THE JOT:RN.\L OF Ilro~oo~rar. CH~M~TRY VC>l. 246, No. 15, Issue of August 10, pp. 1866~‘ail, 1971

Printed in C’.S.it.

The Regulation of Rabbit Skeletal Muscle Contraction

1. 131OCHEMICAI, STUDIES OF THE INTERACTION OF THE TROPORIYOSIn’-TROPOTI~ COMPLEX WITH ACTIS Ai2TD THE PROTEOLYTlC l?RAGMEKTS OF MYOSLN

(Received for publication, March 19, 1971)

JAMES h. SPUDICH* AXD Susm WATT

From the Aleclical Research Council Laboratory of Molecular Riology, Cambridge, England

SUMMARY

Actin purified by a new, simple, and rapid puriIication procedure activated the ATPase activity of both heavy meromyosin and Subfragment 1 of heavy meromyosin, and this activation was not inhibited by the removal of Cazf. Preparations of tropomyosin-troponin inhibited (by 85 %) both the acto-heavy meromyosin and acto-Subfragment 1 ATPases in the absence of, but not in the presence of, Ca2+. This inhibition was shown to result from binding of the tropomyosin-troponin complex solely to actin and in a ratio of about 1 mole of tropomyosin-troponin to 7 moles of actin.

The contraction of striated muscle involves the interaction of two types of filameuts, thick filaments consisting mainly of myosin and thin filaments consisting primarily of actin. Upon stimulus by :I nerve impulse, Ca zf is released from the sarco- plasmic reticulum and diffuses into the myofibrils, initiating a relative sliding of the my&u andactin filaments past one another, which results in contraction. The Ca2+ receptor within the myofibril is believed to be a protein complex called troponin, which has recently been described by Ebashi and his co-workers (see review by Ebashi and Endo (I)). Troponin, together with the prot,ein tropomyosin, is believed to be the regulatory system for muscle contract,ion. Since ‘I’M’ binds to actin (2) and TP binds to TM (3-5), and since both TM and TP have been shown t,o be associated with the thin filaments in muscle (B-S), the

* Present address, Department of Biochemistry and Biophysics, TJniversity of California, San Francisco, California 94122. Holder of a United States Air Force Office of Scientific Research fellow- ship (during the first part of this work) and a National Science Foundation postdoctoral fellowship during the remainder of the time.

1 The abbreviations used are: TM, tropomyosin dimer (70,000 dnltons); .TP, troponin (a complex of at least two proteins); TM.TP, the complex of tropomyosin and troponin believed to be the regulatory unit for Ca 2+-dependent muscle contraction; HMM, “heavy meromyosin” (product of limited trypsin digestion of myosin); Sl, Subfragment 1 of heavy meromyosin (product of limit,ed papain digestiou of myosin); EGTA, ethylene glycol his-@-aminoethyl ether)-N,N’-tet,raacetic acid (a chelator which hzs a much higher affinity for Ca2+ than for Mg2+); DTT, di- thiothreitol; SDS, sodium dodecyl sulfate.

regulatory complex is believed to be part of those thin filaments. The possibility has been considered, however, that TP inhibits contraction by binding to myosin (9, 10). In particular, Kendrick-Jones, Lehman, and Szent-GyGrgyi (10) have suggested from their recent investigations of the regulation of molluscan muscle contraction that in rabbit striated muscle TP might reside on the actin filament during contraction and inhibit contraction in the absence of Ca2+ by association with myosin. This pos- sibility had not been excluded since most inhibition studies have involved an actomyosin complex rather than the separated and purified actin and myosin proteins. Furthermore, even though much more TM .TP binds to actin than to myosin in the presence of Ca*+ (II), the amount of binding of ‘TP to myosin in the absence of Ca2+ has not been measured.

Because of the now obvious importance of the T;\‘I.TP system, we were interested in comparing the structures of pure actin and the actin-TM . TP complex, and our results will be described in a subsequent paper. However, it was first, necessary to establish biochemically (a) that our actin was indeed homogeneous, (b) whether TM’TP inhibits contraction by binding to actin or myosin or both, and (c) what were the best conditions for forma- tion of an actin-TM.TP complex which most closely resembles the in vivo system for study by electron microscopy. Investiga- tion of these three points led us to discover a new, simple purifica- tion procedure for actin which routinely results in homogeneous preparations.

In addition, we have carried out comparative studies with Sl (a proteolytic fragment of myosin which is believed to have one ATPase site and one a&in-binding site (12-14)) and HMM (a proteolytic fragment of myosin which consists of two Sl units joined by part of the rod-like portion of myosin (14-16)). It was of particular interest to discover whether the acto-Sl ATPase is inhibited by TM .TP (without Ca2f) despite the absence of the “two-headed” form of the myosin molecule. In the course of preparation of this manuscript, Eisenberg and Kielley reported that the acto-Sl ATPase is indeed inhibited by TM.TP (17). This report confirms their findings and in addition shows that under the conditions used the kinetics of inhibition is the same as that for HMM.

MATERIALS AND METHODS

Pur$cation of Act&-An acetone powder (10 g) (18) of rabbit back and leg muscle was extracted at 0” (extraction at 0” was found to minimize, but not totally eliminate, extraction of

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TX~TI’, as previously described by Drabikowski and Gergely (19)) for 30 min with 200 ml of Ruffer A (2 mM Tris-Cl, 0.2 mM ATI’, 0.5 111~ fi-mercapt’oethanol, and 0.2 InM Call,; final pH = 8.0 at’ 25’) and filtered through a coarse sintered glass funnel. The residue was washed with 100 ml of Buffer A and filtered, and the supernat~ant fluids were combined and cleared by cen- trif’ugatiou at 10,000 X g for 1 hour (crude extract; about 400 mg of protein). KC1 (to 50 mtif) and ;\IgCln (to 2 mM) were added and the actin was allowed to polymerize for 2 hours. KC1 was then added to 0.6 M (this being the crucial step in the procedure) ; the sohition was stirred gently for 1.5 hours and then \vas cen- trifuged at, 80,000 x g for 3 hours. The pellet was resuspended well in 30 ml of Buffer A and dialyzed with vigorous stirring for 3 days, changing to fresh Buffer A every 24 hours. The G-a&n was clarified by cent,rifugation at 50,000 x g for 3 hours and polymerized in 50 mu KC1 (purified a∈ about 203 mg of protein). dlthough t’hc actin was usually used within 1 week, it was sometimes stored at 4” (with a cryst,a’ of thymol to prevent bacterial growth) for up to 3 weeks without a decrease in the specific activity of the actinactivated HMM ATPase.

IIh/IM equals 0.05 kmole of Pi per min per mg of HMM). The rate increased to 3.3.units per nmole of HM,\I per nmole of actin by lowering the concentrations of KC1 (to 3 mM) and ATP (to 2.5 IIlM). With 50 mM Tris-Cl, 5 m&f 1’2gCl2, 15 InM KCl, and 2.5 rnM ATP, the rate was 0.33 unit per ~nnolr of HMM per nmole of actin.

,F-reparation of BJIIll and SI-HMM and Sl, prepared by digestion of myosin with trypsin and papain, respect’ively (14), werr a generous gift of Dr. A. Weeds. Unless otherwise noted, both were dialyzed overnight a,gainst 5 mM Tris-Cl (pH 7.9), 0.5 m&f EGTA, 1 m&f DTT and stored at -20” in 50% glycerol. hlolecular weights of 340,000 for HMM and 115,000 for Sl (14) were used for the calculations made in the experiments described below.

Preparation of Tropomyosin-Troponin-The TM ‘TP complex was prepared from the residue (stored at -20”) remaining after extraction of t,he actin from the acetone powder at 0”. The procedure was based on those reported by Ebashi and Ebashi (21), Watana,be and Staprans (22), and Hartshorne and Mueller (23). The residue from 10 g of acetone powder was extracted with 170 ml of 10 mM Tris-Cl (pH 8.3)) 0.5 IllM P-mercaptoethar~ol for 3 hours at 25” and sedimented at. 16,000 x g for 1 hour (super- natant fluid: crude TM .TP, 17’7 ml). Ammonium sulfate (35.4 g) was added to the crude T1I.W wit’h stirring at 4” for 20 min. After centrifugation at 16,000 x g for 30 mm, 54 g of ammonium sulfate lvere added to the supernatant fluid (180 ml) at 4”. The precipitate was sedimented at’ 16,000 x g for 30 min, dissolved in 20 ml of the Tris-mercaptoethanol buffer, dialyzed against a total of 2 liters of t’he same buffer (three changes assured thorough dilution of the ammonium sulfate), and cleared by centrifugation at 80,000 x g for 2 hours. This ammonium sulfate fract’ion (359; of l’he protein in t’he crude TM ‘TP extract) represented about :I 3-fold purification over the

The specific activit’y of t,he HbIM or acto-HMM ATPase in the absence of TM. TP was independent (&50/,) of the presence of 0.2 1llM Cazf for all of the IIXM preparations used. Under the conditions of our assay, however, some Sl preparations, but not all, were significantly inhibited (more than 30%) by 0.2 mar Ca”+ when assayed in the presence of purified actin, but not’ when assayed alone. This inhibit,ion was observed wit.h Sl even when HLIX prepared from the same myosin preparation was not inhibited when assayed with t’hc same actin preparation. To avoid t,his complication, most, of the T%. TP inhibition studies described below were carried out wit,11 IIMM.

Assay for Acfin-activated Jfyosin ddenosine Triphosphatase- The rate of hydrolysis of adenosine triphosphate (ATP), cata- lyzed by the rnyosin fragments HMM or Sl, was assayed as follows. The reaction mixture (final volume, 2 ml) cont.ained 12.5 1nhf Tris-Cl (pII 7.9), 2.5 maf MgCL, 5 mM KCl, 0.5 rnnl EGTA (for assays in the presence of Ca*+, EGTA was replaced wit)h 0.2 mM CaC&), actin, TX.TP, and HMh/I or Sl, in that order. The reaction was initiated by the addition of ATP to 3.8 mM. Incubation was at 25” and usually for 15 min, depend- ing on t,he level of enzyme used, before trichloroacetic acid (1 ml of 15%) was added to stop the reaction. After centrifugation for 2 min at 2000 x g, the amount of inorganic orthophosphate in the supernatant was determined according to Fiske and Subba- Row (20). One unit of ATPase is equivalent to the forrnation of 1 pmole of Pi per hour. It should be emphasized that, when the actin-act’ivated ATPsse activity is reported (Figs. 2 and 4 to 8 and Table I), the background act,ivity due to HMM or Sl alone has been subtracted (with the except,ion of Table II).

The initial rate of ATP splitting under the standard assay conditions shown above varied frorn 0.6 to 1.0 unit per nmole of

FIG. 1. SDS-polyrtcrylamide gels of purified actin, TM.‘PI’, TM. and TP. The proteins were purified as described 11nde1 “Materials and Methcds.” Actin (31 pg), TRI,TP (15 pg), TM (12 fig), and TP (12 rg) were applied to 109; polyacr~lamide gels

HMM per nmole of aclin (1 pmole of Pi per hour per nmole of containing 0.2sj; SDS and were electrophorexed :~t 3 ma per tube, as described under ‘Materials and Methods.”

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4568 Regulation of Muscle Contraction Vol. 246, No. 15

TABLE I Requirement for TM and TP fov inh,ihition of acto-HMM

ATPase in absence of Cazi The protein preparations and assay of the acto-HMM ATPase

activity were as described under “Materials and Methods.”

Specific activity of ATPase

Components for ATPase assay Inhibition”

With C&L+ wi%“’

zrnits/nmole HAM /n??&ole actin %

1. Complete systemb . . . . . . 0.97 0.29 70 2. RIinusTP..................... 0.71 0.63 11 3. Minus TM. . . . . . . . 0.61 0.55 10 4. Minus TP, minus TM. . . . . 0.66 0.70 -6

a (1 - without Ca”/with Ca”) X 100. 6 The complete system (see “Materials and Methods”) con-

tained 0.60 mg of HMM, 0.30 mg of actin, and 0.15 mg each of TM and TP in 2 ml of reaction mixture. For Rows 2 and 3, 0.30 mg of TM and 0.30 mg of TP were used, respectively.

H 0.6M KCL WASH)

30 40 50 60

Tube Number

FIG. 2. Sephadex filtration of actin. Actin (40 mg), prepared as described under “Materials and Methods” but without the 0.6 M KC1 purification step (O-O), was filtered through a Pephadex G-200 column (2.5 cm diameter X 80 cm) with Buffer A (see “Materials and Methods”) at a flow rate of about 10 ml per hour (6 ml per tube). Aliquots from various tubes were assayed for their ability to activate HMM ATPase. Sephadex filtration of actin purified with the 0.6 hf ICC1 purification step is shown (a----0) for comparison. Dextran blue (A& and DNP-lysine (3& were added as indicators of column void volume and column bed volume, respectively.

crude TlM . TP as judged by the amount required to inhibit com- pletely the acto-HMM ATPase and, unless otherwise specified, was used as the TM. TP preparation in the experiments described below.

Isolation of Tropomyosin and Troponin-TM and TP were isolated according to Hartshorne and Mueller (23). TP was isolated as the supernatant fluid following pH 4.6 fractionation of the TM .TP preparation. The precipitate was dissolved in 2 mM Tris-Cl (pH 7.9), 1 mM DTT, and 0.2 M KC1 (1 mg of protein per ml), residual TM. TP was precipitated by addition

molarity HMM or S1(x106)

FIG. 3. Relationship bet.ween ATPase activity (without actin) and concentration of HMM and Sl. HMM- and Sl-ATPase activities were measured by the standard conditions (with EGTA) described under “Materials and Methods.” For some of the Sl-ATPase det,erminations (@----e), Sl was used which was prepared by digestion of myosin with a soluble papain prepara- tion; its molecular weight was assumed to be the same as that obtained by digestion of myosin with an insoluble papain prepara- tion (see “Materials and Methods”).

of 31.4 g of ammonium sulfate to 100 ml and was sedimented by centrifugation, and TXI was isolated as a gelatinous precipitate formed upon adding another 7.7 g of ammonium sulfate to the supernatant fluid. The TM and TP preparations were then dialyzed thoroughly against 2 mM Tris-Cl (pH 7.9), 1 mM DTT; yields of TM and TP varied from 17 to 29% and 26 to 51%, respectively.

Polyacrylamide Gel Electrophoresis-Protein preparations were electrophoresed on 1O7o polyacrylamide gels containing 0.2% SDS, as described by Laemmli (24). The samples were heated at 100” for 1 min in the presence of 2yo SDS and P-mercaptoetha- no1 and then applied to the gels. When lower concentrations of SDS were used the TM was not totally disaggregated, as judged by its mobility on the gels.

The SDS gels were calibrated for molecular weight determina- tions (25) with P-galactosidase, bovine serum albumin, oval- bumin, chymotrypsinogen, and tobacco mosaic virus protein as standards.

Other &lethods-Protein concentration was determined by the methods of Lowry et al. (26). Unless otherwise noted, all manip- ulations with the various protein preparations were carried out at O-4”. EGTA was kept as a 10 mM solution adjusted to pH 8 with NaOH.

RESULTS

Purijication of d&in-Actin purified as described under “Ma- terials and Methods” was homogeneous as judged by elec- trophoresis on SDS-polyacrylamide gels (Fig. 1). Its molecular weight calculated from its relative mobility (see “Materials and Methods”) was 48,000 daltons. The purified actin showed a requirement for both TM and TP for maximal inhibition of the acto-HMM ATPase in the absence of Ca2+ (Table I; the low levels of inhibition by TM or TP alone probably reflect con-

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II

J 5

molarity HMM or Sl (XI@)

Fro. 4. ltelationship bet,ween actin-activated ATPase activity alld concentration of HMM and Sl. Acto-HMM and acto-Sl ATPase activities were measured by the standard conditions (with EGTA) described under “Materlab and Methods.” Points with a line through them (e.g. 0) refer to determinat,ions using Sl, those without (e.g. 0) refer to determinations using HMM. d and 6 4.4 PM actin; 0, 2.2 p~ actin; A and 8, 1.1 PM actin; A and f 0.55 tiu~ act in. 8, Sl prepared by digestion of myosin wit 11 a soluble papaillprepxrai ion (see legend to Fig. 3.)

t.amination of one by the other (see Fig. 1)). In the absence of

added TM and TP, none of the purified actin preparations were dependent on Ca2+ for activation of HMM-ATPase; with actin prepared without the 0.6 M KC1 purification step, however, removal of Ca2f resulted in inhibition of the a&o-HMM ATPase varying from 10% t)o 60%.

In agreement with Rees and Young (27) Sephadex filtration of actin prepared by the common procedure which involves repeated cycles of depolymerization and repolymerization in 0.05 1\1 KC1 revealed at least 25yG impurities (Fig. 2). Although the actin purified by the Sephadex fractionation described in Fig. 2 showed a single band on SDS-polyacrylamide gels, con- taminating proteins were sometimes revealed on gels when more than 75 mg of protein were applied to the Sephades column. hctin purified by the 0.6 M KC1 wash described under “Materials and Methods” showed very little, if any, contamination by other prot.ein (Fig. 2).

Actin Activation of ATPase Activity of 81 and HMM-In the absence of actin, the ATPase activity per mole of HMM was twice the activity per mole of Sl (Fig. 3). This is consistent with each HMM consist,ing of two Sl units held together by a “tail” (141.

With an actin couceutration ranging from 1.1 to 4.4 PM, the actin-activated .4Tl’ase activity of HMM was also twice that of Sl up to about 1.6 PM HMM (Fig. 4), showing that, under the conditions used, the Sl units joined together in the form of HMM are activated by actin to the same extent as the independent Sl moieties isolated by papain digestion.

The rate of ATE’ hydrolysis per mole of HMM began to de- crease at about 2 ELM HMM, whereas no such decline was ob- served with Sl.

Under the conditions of assay, the activation of HMM by purified actin was independent of Ca2+ (Table II, Row 2). Sim- ilarly, Ca2+ was not necessary for maximal activity of acto-Sl; indeed, the actin-activated ATPase activity of some Sl prepara-

TABLE 11

Inhibition of ado-HMM ATPase by TM. TP in

absence of Ca2+

The protein preparations and assay of ATPase activity were as described under “Materials and Methods.”

Total ATPase activity” Components for ATPase assay l-

1. Complete systemb. . . . . . . 2. Minus TM. TP . . . . . . . 3. Minus a&in.................. 4. Minus actin, minus TM.TP.. 5. Minus HMM.. . . . . . . 6. Minus HMM, minus actin.. 7. Minus HMM, minus TM.TP 8. Minus HMM, minus actin,

TM.TP.. . .

6.7 1.7 :I 5.2 4.9 ...... 1.1 0.9 ...... 0.9 0.9 ...... 0.0 0.0 ...... 0.0 0.0 . . . . . 0.0 0.0 minus

0.0 0.0 -

With Caz+ Without Caz+

5 ATPase activity of HMM alone was not, subtracted from acto-HMM activities.

b The complete system (see “Materials and Methods”) con- tained 0.54 mg of HMM, 0.11 mg of actin, and 0.094 mg of TM.TP

p?tZOleS Pi/h

in 2 ml of reaction mixture.

18

/

0

16-

14 -

.G

5 12- a, 2 g lo-

> $ 8-

with TM.TP prep.

molarity HMM (~10~)

FIG. 5. Activation of acto-HMM ATPase in the presence of Ca2f by a TM.TP preparation. Acto-HMM ATPase activity was measured by the standard conditions (with Cat+) described under “Materials and Methods.” Actin was used at a concentra- tion of 0.61 PM for all determinations. 0, with equal weights of a TM.TP preparation and actin; l , without TM.TP.

tions was inhibited by Ca* (see “Materials and Methods”).

Thus, HMM was used in most of the studies described below. Inhibition of Acto-HMiif ATPase by TM. TP in Absence of

C&-Addition of the TM .TP preparation made the acto-HMM ATPase Ca2+-dependent (Table II, compare Rows 1 and 2), but had no significant effect on the activity of HMM alone (compare Rows 3 and 4). After subtraction of the ATPase activity of HMM alone from that of acto-HMM, the inhibition by TM.TP is 86% (Row 1, compare with and without Caz+). Without

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‘:K ( , ; ;,: f!b

000 0.02 004 0.06 008 0.10 0.12 TM .TP (mg)

FIG. 6. Titration of actin with TM.TP. Percentage inhibition of ATPase was determined by comparing the rate (R) of ATP hydrolysis using TM.TP-scto-HMM in the presence and absence of Ca2+

[(I - (~+TM.TP,-C~~+/R+T~.TP,+~~~+)) X 1001 .

TM.TP (mg)

FIG. 7. Kinetics of inhibition of acto-HMM ATPase by TM.TP. fl and @, percentage inhibition of ATPase was determined by comparing the rate (R) of ATP hydrolysis with TM.TP-acto- HMM in the presence and absence of Ca*-‘-

[(l - (R+TIII.TP.-C;,2+IR+,~~.TPI+Cs2I)) x 1001.

0, 0, and 0, percentage inhibition of ATPase was determined by comparing the rate (R) of ATP hydrolysis without Ca2+ in the presence and absence of TM’TP

[(I - (R+TM.TP,-Cn2+/n-T~.TP.-Ca2+)) x 1001.

Actin was used at a concentration of 0.4 /IM for all determinations. Cl and jZ, 0.4 PM HMM; l ,1.6 PM HMM; 0 and @, 2.5 PM HMM.

HMM, the TM..TP and actin had no effect on the rate of ATP hydrolysis (compare Rows 5 to 7 with Row 8).

In t,he presence of Ca zf, the TM .TP preparation activated the acto-HMM ATPase (compare Rows 1 and 2). The activation of HI\111 alone, if significant, was less t,han that of acto-HMM (see Rows 3 and 4).

30 IM.TPlmgl

FIG. 8. Kinetics of inhibition of acto-HMM ATPase and acto- Sl ATPase bv TM.TP. Percentage inhibition of ATPase was determined by comparing the rate-(R) of ATP hydrolysis with Ca2+ in the presence and absence of TM.TP

[(I - (R+TM.TP,--C~+IR-TM.TP.-C~~+)) X 1001 Actin was used at a concentration of 4.2 !aM for all determinations.

Activation of Acto-HMM ATPase by TM. TP Preparation in Presence of Cazi--As noted above, the TN. TP preparation (i.e. the ammonium sulfate fraction prepared as described under “Materials and Methods”) activates the acto-HMM ATPase in the presence of Ca*+. The activation at various concentrations of HMM is shown in Fig. 5.

The specific activity of the acto-HM1\I alone was 0.6 pmoles of Pi per hour per nmole of actin per nmole of HMM up to about 1.5 quiz HNM (0.61 par actin used) and then began to decrease with increasing concentration of I-INN. This compares well w-ith the rate in the absence of Ca2+ (see Fig. 4). As the con- ccntration of I-DIM was increased further, the ATPase activity (expressed as micromoles of Pi per hour per nmole of actin) steadily increased, but at a slower rate than that at low HMM concentrations, resulting in a biphasic curve (Fig. 5). The first slope is about twice the second slope, and the latter is equal to that given by acto-Sl (compare with Fig. 4).

Very different kinetics was obtained when the TM.TP prepara- tion was present. The rate of ATP hydrolysis increased with increasing I-In/I&I concentration up to 8 PM HLTM. When less than 1.5 FM H;\IX was used, the ATPase activity in the presence of TM .TP was 40% higher than that with acto-HMM alone, but the percentage activation by the TM .TP preparation in- creased with increasing HMM concentration, reaching a value of 2oo7O at 8 pM HMM.

The extent of activation varied from one TM .TP preparation to another. Some TM.TP preparations did not activate t’he acto-HMM (+Caz+) ATPase at all while they inhibited normally in the absence of CB~+.~ Thus, it seems likely that this activation

2 In two experiments the TM.TP was bound to actin, sedi- mented out of 0.4 M KCl, removed from the actin in 0.8 M KCl, and dialyzed to remove the salt,. The resultant TM.TP prepara- tion inhibited the acto-HMM ATPase 85% (0.6 mg of TM.TP per mg of actin) in the absence of Caz+. This inhibition was completely relieved by the addition of Ca2+, and no activat,ion (~57,) was observed.

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is due to an unknown component contaminating the ‘l’M.TP preparations, but further work is necessary to clarify this point.

Titration of Actin with TX. TP-The amount of TX’TP required for maximum inhibition of the acto-HMM ATPase was linearly related to the coucentrat,ion of actin and completely independent of IINM concentration eren when HMll was in $-fold molar excess over actin (Fig. 6). This shows that the TM.TP exerts its eflect by a binding solely to actill and that relatively large amounts of IIMM do not compete with this binding. With this TBI.Tl preparation, 0.66 mg of Th!l.TP was required for masimum inhibition of 1 mg of actin. This ratio probably represents a high estimate due to impurities preseut in the TM. TP preparation (see below).

It is important to note t,hat in Fig. 6 percentage inhibition was determined by comparing the activity of the TN ‘TP-acto-IINM ATP:lse with and without CaPf. I f the activity of the acto- HMM ATPase (without, Ca*+) was compared to that of the T_\I “I’P-acto-HMM (without Ca*+), then the kinetics of inhibi- tion by TBI.TP was somewhat dependent on HMM concentra- tiou (Fig. 7) as a result of the activation of acto-HXIM (with Cn2+) by the TM. TP preparation (see Fig. 5).

Inhibition of Acto-Sl ATPuse by TX. TP-&to-S1 was in- hibited by T,M. TP with the same kinetics and bo the same extent as acto-HNM (Fig. 8). Thus, the “two-headed” form of the myositl molecule is not required for the Ca2+-sensitive control of contraction by TM.TP.

With this TM .TP preparation (different from that used in Fig. 6), 0.47 mg of Tl\I .TP per mg of actin was required for maximum inhibition, a value similar to but somewhat lower than that obtained from the esperiment shown in Fig. 6. With other T_\I.TP preparations, as little as 0.40 mg of TM .TP per mg of actin was required for maximum inhibition (857;), higher ratios presumably reflecting impurities in t,he TM ‘Tl’ preparation.

DISCUSSION

-\ new purification procedure for actin has been described based on our early observatioiis that few, if any, proteins associate with actiu in 0.6 M KCI. This method is rapid and simple, and yields in a single preparation a larger quantity of homogeneous sctin than obtainable by conventional Sephadex filtration.

The observation that the amount of TM. TP needed for maxi- mum inhibition of the acto-HMM ATPase depends only on the actin concentration even when a ‘I-fold molar excess of HMM was used shows that TM .TP inhibits the ATPase in the absence of Cn*+ by binding to actin and not to HMX. Thus, it is the structure of the actin-TM TP complex which is of interest in trying to ascertain how the TM .TP exerts its regulatory effect.

The conditions used in this biochemical study served as a basis for those which we used in subsequent experiments in which we compared the structure of pure actin to that of the actin-TM.TP complex. A report of the conclusions of these structural studies is in preparation.

The above biochemical studies show that no more than 0.4

mg of Th% . TP per mg of actin is required for maximum inhibi- tion of the acto-i%MM or acto-Sl ATPase. This agrees well with binding ratios established by other investigators using other methods (11, 28). I f the TM.TP complex consists of 1 mole of the tropomyosin dimer (70,000 daltons (29, 30)) and 1 mole each of two troponin moieties (approximately 20,000 daltons and 40,000 daltons3), then t,he molecular weight of the complex would

3 The two major consistent components in our TP preparations

be about 130,000. Since the molecular weight of actin is about 48,000 (see above and Reference 27), a weight ratio of 0.4: 1.0 corresponds to a mole ratio of about one TM.TP complex for every seven G-actin monomers.

dcknowledgments--The authors wish to thank Dr. Hugh E. Huxley for many helpful discussions and support of this work and Dr. A. G. Weeds for his generous supply of most of the HMM and Sl preparations used.

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have molecular weights of about 40,000 and 20,000, as judged by SDS-polyacrylamide gel electrophoresis (see Fig. 1). Both of t,hese two components, TP I and TP II, respectively, are believed to be required, in combination with TM, for Ca2+-dependent regulation of actomyosin ATPase activity; the exact, stoichiom- etry of the TM.TP complex is uncertain, but is near 1 TP I: 1 to 2 TP II:1 ThI (S. Ebashi, T. Wakabayashi, and F. Ebashi, manu- script in preparation).

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James A. Spudich and Susan WattMYOSIN

COMPLEX WITH ACTIN AND THE PROTEOLYTIC FRAGMENTS OFSTUDIES OF THE INTERACTION OF THE TROPOMYOSIN-TROPONIN

The Regulation of Rabbit Skeletal Muscle Contraction: I. BIOCHEMICAL

1971, 246:4866-4871.J. Biol. Chem. 

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