. For actomyosin (and also myofibrils) changes in the free Mg2+ concentration (in excess of ATP) between O-1 mM and 10 mM act to modify the magnitude of activalying in the range tion by calcium (Fig. 4), the optimum free Mg2+ concentration
0.2 mM
t0
2 mM.
REGULATION
I 0
I 7
IN
SMOOTH
I 5
I - loo6w+
MUSCLE
I 4
J
I
Fx. 3. Calcium-activation of gizzard actomyosin (&&AM, ---e---a--) and myofibrils of 1 mx. Note similarity between the actina-) at a free Mg2+ concentration (MYF, ---Icurve for myofibrils is shifted aotivated ATPase activities in the presence of Ca 2 + . The activation towards that obtained for actomyosin when these are assayyod in t,he prssencp of high concentrasee Discussion). tions of Mg2+ (10 rnq -n---Q--;
-log luci* : YIG. 4. The effect of changes in Mg2+ concentration on the calcium-activation of gizzard actomyosin (MgAM). In all experiments the ATP(Na) concentration was 1 maI while the total Mg2 + concentration was varied from O-2 rnM to 11 mM. Optimum activation takes place between 0.2 atid 2 rnM free Mg2+ concentration, while almost complete inhibition occurs at 0.2 mM total magnesium resulting from the relaxing effect of the ATP anion (Sobieszek & Bremel, 1976). The tota. magnesium concentrations were, in IIIM :~~~~~~~~2;-x-x-~1~O;~~~-----~-~
1.2; --.‘.----A--,
1.5; ---m-o--,
2.0; -n----o-,
3-o; --m-m---,
11.0.
Figure 5 shows the effect of changes in pH at the normal assay temperature of 35°C and demonstrates an optimum around neutrality. Increasing t’he temperature to 37°C at pH 7-O acts to elevate the ATPase activity to approximately double t’hat at 25°C (Table 1). From gel electrophoresis in the presence of sodium dodecyl sulphate and sedimentat’ion experiments (Sobieszek & Bremel: 1975) purified vertebrate smoot8h muscle 6
PH
FIQ. 5. Inlluence of pH on the ATPase activity (----a-) andabsence (EGTA, -O---O-;
of gizzard actomyosin EDTA, ----A---A-)
(MgXM) in the prewnw of (‘aZ+.
actomyosin shows only the three primary contractile proteins, actin, myosin (with its two light chains) and tropomyosin (Fig. 6 (a) and (b)). Densitometry of photographic negatives of stained gels with corrections applied for the relative stainability of each protein component (Sobieszek & Bremel. 1975) yields proportions for m.yosin : actin : tropomyosin of 2.5 : 1 : 0.3. (b) Loss of calcium-sensitivity For Mg-actomyosin, both the calcium-sensitivity and the ATPase activity in the presence of calcium decay, after 7 to 10 days at 4”C, to about 50% of their original value and calcium-sensitivity is completely lost after a few weeks. Actomyosin produced by precipitation overnight with Ca2 + (1 mM), CaAM, or with Ca2 + plus Mg 2 + , CaMgAM, exhibits rather different properties. In bot,h cases, calciumsensitivity was considerably reduced (Table l), although the absolute activity was comparable to that for Mg-actomyosin. However, while calcium-sensitivity is rapidly lost in these actomyosins during the preparation, they retain, in contrast to Mgactomyosin, almost full MgATPase activity for at least one week. Calcium-sensitivity is also irreversibly lost on brief treatment of Mg-actom,vosin with trypsin or papain : this effect is discussed again in a later section.
(c) Evidence for rnyosin-linked cuEciunl-.sen~s~t~T:jt?/ (i) Recombination of gizzard proteins Using a new and relatively simple procedure (Sobieszek t Bremel, 1975; and Fig. 1) myosin may be readily obtained in high yield and purity from gizzard muscle (Fig. 6(d)). We further describe here (see Materials and Methods) techniques for actin and tropomyosin obtaining native thin filaments from gizzard, containing (Fig. 6 (i)) and a,lso gizzard F-actin. The latter F-actin shows the characteristic thin filaments in the electron microscope and runs as a single band on sodium dodecyl sulphate gels (Fig. 6 (g) and (h)). (j) Rabbit F-actin. (k) Complex of gizzard S-l and gizzard F-a&in. S-1 and Y-aotin were mixed together at low ionio strength and pelleted by low-speed centrifugation. (1) Hybrid aotomyosin from rabbit myosin and gizzard thin filaments. The preparation8 were mixed at about 0.3 M-KCl, dialysed against 0.06 M-KC1 and pelleted by low-speed centrifugation.
84
A. SOBIESZEK
AND TABLE
AT Pase activity
and Ca’ + -sensitivity
J. V. SMALL 2
of recombined gizzard ~re~arf~~i~)~~~-~-
Preparation
Ratio
EGTA
ca= ’
Myosin + F-actin Myosin + thin filam. S-1 + F-e&in
2.5:1 2.6: 1.3 2.5:1
20.6 21.5 180
62.3 137 194
t Expressed
Ca* + -sensit,ivity 0.67 0.84 0.07
in nmol . mg myosin - 1 . min - I.
Table 2 shows the ATPase activities for mixtures of gizzard myosin with gizzard F-actin and with native thin filaments, In each case: the combination shows 70 to 80% calcium-sensitivity. By comparison, gizzard myosin subfragment. 1 produced by papain digestion and which lacks the L,, li g ht, chain (see further below) shows an actin-activated ATPase activity that is insensibive to calcium. (ii) Cross-reactiojzs
between gizzard and skeletal muscle proteins
The actin competition test devised by Lehman et al. (1973) provides a simple test for the presence of a myosin-linked regulatory system. This test relies on the assumpt’ion t#hat unregulated actin (for example ra,bbit F‘-actin) added to a myosin-regulated actomyosin will not affect calcium-sensitivity, since in the absence of calcium it is the myosin that is inhibited and actin cannot activate its ATPase activity. Conrerselv. for an a&in-linked system where there is no myosin control, unregulated P-actin will act,ivat)e the myosin ATPase and act t.o decrease or abolish the calciumsensitivity. ilpplied to gizzard actomyosin this t’est indicates the existence of a myosin-linked control system; thus, the addition of F-actin to MgAM has litt,lrl effect on either the ATPase activity or the calcium-sensitivity (Table 3). The same result, was formerly obtained using acbomyosin prepared with ammonium sulphattb (Bremel, 1974). Table 3 also shows the effect of mixing gizzard myosin and subfragment 1 with rabbit F-a&in. The result is essentially the same as with gizzard F-actin (cf. Table 2). Calcium-sensitivity is exhibited by the intact myosin molecule wit,h F-actin hut, is ;tbsent in t,he mixbure of rabbit F-a&in with gizzard S-l.
TABLES
ATPase
of hybrid uctomyosins formed activity and Ca” -ser&tivity from rabbit skeletal P-act& and gizzard preparation.st Protein-toF-actin mass ratio
Prepamtion
MgAM Myosin 8-l
control + F-ectin + F-actin + F-a&in
7 Expressed
in nmol.mgS1
3.8:O 3.8:1 2:l 2:l (protein
EGTA per mg of protein 11.3 11.2 30.2 140
or myosin).miIl-‘.
myosin 17.5 21.5 45.3 209
ca2 + per mg of protein 181 143 67.6 146
myosin 279 274 101 219
Ce2 + -sensitivity
0.94 0.92 0.45 0.04
REGULATION
IN
SMOOTH
x5
MUSCLE
TABLE 4
ATPase activity and Ca’+ -sensitivity of hybrid actomyosina formed from rabbit skeletal myosin and gizzard preparationst
P-AM
control + myosin Thin fil. + myosin i l&pressed
EGTA per mg of
Protein-to myosin mass ratio
Preparation
protein 36.2 81.1 308
3.8:O 3.8: 2.5 2:l
in mnol’mg-’
(protein
CL32+ per mg of
actin 01.7 341
protein 152 153 305
actin 385 642 -
Caa+ -sensitivity
0.76 0.47 0
or aotin).min-‘.
The absence of any regulatory system associated with the thin filaments of gizzard was indicated by experiments in which skeletal muscle myosin was mixed with gizzard thin filaments. It can be seen in Table 4 that the gizzard thin filaments activate rabbit myosin equally in the presence or absence of calcium. In contrast, and as already indicated (Table 2), the same thin filaments give a fully calciumsensitive actomyosin when combined with smooth muscle myosin. Further, consistent with the absence of any thin filament regulation was the observation that skeletal muscle myosin added to actin-rich smoot,h muscle actomyosin (P-AM, see Fig. 1 and Fig. 6 (c)) resulted in an increase in actin-activated ATPase activity in the a,bsencr of calcium t’o the level of act’ivity of the control actomyosin with calcium present (Table 4). (iii) (Yalcium-binding Gizzard myosin and actomyosin bind calcium with high affinity over the range of concentration where the activation of the ATPase activity occurs and for a Mg2f concentration of 2 InM (Fig. 7). The amounts bound (see Table 6) at 60 to 90 PM free Ca2+ concentration, correspond t’o 3.4 nmol Car2+ per mg myosin and 2.4 nmol Ca2+
7 Pro. 7. Dependence (,MgAM, - -m--m--)
6
5
of calcium-binding of gizzard myosin (--C---C-) on the free Crt2+ concentration at 2 mM.MgCl,.
4
and actomyosin
86
A.
SoHItcsS%15li
.\SI)
.I.
\‘.
SM,\i.I,
per mg actomyosin. Since :tc:t,om~wsin is composed of about N(i”,, (iv/\V) of’ 1r1y0si1,. the value for actomyosin may by accounted for I)y the binding of’ calcium by. myosin (Fig. 7). This is consistent with t’he correspondingly insigniticant, amounts of calcium bound by gizzard thin filaments and gizzard IT-actin (l’ahlc 6). Associated with th(s lower pCa requiwd for their nctivat.ion. myofibrils IY(W found t.o I)intl calcium irt, correspondingly lower Ca2 + concentrations than for actomyosin. It may be not’ed that optimum calcium-binding oc :lrs at’ an apparently higher Ca2 + concentration than required for t’he full actjivation of actin-activated STPaw activity. However. when ATPase activity mca,surement,s were made at the 14-fold lower CaEGTA buffer concentration used for t,hc calcium-binding experiments, full activabion was achieved only at 60 to 90 PM free Ca2+ concentration as compared to 20 pnf for the same preparabion under standard conditions (Fig. 3). The optimum calcium-binding at 60 to 90 PM (Fig. 7) thus correlates directly with the ATPaw activity measurements made under the same conditions. We conclude that a,t lo\\calcium-buffer concentrations the CaEGTA stahiliby constant’ is somewhat dependent on the total Ca2+ concentration. The figure obtained for myosin is approximately t~he same as that obtained for scallop (Kendrick-Jones et al.. 1970;1973 : Szent-Gyorgyi st al., 1973) and corresponds to nearly two calcium ions bound per molecule. A IOO-fold change in the free Mg2’ concentration from 0.1 mM to 10 mM caused only A t’uofold change in the amount of calcium bound by myosin demonstrating a high specificity of thcb binding site for calciuni .
As indicated above (see Table 3), gizzard myosin 8-l produced by papain digestion forms a calcium-insensitive actomyosin when combined with rabbit F-actin. In agreement with the data of Kendrick-dories (1973). we find that this S-l lacks tho L,,, light chain (Fig. 6 (e)). To establish if there was a more specific correlation between the degradation of this light chain and the loss of calcium-sensitivity, we assayed the digestion by papain of gizzard actomyosin for different enzyme-ho-prohein ratios. The results are shown in Tahlt~ 5 and Figure 8 (a) t’o (d). As shown in Figure 8(d): papain causes ext’rnsivt:
EDTA EGTA Calcium Calcium-sensitivity a Expressed in b After 25 min c After 30 min fl Expressed in e Subjected to
TrypsinC
Papainb
Enzyme Enzyme to MgAM ratioa
Conhole
16
80
400
31.5 45.9 134 0.66
31.3 62.6 150 0.58
29.6 63.7 157 0.59
37.4 95.4 195 0.51
nmol . mg protein - 1 . min -- I. digestion. digestion. ng mg - I. iodoacetic acid as for papain digests.
( ‘ontrol
8
21.1 20.0 16.6 19.0 122 160 0.86 0.88
40
?o(J
21.6 14.6 40.7 0.36
33.4 22.3 23,3 0.04
REGULATIOX
IN
SMOOTH
MUSC’LE
ti;
degradation of the myosin heavy chain without noticeably affecting the light chaitl components. Further, as long as the L,, myosin light chain is present! the preparations st,ill exhibit calcium-sensitivity. Unfortunately, for the concentration of papain required to split myosin fully to S-l this light chain is degraded and. as alread! indic;ltrd. t,he calcium-regulatory property is lost. While not conclusive. these finding+ arc consistent’ \vith the L,, light chain being necessary for calcium-sensitivity. Rit)h trypsin the digestion of actomyosin follow-s a mnrked1.v different courw (Fig. 8(r) to (h)): the light chains being affected much more rapidl- than the heav>chain components. The degradation of the L,, light chain in t’his case is associatctl I\ ith t,hr complete loss of actin-activated ATPase activity (Table 5). AMorc direct evidence for the involvement of the L,, light chain in the calciumregulat’or- process has come from the demonstration (Sobieszek. unpublished results) that R phosphorylation of this light chain is triggered by Dhe same Ca2 + concentrat8ioll wquiwd to activat,e the BTPast activity of actomyosin (see Discussion).
4. Discussion (a) General Whiltb the numerous physiological and pharmacological investigat,ions of vertebrate smooth muscle (see reviews by Somlyo & Somlyo. 1968,197O; Biilbring et al., 1970; Phil. Traru. Roy. Xoc. ser. B. (1973). vol. 265, pp. 1-231) attest to its importance in the performance and control of diverse physiological funct’ions rat,her little has been established, in comparison to skeletal muscle, either about the contractile process or its wyulation in this muscle type. In addition to furthering our understanding of IION smooth muscle operates, biochemical studies of this muscle type will clearly complement dat’a from other muscle systems about the fundamental process of cont,raction. Furthermore, the!- promise bo furnish significant information relating to the, operation of non-muscle a&omyosin-containing systems which have been shown to possess actomyosins and myosins t’hat exhibit’ similar properties t#o those of smoot’h muscle (Adelstein & Conti. 1974). (b) I~ertrhrute smooth muscle actomyosinz \Vts describe here a method! as modified from an earlier study (Sobieszek & Bremel, 19i5). for obtaining actomyosin from a’ vertebrat,e smooth muscle which exhibits a calcium-sensitivity and acbivit’y comparable to tha’t shown by the washed, parent myofihrils. The actin-activat,ed ATPase activities that we have obtained for gizzard actomvosin (0.25 prnol Pi/mg protein at’ 25°C and 0.47 PmoI Pi/mg protein at 37°C) now compare realistically wit’h those obtained for striated muscle myofibrils (Weber. 1969: Port,zehl rt al.. 1969) and a’ctom,vosin (Kenttrick-Jones it al., 1970; Murphy, 19i 1) and at physiological temperatures exceed t’llilt obtained for a molluscan smoot,h muscle I Hendrick-Jones et (1.1..1970). Wr have noted that the Ca2+ concentration required for activation is normall>, low,er for myofibrils than for actomyosin and we have attributed this difference bo the modifying effect of the high concentrations of Mg’ + used for actomyosin precipitation. However, while high Mg2 + concentrations clearly shift t.he activation curve for myofibrils the activation of P-&4M (Fig. 2). obtained wit,hout magnesimn precipitat,ion, also occurs in th(l same range of Ca2+ concent,rations as for MgAM. On t’his basis the shift in activation of a.ct’omyosin with respect to myofibrils ma?.
.
FIG. 8. Mild proteolytic digestion of turkey gizzard act,omyosin (MgAM) by papain ((a) ttr. (d)) and trypsin ((0) to (h)). The actomyosin-to-enzyme (papain and trypsin) ratios and the calcmmsensitivity of each actomyosin preparation are given in Table 5. Note the complete removal of the L,, light chain of myosin dhring trypsin digwtion (h) and its presence in the gels of the [‘apain digests.
REGULATION
IN
SMOOTH
TABLE
Calcium
binding
MUSCLE
6
by gizzard preparation-st M&L
b’M) 2.0
Preparation
0.1
Myosin MgAM Xyofibrils C&AM CaAM - TM Thin filaments F-actin Myosin rod:
5.1
3.1 ‘P.4 2.2
2.0 1.3
1.0
2.5 2.7 w07 0.06 II.6
1.3 1.3
10.0
04
t Expressed in nmol of calcium per mg of protein at HO to 90 pM of CaZ+ and at 3 different Mg= + concentrations of 0.1, 2.0 and IO.0 mM. For other condit’ions see Materials and Methods. $ At, pH about 6.
be simply due to the more compact state of actomyosin, produced either by precipitation or centrifugation, which gives rise to some limitation of diffusion during the ATPase assays. In all cases, however, the steepness of activation, reflecting the degree of co-operativity, is the same for both myofibrils and actomyosin, and we may presume from t,his that the fundamental properties of activation are retained by the actomyosin. The reduced actin-activated ATPase activity of actomyosin at high Mg2+ concentrations (Fig. 4) is probably due to the associated higher rate of precipitation of actomyosin during the assays, which could result in a steric limitation of the number of active sites. At high Mg2 + concentrations there is, in addition to a decrease in activation, a shift in the calcium-activation curve (Figs 3 and 4) and also a decrease in calcium-binding (Table 6). This would suggest that at high Mg2+-to-Ca2+ ratios. magnesium successfully competes with calcium for the calcium-binding site on myosin. This is compatible with the findings that in fresh Mg-actomyosin the ATPase activity is inhibited up to 5Oo/o by magnesium (EGTA, Table 1) relative to t,he activity in the absence of both magnesium and calcium (EDTA, Table l), and for Ca-actomyosin, which is calcium-insensitive at 1 mM free Mg2 + concentration, some calcium-sensitivity map he restored by reducing the free Mg2+ concenDration to 0.1 mM. (c) Calcium
regulatiolL
in smooth ~muscle
9s demonstrated in the case of molluscan muscles, the actin-activated ATPase activity of vertebrate smooth muscle myosin is sensitive to Ca2+ with F-actin alone, that is in the absence of any a.ctin-linked regulatory components. Since the combination of thin filaments or F-actin from smooth muscle with rabbit skeletal muscle myosin produces a calcium-insensit’ive actomyosin. we may conclude that, in vert’ebrate smooth muscle, regulation by calcium is a specific property of the myosin molecule. Consi&ent with this conclusion is the absence of any troponin-like proteins either in smooth muscle actomyosin or myofibrils (Sobieszek $ Bremel, 1975) or on t’he thin filaments (Fig. 6(i)). The finding of myosin-linked calcium-regulation in vrrt’ehrate smooth muscle indicates t’hat this type of system is more widespread than
!I0
x. SOHIERZEI< .-\SI) ,I. \‘. S~lAI,I,
f)rrviously su~~~osed and, in particular. not, restricted to invertebra&. At preserlt 21 correlation between muscle t,ypr. for example smooth or striated. and the type of‘ regulation system is not obvious and indeed. for molluscs, bot,h smooth and striat,ed muscles are regulated by- myosin. One inttrest,ing possibility raised bp these results on vertebrate smooth muscle is that the various non-muscle actornyosin-containitl~ syst’ems (see review by Huxley, 1973a; Adelstein & C’onti, 1974) including, for exa,mplc. slime mold. amoeba. blood plat’elets and fibroblasts may also be regulated by nryositr. This suggestion derives from the noted similarit,y between the myosins of thest systems and that of vertebrate smooth muscle. in light chain composition and ill solubility and assembly properties (Adelman & Taylor, 1969 : Sbramolvitz et (11.. 1973 ; Hinssen 8r D’Haese, 1974: Bdelst’ein $ Comi. 1974). Indeed. we might expect that in systems where the contract’ilc elements are continually assembled and disassembled according to need. a myosin system composed of fewer probein components would be a dist,inct’ advantage. In molluscan muscles it has been shown that one of the myosin light chains. specifically that released by EDTA, is intimately involved in the calcium-regulat.ory process. Thus, t.he removal of this light, chain result’ed in a desensitizat,ion of myosin. \\.hile its recombination with desensitised myosin caused a restoration of calciumsensitivity. The light chains of vertebrate smooth muscle myosin are not so readil! released and we have, as yet, been unable to define conditions under which any of the light chains can be reversibly removed. However, some other recent experiments (Sobieszek, unpublished results) have provided strong evidence for the involvement of at least one of the smooth muscle myosin light chains in the regulation process. Thus. it has been shown that the triggering by calcium of the actin-act,ivated ATPase of smoot,h muscle actomyosin is associated with a parallel phosphorylation. at the same Ca2 + concentration, of the 20,000 molecular weight (L2,,) light chain of myosin. From this and a demonstrated correla,tion between the calcium-sensitivity of act,omposin and the degree of phosphoryla,tion. it is clear that the phosphorylation of the L,, light chain is int’imately associated xvith calcium regulation. The concomitant loss of calcium-sensitivity with the digestion of the L,, light chain by papain or trypsin is further consistent with t,his conclusion. A phosphorylat8ion of a myosin light chain has been shown to occur in other systems. including striated muscle (Pires et al., 1974: Adelstein et al.. 1973) but., although not, overlooked (Adelst,ein & Conti, 1974), a link ljetween this and calcium-sensit,ivity was not’ demonstrated. How the L,, light chain and whether the L,, li g ht, chain of smooth muscle myosin operate in the regula.tion process has yet bo be defined. In scallop myosin t#here appea’m to be only one regulatory “EDT,A” light, chain per molecule and, on this basis. son~t’ with the same light chain. co-operativitp between the two heads. each interacting smooth muscle the has been postulated (Szenb-Gyorgyi et al.. 1973). .I n vertebrate available data, suggests that there a,rr at, least, two L,, light chains (Sobieszek & Bremel, 1975) and that two light chains per molecule become phosphorylated in t’he presence of calcium (Sobieszek, unpublished results). Since: in addition, t,he amount of calcium bound by myosin corresponds to about two calcium ions per molecule, it is not unlikely that the tn-o heads (S-l) of myosin are independently activated 1)) calcium. Presumably. this may occur via conformat’ional changes resulting from the itself being dependent phosphorylation of the L,, light, chain, the phosphorylation on calcium. As far as the L,, light chain is concerned, it would appear that this light, chain may be necessary for the hydrolytic activit!, of mvosin. An indication of this
REGULATION
IS
SMOOTH
MUSC!LE
9I
comes from the parallel loss of the L,, light chain (in addition to the L,, light chain) and of actin-activated ATPase act,ivity with trypsin digestion (Fig. 8 and Table 5). The role that tropomyosin plays in the operation of myosin-linked systems is unclear. In striated muscle it serves to propagate the calcium-induced changes on troponin in a co-operative manner along the thin fila.ment, (Bremel & Weber. 1972 : Bremel et u.Z., 1973). In the absence of troponin the same changes clearly cannot occur. I)utj nevertheless from X-ray diffraction evidence (Vibert et nl.. 1972 : Huxley. 1973h : Haselgrove. 1973) a similar conformational change. taken as a shift in the position of tropomyosin. occurs on activation of smooth muscle as it does in striat,ed muscle. That, tropomyosin is bound to nctin in smooth muscle is indicated 1)~ its parallel cbxt,raction and co-sedimentation with actin in the thin filament,s. As we hare shown (see also Sobieszek & Bremel, 1975). t,ropomyosin can be selectively removed from smooth muscle actomyosin by ammonium sulphate precipitation (2524, saturation) and, depending on the type of actomq-osin. A variable amount of’ actin-activated ATPase is lost (see Table 1). Since ammonium sulphatc may act. in general. to decrease the activit’y of total act’omyosin (i.t,. whc,n precipitating all components a.t 6001;~saturation: Sobieszek & Bremel. 1975) it would appear that t,his variability result)s more from differing susceptibilities of actomyosin (e.g. MgAM or C!aAM) to ammonium sulphate t’han t)o the loss of tropomyosin. This is more dircctl). indicat#ed hy the effect of tropomyosin removal on ci~lcium-insc,nsit’ive Ca-actomyosin (&AM). whirh rcxxults in a relat’ivclly minor loss of actin-act,ivnttLd .4TPase activit!, (Table I ). While these lat,ter results would t’end to suggest t’hat t’ropomyosin plays a, minor role in t,hr activation of ATPasr activity, the result’s from the rrcomhination experimentb rTahle 2) may be more rrlwant. These cxpcriments showed that the comhination of’ gizzard myosin with gizzard thin filnment,s. composed of actin and tropomyohill. was twice as active as that with P-actin prepared directly from the thin filaments. A1 similar r+l%ct~ \\.as found in the case of stria,ttld muscle from Lirrrulus (Lehman & Szent-(:yorgyi. 1972). The possihilitg thus exists. that tl'(JpOmyOSiti acts. as in striated muscle. t 0 “turn on” the t*hin filament in a co-operativca manner when act,ivatiotl t:l kes plwre. Indeed. the steepness of the activation curves that L\‘e hare obtained fol snioot8h muscle (Pigs 2 and 3) suggests a co-operative a&n-myosin intera,ction in smooth muscle similar to that’ in striat’ed musclr (Bremel et ccl., 1973) and. on this I,asir. I\ t’ might, ttxpect this co-operat’ivity to he due t,o the presence of tropomyosin on thus thin filamt~nts. We should. however. not exclude the possibility that, myosin alone II~;I~ he thcl component responsible for the c.o-opcrativity seen in the actinmyosiu interaction. This possibility is suggesttad 1,~ the observation that the steepness of the ~.aIciutii-act’ivat’ion curve’ for ni.yosin phosphorylation is the same as for a&omyosin ;\TPase activity and. furthermorr. calcium is needed only for myosin phosphorylation and not for its lrydrolyt’ic actiritj(Sobicszek, unpublished results). Studieh art in progress to cla.rify the specific roles of’ calcium and myosin phosphory. lation in the regula,tion of the ac*tirit’- of rcrtebratc, smooth musclt). Thlc work represents an outgrowt,h of investigations I)egun in collaboration w4th Dr 12. D. Hremel at the Department of Anatomy, l)uke Universit,y Medical Center. \Ve thank Dr Brelnel for stimulating discussions and are indebt,ed to Dr M. K. Reedy for I& support during the early stages of this work (National Institutes of Health grant 1 ROlAM14317). We also thank Professor K. Marcker for his positive at,titude in making worn n\~ailahlo ill t Ile Mnlecular Bio1og.y Inntitlltcx. This \wrk \~as drnle (luring the tenure
92
A. SOHIESZEK
dND
J. V. SMALL
of a research fellowship to one of the authors (A.S.) from the Muscular Dy&rophy .Aewciu. tion Inc. and was supported by grants from the Volkswagen Foundation and the Muscular Dystrophy Association Inc. We thank MS Tove Wiegers for excellent, technical assistanve and Miss Aase Sorensen for t,ypinp. REFERENCES Abramowitz, J., Malik, M. N., Stracher, A. & Detwiler, T. C. (1973). Gold &w&q Harbor Symp. Quant. Biol. 37, 595-598. Adelman, M. R. $ Taylor, E. W. (1969). Biochemistry, 8, 4976-4988. Adelstein, R. S. & Conti, M. A. (1974). In Exploratory Concepts in Muscular lly&oph,?y (Milhorat, A. T., ed.), vol. 2, pp. 70-78, Excerpta Medica, Amsterdam. Adelstein, R. S., Conti, M. A. & Anderson, W. (1973). Proc. Xat. A4carl. Sci., U.S.A. 79, 3115-3119. Bremel, R’. D. (1974). Nature (Londo?~), 252, 405. 407. Bremel, R. D. & Weber, A. (1972). Nature New Biol. 238, 97-101. Bremel, R. D., Murray, .l. M. & Weber, A. (1973). Cold Spring Harbor Symp. Qua&. Bill. 37, 267-275. Biilbring, E., Brading. A. F., Jones, A. VV. & Tomita, T. (1970). Editors ofSmooth. -1fu,scle, Arnold, London. Driska,, S. & Hartshorne, D. J. (1975). Arch. Biochem. Biophya. 167, 203-212. Ebashi, S. & Endo, M. (1968). Prog. Biophys. Mol. Biol. 18, 123-183. Etlinger, J. D. & Fischman, D. A. (1973). Cold Spring Harbor Symp. Quant. Biol. 37, 51 l-522. Fairbanks, G., Stock, T. L. & Wallach, D. F.-H. (1971). Biochemistry, 10, 2606-2617. File, R,. S., Bohr, D. F. & Riiegg, J. c’. (1965). Science, 147, 1581-1583. Fiske, C. H. & SubbaRow, Y. (1926). J. Biol. Chem. 66, 375-400. Haselgrove, J. C. (1973). Cold Spring Harbor Symp. Quunt. Biol. 37, 341-352. Hinssen, H. & D’Haese, J. (1974). J. Cell Sci. 15, 113-129. Huxley, H. E. (1973a). Nature (London), 243, 445-449. Huxley, H. E. (1973b). Cold Spring Harbor Symp. Quant. Biol 37. 361-376. Kendrick-Jones, J. (1973). Phil. Trans. Roy. Sot. ser. B. 265, 183-189. Kendrick-Jones, J., Lehman, W. & Szent-GyGrgyi, A. G. (1970). J. MoZ. Biol. 54,313-326. Kendrick-Jones, ,J., SzentkirBlyi, E. M. & Szent-GyBrgyi, A. G. (1973). Cold Spring Harbor Symp. Quark Biob. 37, 47-53. Lehman, W. & Szent-Gyijrgyi, A. G. (1972). J. G’erc. Physiol. 59, 375-387. A. G. (1973). Cold h’pring Harbor Lehman, W., Kendrick-Jones, J. & Hzmt-Gyargyi, Symp. Qua&. Biol. 37, 319-330. Margossian, S. S. & Lowoy, S. (1973). J. ,IloZ. Biol. 74, 313-330. Murphy, R. A. (1971). Amer. J. Physiol. 220, 1494&1500. Needham, D. M. & Shoenberg, C. F. (1968). In Handbook of Physiology (Code, Ch. I”‘., ed.), vol. 4, sect. 6, pp. 1793-1810, Amer. Physiol. Sot., Washington. Ogawa, Y. (1968). J. Biochem. (Tokyo), 64, 255-257. Pires, E., Perry, S. V. & Thomas, M. A. W. (1974). FEBS I,etters, 41, 292-296. Portzehl, H.? Zaoralek, P. & Gaudin, J. (1969). Biochim. Biophys. Acta, 189, 440-448. Sobieszek, A. & Bremel, R. D. (1975). Eur. ,7. Biochem. 55, 49-60. Sparrow, M. P., Maxwell, I,. C.. Riiegp. .J. C. dz Bohr, D. F. (1970). Amer. J. Phyviol. 219, 1366-1372. Spudi& J. A. & Watts, S. (1971). J. Biol. Chem. 246, 4866--4871. Somlyo, A. P. & Somlyo, A. V. (1968). Pharmacol. Rev. 20, 197-272. Romlyo, A. P. & Somlyo. A. V. (1970). Ph,armacoZ. Rev. 22, 249-353. E. M. B Kenrlrick-Jones, .J. (1973). J. Mol. Biol. 74, Rzent-Gyijrgyi, A. G., SzentkirBlyi, 179-203. Vibert, P. J., Haselgrove, J. C., Lowy, J. d Poulsen, F. Ii. (1972). J. Mol. HioZ. 71, 757-767. Weber, A. (1969). J. Gen. Physiol. 53, 781--791. Weber, A. & Herz, R. (1963). .7. BioZ. Chem. 238, 599-605. Weber, A. & Murray, J. M. (1973). Physiol. Rev. 53, 612-673. Weber, .4., Herz, R. 8: Reiss, I. (1969). Biochemistry, 8, 2266-2270. Wnher, K. & Oshorn, M. (1969). .I. Biol. Chem. 244, 4406-4412.