Exchange of adenosine diphosphate bound to actin in superprecipitated actomyosin and contracted myofibrils

J. Mol. Biol. (1966) 15, 515-538

Exchange of Adenosine Diphosphate bound to Actin in Superprecipitated Actomyosin and Contracted Myofibrils ANDREW G. SZENT-GYORGYI AND GWEN PRIOR

Department of Oytology, Dartmouth. Medical School, Hanover, New Hampshire, U.S.A. (Received 23 July 1965, and in revised form 1 Novemher 1965) Up to half of the ADP bound to actin passes into the medium from superprecipitated actomyosin. The presence of ATP or other nucleotide triphosphates is necessary for release to take place. Superprecipitation is also a required condition. If superprecipitation of actomyosin is delayed or inhibited by the omission of magnesium in the presence of the various nucleotide triphosphates or by lowering the temperature, the release is also delayed or inhibited even though ATP or nucleotide triphosphates are present in the medium in great excess. The reaction is an exchange reaction and the actin-bound ADP is replaced by ADP formed from the ATP of the medium. In a system where the actomyosin contains [3H]ADP and the medium [14C]ATP, the loss of [3H]ADP from the actomyosin precipitate is compensated for by the incorporation of [14C]ADP throughout the whole course of the reaction. The same sites can undergo a reaction more than once and a major portion of the newly incorporated [14C]ADP reexchanges at about the same speed as the unreacted sites containing [3H]ADP. The change on actin leading to the availability of its bound ADP is a transitory one and the ADP newly incorporated into super-precipitated actomyosin cannot serve as cofactor for the creatine kinase-phosphocreatine system. Exchange takes place also on "natural" actomyosin and, though with a slower speed, on washed myofibrils. The exchange reaction with actomyosin starts with a rapid burst, then proceeds at a slower rate. The experiments are interpreted to mean that myosin and ATP in conditions of superprecipitation and contraction impose a change on actin which leads to a loosening of the bond between actin and its bound ADP. The evidence would indicate that repetitive, cyclic reactions occur at the sites of actin and myosin interaction. The rapidly exchanging fraction of ADP may represent the maximum number of actin and myosin sites in contact in an actomyosin precipitate. The slower exchange would be limited by the time required for the completion of the previous cycle and for the unreacted actin sites to come into close proximity with active sites on myosin.f

1. Introduction An essential feature of the sliding theory of contraction is that shortening is the result of repetitive cyclic changes at the sites of interaction between actin and myosin (Huxley, H. E., 1960). Thus contraction of muscle is more complex than contraction of polymers. Whereas in synthetic polymers a change in length is proportional to conformational changes, no such simple relationship is expected in the case of muscle.

t Part of this work was presented at a Symposium on Muscle, held at the University of Alberta in June 1964. Figures 1, 3, 5(a) and 7 appeared in the Proceedings of the symposium. Some of the results have been reported at the Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, 1965. 515

516

A. G. SZENT-GYORGYI AND G. PRIOR

The conformational change at any time may involve only a restricted small region of a few of the molecules making up the filaments. Most importantly, any such change must be of a transitory nature, and should occur a number of times before contraction reaches its maximum. The extent of shortening depends on the number of these cycles, and one does not expect a conformational difference between a slightly or extensively contracted muscle. Hanson & Huxley (1955) suggested several possible mechanisms of sliding in their original proposal. Some of these possibilities have since been elaborated in detail and may be summarized as follows: repetitive shortening or oscillation of the crossbridges which represent the linkage between actin and myosin (Huxley, A. F., 1957; Davies, 1963); a cyclic localized change in the length of one of the filaments, preferably actin (Asakura, Taniguchi & Oosawa, 1963a); interaction of electrostatic forces involving no conformational change (Spencer & Worthington, 1960). At present no direct evidence is available to show which if any of these possibilities is correct. Indeed, since the expected conformational change at any moment is small, and since contraction has been observed only in structured systems in which actomyosin is in the insoluble phase, a direct test faces rather formidable experimental difficulties. The properties of actin allow indirect approaches to check whether or not this protein is undergoing a conformational change in the contraction of actomyosin or myofibrils; and to determine to what extent such a change is a cyclic one. Actin can exist in a globular (G-actin) form and in a fibrous (F-actin) form (Straub, 1943). G-actin contains ATP whereas F-actin contains ADP as its bound nucleotide (Straub & Feuer, 1950; Laki & Clark, 1951; Szent-Gyorgyi, A. G., 1951; Mommaerts, 1952a). The ATP associated with G-actin may participate in enzymic reactions (Straub & Feuer, 1950; Laki, Bowen & Clark, 1950; Laki & Clark, 1951; Strohman, 1959) and exchanges with ATP in the medium (Martonosi, Gouvea & Gergely, 1960a). The ADP of F-actin is protected from enzymic attack (Strohman, 1959) and does not exchange with ATP or ADP of the medium (Martonosi et al., 1960a). Some of the SH groups in F-actin are also protected (Barany, 1956; Katz & Mommaerts, 1962). These differences between the two forms of actin can be utilized to test the proposition, originally advanced by A. Szent-Gyorgyi (1943) and entertained since by many others (Straub & Feuer, 1950; Mommaerts, 1951; Hanson & Huxley, 1955; Barany, 1959; Carlson & Siger, 1960; Podolsky, 1962; Asakura et al., 1963a) that depolymerization or some other type of altered conformation of actin is an essential part of contraction. This notion is supported to some extent by the observation that mechanical forces, such as sonic vibration, will lead to a rapid exchange of the actinbound ADP with ATP, accompanied by a dephosphorylation of ATP many times in excess of the stoichiometric nucleotide-actin ratio (Asakura, 1961a; Asakura et al., 1963b; Barany & Finkelman, 1963), a reaction interpreted as a conformational change converting actin from a helical into a linear polymer (Asakura et al., 1963a). The available evidence suggests that in washed myofibrils and also in conditions approximating the relaxed state, actin is present as F-actin. This evidence is based on the nature of bound nucleotides in muscle (Perry, 1952; Biro & Nagy, 1955; Biro & Miihlrad, 1960; Seraydarian, Mommaerts & Wallner, 1962), their resistance to creatine kinase action (Perry, 1954), SH reactivity (Barany, 1959), and electron microscopy (Hanson & Lowy, 1963; Huxley, H. E., 1963).

EXCHANGE OF ADP ON ACTIN IN CONTRACTION

517

Martonosi, Gouvea & Gergely (1960b) reported little or no release of the actinbound ADP in superprecipitation of actomyosin. These negative results, the slow equilibration of actin-bound ADP with pool ATP in living mu scle and the apparent lack of correlation of the equilibration time with exercise or with repeated contractions, were interpreted by these authors to mean that actin structure does not change significantly in contraction. Incorporation of lab eled ATP into myofibrils or actomyosin was also found to be small (Biro & Mtihlrad, 1960; Moos, 1964; Noda & Bono, personal communication). On the other hand, Barany & Finkelman (1963) interpreted their findings on F-actin containing Ac"\1P as evidence that a conformational change in actin is caused by superprecipitation. Previously we have presented observations showing a significant release of ADP bound to actin which was dependent on superprecipitation of actomyosin (SzentGyorgyi, A. G., 1965; Szent-Gyorgyi, A. G. & Prior, 1965). In this paper these experiments will be described in detail and discussed further. It will also be demonstrated that the reaction is an exchange reaction and that it can occur extensively in myofibrils. Evidence will also be presented that the changes in actin resulting in nucleotide exchange have the characteristics of repetitive cyclic reactions.

2. Preparations Rabbit myosin prepared a ccording to A. Szent-Gyorgyi (1951), was reprecipitated twice and clarified by a 45-min cen t r ifuga t ion at 60,000 g . Nucleic acid con ta m ination was remov ed from 0·5 to 1'0% m yosin so lut ion by p assa ge through a DEAE-cellulose co lumn in 0·3 M-NaCl containing 0·02 II{ phosphate buffer (pH 7,0). Myosin came out with the front, impurities conta in ing nucleic acids being r etained. The column was r egenerated by washing with 0·1 N -N a OH , water and exce ss buffer and then re-used. Myosin was stored for p eri ods of not m ore than 2 weeks at O°C. H omogeneity of the preparations was checked b y ultracen trifugation. Actin was extracted at O°C fr om ac etone-dried powder of rabbit muscle which was prepared b y a slight modifica tion of Straub's original procedure (Szent-Gyorgyi, A., 1951), and was purified by ccntrifugation at 100,000 g (Mommaerts, 1952b). The first ext r a ct and also the depolymerized a ctin obtained from the first pellet were cent r ifu ged at 100,000 g for 45 min t o remove fast sedimenting material. The a ctin obtained aft er on e cy cle of depolymerization-polymerization using 0·1 M.NaCI was st ored in p ellet form at O°C for less than 10 days. Actin was labeled with [3H)ATP or [14C)ATP on the day of the experiments. The pellets were rinsed with 10 mM-tris-HCl (pH 7,6), then homogenized with a Teflon homogenizer in 0·05 mx labeled ATP in 10 mM-tris-HCI (pH 7'6). Actin was stirred gently in the cold for 2 hr, and in some ca ses sonicated with an MSE soni cator. Excess ATP was removed by AGI-X2, 200 to 400 mesh resin (Strohman & Samorodin, 1962). The actin prep arations obtained were equilibrated with labeled ATP to the extent of 60 to 95% and cont a ined 1 mole of ATP in 62,000 ± 5,000 g protein. A ctin was polymerized in 0·1 M·NaCI and mixed with myosin at 0·35 ionic strength in a ratio of 1 g actin to 6 g myosin, exce pt when otherwise m ent ion ed. The a ctomyosin formed was r eprecipitated 3 times from 0·6 M-NaCI with 15 vol. of 10 mM-imidazole-HCI (pH 7,0) . R eprecipitation did not significan t ly alter the s pecific ac t ivity of a ctomyosin. The suspens ion obtained was gently homogenized by hand with a Teflon homogenizer and immediately us ed for t he experimen ts. The a ctomy osin suspension was ne ver store d for longer th an a few hours. N aCI was us ed in all ex periments, since K CI interfere s with the colorimet r ic protein determinations. Rabbit myofibrils were obtained from glycerol-extract ed psoas muscle (Szent-Gyorgyi , A., 1951). The muscle strips were stored in glycerol-water (50: 50) at - 25°C for m ore than a. m onth, homogenized for 1 min an d wash ed twice with 40 mM-NaCI (pH 7'0)-

518

A. G. SZENT.GyORGYI AND G. PRIOR

10 msr-imidazole-d mM.MgCI2 • "Natural" actomyosin was prepared according to A. SzentGyorgyi (1951) and reprecipitated 4 times. Creatine kinase was prepared according to Kuby, Noda & Lardy (1954), with omission of the last crystallization step, and stored in lyophilized conditions. Phosphocreatine was synthesized according to Ennor & Stocken (1948a). Labeled ATP was obtained from Traeerlab Co. and Schwartz BioResearch, Inc. ATP, ADP, AMP, ITP, UTP, CTP, GTP, were purchased from Pabst Laboratories.

3. Methods Release of bound nucleotides was determined by measuring the amount of labeled ADP in the medium. An actomyosin suspension, concentration "" 0·1 %, containing labeled ADP on actin, was incubated with non-radioactive ATP or other nucleotide triphosphate. At various times the protein was removed by Lrnin centrifugation in a clinical centrifuge and the radioactivity, protein content (Lowry, Rosebrough, Farr & Randall, 1951), PI (Fiske & Subbarow, 1925) or creatine content (Ennor & Stocken, 1948b) of the supernatant solution were measured. The values for protein and radioactivity in blanks which were not treated with ATP amounted to less than 5% of the. total protein and total radioactivity, and were subtracted from the experimental observations. Activities were adjusted in such a way that at maximum release about 2000 to 3000 cts/min were measured in a Packard Tri-Carb scintillation counter. 1 to 2-ml. samples containing less than 0·13 M-NaCl were mixed with 5 vol. of scintillation solution which was prepared by dissolving 100 g naphthalene, 7 g 2,5-diphenyloxazole and 0·3 g 1,4·bis2-(4-methyl-5-phenyloxazolyl).benzene in 1 liter dioxane and counted at 3°C for 10 min. In exchange studies, the bound ADP was initially labeled with tritium. The medium contained low concentrations of [14C]ATP together with phosphocreatine and creatine kinase. After incubation for various times, free nucleotides from the centrifuged actomyosin precipitates were removed by washing them 7 to 8 times with about 30 vol. of 40 lllM:-NaCl-10 mer-phosphate buffer (pH 7·0). Free nucleotides were already effectively removed by the fifth washing. For the individual measurements about 20 to 30 mg washed protein was resuspended in 4 ml, washing solution and the nucleotides extracted with 2·5% perchloric acid. The precipitate was redissolved by addition of 1·1 ml. cone, H 2S0 4 at 100°C and protein determined by the Kjeldahl procedure. Perchloric acid from the supernatant liquid was precipitated by neutralization with KHC0 3 and the ADP concentration per unit protein was obtained both from ultraviolet absorption spectra using a molar extinction coefficient of 1·54 X 104 at 260 miL and from radioactivity. Samples from the supernatant liquid were counted at 3 different dilutions in double labeling conditions. Each sample was counted alone and with internal standards consisting of [3H]ATP and [14C]ATP. The extent of bound [3H]ADP and of [14C]ADP was calculated at each point. When kinetics of exchange were studied, the values at each point were obtained from 4 to 5 mg protein. Creatine was measured from the first supernatant liquid. The superprecipitated actomyosin was washed as above and dissolved in 0·6 M-NaCl, and for portions of the solution, protein (Lowry et ai., 1951) and radioactivity were determined. Bound ADP values were obtained only from 3H and 14Ccontent, since the concentrations were too low for accurate ADP determinations from ultraviolet spectra. The bound nucleotides in "natural" actomyosin and myofibrils cannot be readily labeled previous to the experiments. With these materials, total nucleotide content was determined from ultraviolet spectra, newly incorporated ADP from radioactivity, protein by Kjeldahl's method. Superprecipitation was followed by the increase in turbidity at 600 uu: using a Zeiss spectrophotometer with a Varicord recording attachment. Extent of superprecipitation was also qualitatively estimated from the reduction in volume of the actomyosin suspension after centrifugation. Bound nucleotides were analyzed by descending paper chromatography in a solvent consisting of isobutyric acid and 0·5 M-NH 40H (5: 3 vjv) (Magasanik, Vischer, Doniger, Elson & Chargaff, 1950). Nucleotides were adsorbed on Norit A from the neutralized

EXCHANGE OF ADP ON ACTIN IN CONTRACTION

519

perchloric acid extracts, eluted from the charcoal with ethanol-NH"OH and concentrated in a flash evaporator. A better separation of ATP from ADP was obtained using trichloroacetic acid precipitation, followed by repeated ether extractions previous to adsorption on Norit,

4. Results (a) Release of ADP bound to actin Addition of ATP to an actomyosin suspension at low ionic strength in the presence of Mg 2 + leads to a rapid superprecipitation which is completed within a few seconds. ATP hydrolysis proceeds at an accelerated rate until completion, and at the same time ADP which was initially bound to actin is released into the supernatant solution. Figure 1 shows the time curve of ATP hydrolysis, protein solubilization and release of nucleotide. In order to measure the free nucleotides in the solution, the

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counts from radioactive material were corrected by assuming that the protein solubilized was actomyosin and that the dissolved actomyosin retained its bound ADP. In most cases the correction amounted to less than 5% of the counts above the amount of the ATP-free blanks, and only the corrected values are given in later Figures. In all the experiments reported here, determinations of radioactivity, protein and phosphate or creatine were all obtained at each point of the time curve.

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Release of bound ADP started with an initial burst and by the time of the first measurement (0·5 minute) nearly 20% of the bound nucleotide was released. The release then proceeded more slowly and in this experiment stopped at a level below 50% in about eight minutes. Hydrolysis of ATP was completed in about 16 minutes under these conditions. The initial burst varied between 5 and 25% depending on conditions, provided superprecipitation was rapid. The presence of ATP or nucleotide triphosphate in the medium was required for the release to proceed. ADP or AMP could not support the reaction in actomyosin preparations (Fig. 7(b». These preparations were washed free of adenylate kinase, as checked by their inability to liberate inorganic phosphate from ADP. We have been unable to obtain release which exceeded 50 to 60% of the actinbound nucleotides. In presence of phosphocreatine and creatine kinase, the ATP concentration required for maxium release could be greatly reduced. ATP concentrations of 0·02 to 0·1 mM in the presence of the ATP regenerating system were enough to give maximum release, though at a slower rate. (b) Oontrol experiments

These controls were designed to check that the nucleotide released was actinbound nucleotide and that the tight binding was retained in actomyosin. All actin preparations were checked for their nucleotide content after labeling procedures were completed and were found to contain 1 mole of ADP in 62,000 ± 5000 g of actin. There was no excess nucleotide to account for the release observed. Two of the F -actin preparations were tested for the release of bound ADP without myosin at 0·15% protein concentrations in 0·04 to 0·15 M-NaCI in presence and absence of ATP. After incubation for 30 to 90 minutes, the preparations were centrifuged for 180 minutes at 100,000 g and protein and radioactivity were measured in the supernatant liquid. About 20% of the radioactivity and 5 to 15% of the protein were recovered in the supernatant liquid. The presence of non-radioactive ATP had little effect. These results agree with the reports in the literature that the ADP bound on F-actin is protected and does not exchange with ATP in the medium. While these observations indicate the relative inaccessibility of ADP in F-actin, independent evidence is required to show whether or not the bond between actin and ADP remains stable in actomyosin. Reprecipitation of actomyosin five times leads to little or no loss in its specific activity, and the pattern of release was found to be independent of reprecipitations (Fig. 2), indicating that ADP was bound to actin firmly enough to withstand reprecipitations and extensive washings. In the coupled enzymic reaction: creatine kinase

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FIG. 2. E ffect of repr ecip it a t ions of actomyosin on re lease of nucleot ide. 0·11 % actomyosin in 50 mM-NaCI; 10 m M-imidazole-HCI (pH 7,0); 4-3 mM-MgCI2 ; 3·3 mM-ATP at 23°C. e, 1st; 0, 2nd ; 6. , 3r d ; D, 4t h ; _, 5th preci pitat e.

can be checked . Th e results ar e shown on Fig. 3. The lack of significant creatine liberation in the ab sence of added free ADP or ATP signifies that 98% or more of the bound ADP is protected in actomyosin. Free ATP or ADP add ed to tho complete syste m can be readily detected by liberation of crea tine in concentrations as little as 5% of the ADP concentrat ion introdu ced with actomyosin. Th e lack of crea ti ne libera ti on indi cates tha t the specific protective action exerted by F-actin is retained by actomyosin. To redu ce the possibility of free actin pa ssing into the supernat ant solut ion, myosin was added to act in in excess in all experiments . The myosin to actin ratio was kept 6 : 1 w/w, well over the combining ratio of about 4 : 1. An increase of the ratio t o 12 : 1 did not cha nge the percentage or the time course of nucleotide release. The protein concentrations for the experiments were usually ar ound 0·1 %. The maximum release at these concentrations amounted to about 40 to 50% of the total ADP bound to actin. At higher prot ein concent ra t ions the apparent ext ent of release was less; at t hese concentra t ions ATP was also exha usted sooner and the time ava il. able for release reaction was shorter . Since superp recipit at ion requires a low ionic strength and exte nsive solubilization of act omyosin has t o be avoided , the am ount of pho sphagen which can be introduced is limited. The more rapid depletion of ATP ap pears to be the main reason for t he more limited release at higher pro tein concentrations. If the release is plott ed against time at vari ous protein concent rations, usin g only those points at which ATP is st ill found in the supernatant solution, the relea se is independent of protein concentration (Fig. 4). The correction for the protein in solution is important in such a st udy, since at lower protein concent rat ions the fraction of protein dissolved is larger a nd the corre cti on becomes significant. 36

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(c) Superprecipitation and release of bound ADP

Both superprecipitation and release require nucleotide triphosphate. The correlation of release with superprecipitation can be clarified only if conditions are found in which superprecipitation is inhibited though ATP or other triphosphorylated nucleotides are present. Such conditions were approximated by variation of temperature, and by the omission of magnesium chloride. Superprecipitation is greatly suppressed at lO°C at relatively high ATP concentrations, as measured by turbidity and by centrifuged volume. At lower ATP concentrations, superprecipitation can proceed. The inhibition of superprecipitation is not the "clearing response" (Spicer, 1952; Maruyama & Gergely, 1962a,b) since at the low ionic strengths employed, actomyosin stays in suspension and can be readily removed by centrifugation. With ATP alone, superprecipitation and release were both delayed by about 10 minutes, presumably the time required for the ATP level to drop sufficiently. The onset of superprecipitation is further delayed in the presence of phosphocreatine; and the delay may last for 90 minutes or more. The increase in turbidity is small and incomplete. The release of bound ADP also proceeds slowly and is limited (Fig. 5(a)) and is less than 10% in 90 minutes at lO°C. In a number of cases there was an initial faster release of about 10% or less at lO°C which then continued at a slow rate characteristic to this temperature (Fig. 5(b)). A shift from 10 to 23°C at any point will cause rapid superprecipitation, accelerated release of nucleotides and rapid liberation of creatine. Superprecipitation and ADP release depend on similar factors in these experiments. The fact that release is limited and proceeds only very slowly at lO°C though ATP is present is perhaps the most convincing control for the tightness of the bond between ADP and actin and that the reaction measured is a specific one. It should be noted that the release reaction continues after superprecipitation has been completed, although the superprecipitated state by itself does not necessarily ensure the continuation of ADP release. In the samples which were cooled to 10 from 23°C, superprecipitation was not reversed although release was strongly inhibited. The reactions responsible for the continued release have the same temperature dependence as the initial burst of release and the onset of superprecipitation. The chemical reactions associated with the superprecipitated state may continue long after the visible changes of superprecipitation have been completed; they may also be interrupted without any macroscopic alteration in the superprecipitated state. ATP is not specific in causing superprecipitation. In the presence of magnesium, other purine and pyridine nucleotide triphosphates are also effective (Spicer & Bowen, 1951; Ranney, 1954; Bergkvist & Deutsch, 1954; Portzehl, 1954; Hasselbach, 1956). As judged from turbidity measurements in 4 mM-MgCI 2 , superprecipitation proceeds at about the same speed and to about the same extent with ATP, CTP and UTP (Fig. 6). CTP and UTP also lead to a rapid and extensive release, and are hydrolyzed relatively fast (Figs 7 and 8). With GTP and ITP, both superprecipitation and release were slower and incomplete; Pi liberation was also somewhat slower. Omission of magnesium reduced the rate and extent of all these reactions. ATP and UTP were relatively the most effective in eliciting superprecipitation and leading to release of ADP in the absence of magnesium ions. CTP caused only a slight change in turbidity; with GTP and ITP superprecipitation did not proceed at all. Release was very slow and barely measurable with GTP and ITP, and somewhat faster

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with CTP. In absence of magnesium ions, hydrolysis of UTP and ATP is the fastest, hydrolysis of CTP is considerably slower, that of GTP and ITP the slowest. The order of effectiveness of the various nucleotide triphosphates agrees well with previous measurements of Hasselbach (1956) in measuring their effect on superprecipitation and on contraction of glycerol-extracted muscle fibers. These results indicate that there is a correlation between superprecipitation and release of ADP bound to actin. Conditions which favor superprecipitation also favor release, and for rapid and extensive release the superprecipitated state appears to be a requirement.

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FIG. 8. Release of ADP from actomyosin with GTP, UTP, ITP and CTP in presence and absence of magnesium. Conditions as in legend of Fig. 6. 0 and O. 4·3 mM.MgCI 2 ; • and . , no magnesium; ----, P, hydrolyzed; - - , ADP released %.

(d) Evidence for exchange reaction

The experiments presented indicate that superprecipitation leads to a loosening of the bond between actin and ADP associated with actin. The experiments. however, do not exclude the possibility that the small amount of excess protein solubilized in the presence of ATP is actin, and the release measured may partly represent dissociation of actin from myosin. Since it is known that nucleotide-free actin preparations are unstable (Asakura, 1961b), denaturation of actin during the release reaction has also to be excluded. A demonstration that the release in fact is an exchange reaction in which the actin-bound ADP is replaced by ADP formed from ATP of the medium leads to a more straightforward interpretation and eliminates such possibilities. Exchange reactions can also be investigated with "natural" actomyosin and myofibrils which are not amenable to release studies due to the difficulties of specific labeling of the actin-bound ADP in these preparations. DEAE-cellulose treatment of myosin yields a preparation free of nucleic acid or nucleotide contaminations. The absorbancy of the perchloric acid extracts of 0·5% myosin preparations was less than 0·01 at 260 mfl-' Furthermore, myosin preparations incubated with ATP show no significant uptake of ATP or ADP when washed as previously described. Therefore the nucleotides present in a perchloric acid extract of an actomyosin prepared from purified myosin and actin must be derived solely from the actin. Actomyosin preparations, containing [3H]ADP on actin, when incubated with non-radioactive ATP showed a 25 to 35% loss oflabeled ADP without significant loss of the over-all bound nucleotide content (Table 1). The bound ADP released was replaced by nucleotide in the medium. The total bound nucleotide

EXCHANGE OF ADP ON ACTIN IN CONTRACTION TABLE

527

1

ADP release and nucleotide content of actomyosin JLmole nucleotidejg

Ultraviolet

[3H]ADP

[3H]ADP-actomyosin [3H]ADP-actomyosin

+ ATP

2·30 2·19

1·94 1·29

[3H]ADP-actomyosin [3H]ADP-actomyosin

+ ATP

2·37 2·24

2·01 ] ,52

Conditions: 0·]% and 0·16% actomyosin in 3mM-ATP, 40mM-NaCl, 4 mM-MgCl 2 , 10 mu-imidezole-Hol (pH 7). 23°C for 30 min and 45 min.

content stayed close to the value of 2·3 p,moles/g expected from the 1 : 6 wlw actin to myosin ratio. The exchange reaction can be demonstrated quite unambiguously with doublelabeling techniques. In these experiments the actomyosin contained ADP labeled with 3H, the medium contained ATP labeled with 14C. The concentration of ATP of the medium was kept low and did not exceed the molarity of actin by more than 10· to 20-fold, and it was kept constant by the presence of excess phosphocreatine and creatine kinase. In these conditions the reaction was somewhat slowed down, making it easier to follow its time course. ATPase activity was reduced considerably, due to low substrate concentration, which prolonged the time during which ATP was available and the reaction could proceed. A typical experiment is summarized on Table 2. One-half of the original [3H]ADP was released from the protein and an equivalent amount of [14C]ADP was incorporated into actomyosin. The total nucleotide content stayed constant as measured independently from absorbancy, regardless of whether unlabeled or [14C]ATP was used in the medium. With myosin alone, only incorporation of ATP could be studied, since the absorbancy of perchloric acid TABLE

2

Exchange of bound nucleotide at 23°0 JLmole nuclcotidejg Ultraviolet [3H]ADP [14C]ADP [3H]ADP

[3H]ADP-actomyosin [3H]ADP-actomyosin [3H]ADP-actomyosin

+ cold ATP + [14C]ATP

Myosin + [14C]ATP Myosin fraction I (82%) Myosin fraction II (18 %)

2·02 1·87 ] ,95

2·02 0·99 0·98

1·0

+ [14C]ADP

2·02 0·99 ] ,98

0·05 0·03 0·14

Conditions: 0·] % actomyosin or myosin in 0·02 mM.ATP, 10 msr-phosphocreat.ine, 36 JLg/mJ. creatine kinase, 40 mM·NaCl, 4 mM.MgCI., 10 msr.imidazole-Hfjl (pH 7) for 90 min at 23°C.

528

A. G. SZENT·GYORGYI AND G. PRIOR

extracts was too low to be measured accurately both before and after incubation with ATP. Incorporation of ATP into myosin was limited; the value obtained was about one-twentieth of the amount incorporated into actomyosin. If the fraction (fraction II, representing 18% of the total myosin) which precipitates at 0·25 ionic strength was removed, the radioactive material associated with myosin (fraction I) decreased further. Such a procedure is known to remove actomyosin impurities. The small incorporation observed with myosin can be accounted for by an actin contamination amounting to less than 0·5%. The bound nucleotides containing both labels were analyzed by paper chromatography after the reaction was completed and 98% of the radioactivity was found to be associated with ADP (Table 3). TABLE

3

A nalyses of bound nucleotides %ATP

%ADP

% AMP

1

98·3

0·7

Actomyosin (after superprecipitation)

Conditions: Descending chromatography, isobutyric acid-0'5 N·NH.OH (5: 3). Eluted spots were analyzed for radioactivity and for ultraviolet absorption.

It is clear that the ADP released was compensated for by uptake of ADP derived from ATP in the medium in the experiment presented. Since the experiment shows the result when the reactions were at completion, it was of interest to follow the time course of release and of incorporation to see whether or not these reactions can be readily separated. It is seen from Fig. 9 that release and incorporation have a similar

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FIG. 9. ADP exchange with actomyosin. 0·11 % actomyosin containing [3H]ADP; 36 p.g/ml. creatine kinase; 40 mM.NaCI; 20 mM·imidazole-HCI (pH 7,0); 4·3 mM.MgCI2 ; 10 mx.phosphocreatine and 0·04 mM·p·C]ATP at 23°C. Ordinate: bound ADP in the precipitate. Arrow, [3H]ADP at zero time; (-0-0-), [3H]ADP; (-e-e-), [HC]ADP; (-A-A-), total ADP.

EXCHANGE OF ADP ON ACTIN IN CONTRACTION

529

time curve; both start with an initial burst and then proceed at about the same rate, though incorporation appears to lag somewhat behind release. Still, release and incorporation were coupled sufficiently closely that, with the techniques used and in the conditions of the experiments, no clear-cut separation of these steps was obtained. The total nucleotide content stayed constant within the experimental resolution, although incorporation at the end slightly exceeded release. At 12°0 and at low ATP concentrations, superprecipitation is considerable though slow, and the release is not compensated for by incorporation, unless the preparation is warmed to 23°0 (Table 4). If the suspension is centrifuged and resuspended in the same [140]ATP-phosphocreatine medium before warming the preparation, no incorporation takes place. This would indicate a partial dissociation of actin at low temperature, which was then removed by centrifugation, and may be the explanation of the initial release at lower temperatures shown on Fig. 5(b). TABLE

4

Exchange of bound nucleotide at 12°0 JLmole nucleotidejg Ultraviolet [3H]ADP [14C]ADP [3H]ADP

[3H]ADP-actomyosin

+ [14C]ADP

1·55

1·57

Jo44

1·04

0·29

1·33

1·24

0·92

0·32

1·24

[3H]ADP.actomyosin + [14C]ATP, 30 min at 12°C, then 30 min at 23°C

1·43

0·92

0·72

1·64

[3H]ADP.actomyosin + [14C]ATP, 30 min at 12°C Centrifuged + [14C]ATP, 30 min at 23°C

1·26

0·77

0·38

I-l5

[3H]ADP.actomyosin 30 min at 12°C

+ [14C]ATP,

[3H]ADP·actomyosin 60 min at 12°C

+ [14C]ATP,

1·57

Conditions: 0·1 % actomyosin, 0·02 mM.ATP, 10 rmr-phosphocreatine, 36p.g/mI. creatine kinase, 40 mM·NaCI, 4 mM.MgCI2 , 10 msr-imidazole.-H'Cl (pH 7). Myosin to actin ratio, 7: 1 w/w.

(e) Evidence for the cyclic nature of the reaction

Double-labeling techniques allow one to distinguish sites which have reacted to the extent that an ADP exchange took place, from sites which have not reacted. In the experiments described, unreacted sites contain [3H]ADP, reacted sites contain [ 1 4 0 ]ADP . Using non-radioactive ATP on such a preparation, one can determine whether or not a site can undergo exchange more than once, and whether their previous history influences the reactivity of sites. The exchange reaction can be interrupted at any time by removing and washing the superprecipitated actomyosin. If the washed actomyosin suspension is re-introduced in a medium containing [140]ATP, phosphocreatine and creatine kinase, the reaction will proceed again and follow its normal course, to the point where about 50% of the bound nucleotides exchange. When non-radioactive ATP is used in place of [140]ATP, then the exchange

530

A. G. SZENT-GYORGYI AND G. PRIOR Cold ATP added

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Time (min)

FIG. 10. Chase of labeled nuc1eotides from actomyosin. Portion of the actomyosin of Fig. 9 removed at 30 min, washed twice and resuspended in the same medium as in Fig. 9, but containing 0·04 mM non-radioactive ATP.

taking place on 3H_ and 14C-labeled sites can be determined independently. Figure 10 shows the results of such an experiment. A portion of the actomyosin of the experiment shown on Fig. 9 has been removed at 30 minutes, washed and resuspended in cold ATP. Comparison of Figs 8 and 9 indicates that the treatment did not greatly influence the removal of [3H]ADP from actin and that the release proceeds at about the same speed in the samples which remained in [14C]ATP and in samples which were washed and resuspended in non-radioactive ATP. In the chase experiments, the [14C]ADP is also removed, indicating that these sites also exchange with nonradioactive ADP and that the same site can undergo reaction more than once. About one-third of the [14C]ADP is lost 'within 0·5 minute, while the rest is removed slowly with a speed comparable to the removal of exchangeable [3H]ADP. The rapid loss in [14C]ADP at the beginning of the chase experiments will be dealt with in a later communication. The total nucleotide content stayed constant within 10% as measured from ultraviolet spectra of perchloric acid extracts in parallel experiments, indicating that the reaction in these conditions was also an exchange reaction. Similar results were obtained if centrifugation and washing of actomyosin were omitted and [14C]ADP was chased by the addition of non-radioactive ATP in excess of 50-fold over the [14C]ATP present. Most of the [14C]ADP exchanged at a rate similar to the exchangeable [3H]ADP and was removed only slowly. The availability of newly incorporated ADP can also be checked by measuring its catalytic effect on creatine liberation in the creatine kinase-phosphocreatine system, using superprecipitated actomyosin. A preparation in which one-third of the total actin-bound ADP has exchanged and from which free nucleotides have been removed by extensive washings behaves in the same manner as control actomyosin preparations which did not undergo superprecipitation (cf. Fig. 3). Creatine liberation was not catalyzed, indicating that 98% or more of the ADP bound to actin was protected even though the superprecipitated state was retained (Fig. 11).

EXCHANGE OF ADP ON ACTIN IN CONTRACTION

531

, ,2 1· 1

1·0 0·9 0'8

. 0 '7 3- 0'6 . 0'5 .. 0-4 u

~ (5

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20

30

50

Time (min)

FlO. 1I. Creatine liberation with superprecipitated actomyosin. Superprecipitated and washed actomyosin in 40 m~ ·NaCI; 20 mM-imidazole-HCl (pH 7·0); 4·3 m~-MgCl2 ; 10 msr-phosphccreatine and 36 f'g/mI. creatine kinase at 23°C. 0·09 % actomyosin (1,9 P.M bound ADP) without free ADP (- 0 - 0-) and with 0·1 f'~1 (- e - e-), 0·2 f'~ (- .6. - .6.-), 0·5 f'M (- A- A-), 1·0 f'M ( - 0 - 0 - ) and 2·0 p.~ ( -. -. - ) added ADP.

(f) Exchange of A DP in "natural" actomyosin and myojibrils

The prev ious experiments were all performed with purified actin preparations obtained by normal preparative procedures which include acetone treat ment of the muscle residue. It was of some interest to check whether or not the results depend on this particular way of actin preparation . In "natural" actomyosin, actin is ex tracted along with myosin an d the preparation is not treated with organic solvents. The time curve of incorporation of [3H]ADP int o actomyosin in conditions favoring superprecipitation is shown on Fig. 12. The reaction was not followed to completion; it was stopped at a point where an amount of ADP corresponding to more than 30% of the total nucleotides was taken up by actomyosin. The total nucleotide content stays constant and gives a value expected from the actin content of such a preparation. The constancy of total nucleotide content, in spite of the sizeable nucleotide incorporation, proves that the reaction is an exchange. The impurities present in "natural" actomyosin, possibly tropomyosin, making its superprecipitation sensitive to removal of calcium (Ebashi & Ebashi, 1964), do not greatly interfere with the exchange reaction.

532

A. G. SZENT·GYORGYI AND G. PRIOR

.s:

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4

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100

2 120

Time (min)

FIG. 12. ADP exchange with "natural" actomyosin. 0·19% actomyosin; 40 mM-NaCI; 10 msrimidazole-HCI (pH 7·0); 4·3 mM-MgCI2 ; 36 /Lg{mI. creatine kinase; 10 mx-phosphocreatine and 0·02 mM-[3H]ATP at 23°C. -0-0-, Total ADP; -0-0-, creatine liberation.

To judge from previous reports, myofibrils incorporate ADP from medium ATP only to a very small extent, amounting to a few per cent of the total bound nucleotides. It seemed worth while to check whether these negative findings demonstrate the inability of the myofibrils to undergo exchange or whether they may have been a reflection of experimental design. The nucleotide content of glycerol-extracted, washed myofibrils amounted to 4·4 ± 0·2 f'molesfg protein. The value is similar to the values reported from several laboratories (Perry, 1952; Biro & Miihlrad, 1960; Seraydarian et al., 1962). Paper chromatography showed that 95% or more of the nucleotides were ADP. Assuming that all the ADP is bound to actin and that 60,000 g actin binds one mole of ADP, the actin content of the washed myofibrils would be 26% of the total proteins. Extensive incorporation of ADP into myofibrils can take place without a change in the over-all nucleotide content (Fig. 13). At the end of the experiment, paper chromatography of nucleotides extracted from washed myofibrils showed that radioactivity was exclusively associated with ADP. The exchange reaction in myofibrils took place at a rate which was considerably slower than the reaction with actomyosin in comparable conditions. In order to obtain exchange of 30% or more of the total bound nucleotides, ATP must be present for several hours. Solubilization of myofibrils at high ionic strengths prevents the unlimited addition of ATP and phospho-

EXCHANGE OF ADP ON ACTIN IN CONTRACTION

(0)

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100

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180

220

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FIG. 13. . ADP exchange with m yofibrils, 0·13 % myofibril (protein); 40 mM-NaCl; 20 msrimidazole-HCl (pH 7,0); 3 mM.M:gCI2; 36 J-Lg/ml. creatine kinase ; 13 rmr-phosphocreatine. At 90 min additional 13 msr -phosphocr eatlne added. (a) 0 and • • total ADP con tent of myofibrils. (b) D O b.. ATP 0·037 mM.[14C1ATP /Lmole/mI.; • • A. ATP 0·180 mM·[14C]ATP J-Lrnole/mI.; - - . % ADP incorporation of total ADP; ----. cre at in e liberation.

crea t ine. At sufficiently low ATP concentrations, the ATPase activity is reduced by a greater degree than exchange, provided the ATP level is maintained by phosphocreatine. As a matter of fa ct, exchange proceeded at the same rate at ATP coneentrations exceeding the bound ADP by sixfold and by thirtyfold (Fig. 13). If the exchange react ion were a simple equilibrium process, one would have expected the greatest effect at these lower ATP concentrations. It appears then that, with myofibrils, the limitation in the over-all exchange reaction is not the exchange step itself but rather the availability of the sites.

5. Discussion (a) Comparison of results with published data The observation that a significant portion of the ADP associated .wit h F-actin exchanges in superprecipitated actomyosin with ATP of the medium contradicts previous reports on the subject . Martonosi et al . (1960b) concluded : " Thus it is evident that the combination with myosin and the superprecipitation process did not cause any loss of activity in the bound ADP of F-actin." Their actual experiments indicate about 15% loss of bound ADP in an actomyosin preparation which was subjected to superprecipitation twice. Since experimental conditions including

534

A. G. SZENT·GYORGYI AND G. PRIOR

protein concentration, incubation time, phosphate liberation, are not reported, it is difficult to evaluate these results. Maruyama & Gergely (1962a) also obtained negative results, but without giving experimental details. A recent short report by Kitagawa, Martonosi & Gergely (1965) confirms our findings that a significant portion of the ADP bound to actin is released in the medium. Only a limited amount of ADP incorporation was reported when actomyosin or myofibrils were incubated with ATP (Biro & Miihlrad, 1960). The maximum incorporation observed by Moos (1964) amounted to 0·2/-'mole!g with actomyosin and even less with myofibrils. Noda & Bono (personal communication) found a variable amount of incorporation into myofibrils; usually less than 10% of the total bound nucleotides were labeled. The relative slowness of the exchange reaction in myofibrils coupled with the possible depletion of ATP in the medium may explain the limited incorporation observed in these studies. Barany & Finkelman (1963) used sonication to obtain an F-actin containing AMP. They observed that in actomyosin, when superprecipitated by ATP, the AMP bound on actin was replaced by ADP. These authors proposed that in superpreeipitation, actin underwent a conformational change which led to the exchange reaction. In these experiments it is not clear at which point the actin structure was modified and how strongly AMP was bound on actin. In fact, in the experiments reported, AMP was readily removed by simple reprecipitation of actomyosin even in the absence of ATP, indicating an already weaker binding. No control was shown as to what happens when actomyosin was incubated with ATP in conditions where superprecipitation was prevented. Estes & Moos (1965) and Kitagawa et al. (1965) indicated that F-actin alone shows some exchange with medium ATP which is dependent on concentration and increases with decreasing actin concentrations. Such behavior of actin is in agreement with the observation of Oosawa, Asakura, Hoota, Imai & Ooi (1959) that for polymerization a critical actin concentration is required, and would indicate that even at 0·1 ionic strength F-actin co-exists with some actin in the globular form. Since the amount of G-actin co-existing with F-actin is independent of F-actin concentrations, at high actin concentrations the exchange may be a negligible quantity. The actin concentration must be very low, of the order 0·01 %, to show a significant exchange. The possibility exists that some of the exchange reported in our studies takes place on free actin, dissociated from myosin somehow in snperprecipitation. The results, particularly experiments shown in Fig. 9, demonstrate that at 23°C incorporation compensates for release during the whole time course of the reaction, and that there is no significant loss in the nucleotide content of the precipitate during the exchange. These experiments also indicate that actin did not pass into solution in significant amounts during superprecipitation. The relevant actin concentration is the local concentration of actin in the actomyosin suspension or in myofibrils. The local concentration of actin wiIJ not change if the actomyosin suspension or myofibrils are diluted by the suspending medium. While accurate concentrations are not available for the protein content of an actomyosin gel, the centrifuged actomyosin has a protein content of about 2 to 3% (,-...., 0'5% actin) before superprecipitation, which increases to about 20 to 30% (,-...., 5% actin) after superprecipitation. The actin content of myofibrils is about 2 to 3% of the wet weight and increases further in contracted preparations. The local actin concentration is higher by several orders of magnitude than the concentration of F-actin at which exchange is measurable, The

EX CHAN GE OF A D P ON ACTIN IN CO N TR A CT I O N

535

relevant control, if one wishes t o st udy the effect of superprecipitat ion, is an acto myosin which is not sup erprecipitated though ATP or other nu cleotide triphosphate is present. In th ese st udies, superp recipit at ion was suppressed in severa l different ways, and in all cases the release of bound ADP was also strongly inhibited. By changing condit ions t o induce superprecipit at ion , a concurrent rapid release was obtain ed. No such cont rol was readil y available for " nat ural" actomyosin or myofibril s, since at the low ATP concentrations used for incorporation studies, chelati ng agents or lower te mpe ra t ures do not effect ively prevent superp recipit at ion . The evidence that nu cleot ide content agreed wit h actin content in different preparati ons and that the bound ADP in acto myosi n was protected from enzym ic attack also indicate t hat actomyosin formation p er se did not lead to significant labilization of the bond between actin and it s bound ADP. Superp recipitat ion appeared t o be a required condition to facilitate exchange. (b) Interpretation

Those actin monomers in the actin filaments whi ch inte ract with myosin sites undergo a change during contrac t ion and superprecipitation ; this leads to exposure of t he nu cleotide bound t o act in. Th e change in actin struct ure may result in a release of the bound ADP and incorporation of the medium ATP. The bound ATP is t hen dephosph orylated and t he changes in the actin st ruc t ure are reversed. Only a fra ction of t he total act in sites (5 to 20% ) can interact with myosin sites at any given moment due to ste ric considerations . Th e initi al rapid burst of excha nge represents t hose actin sites which are in close enough proximity to myosin sites t o participate in t he rea ction cycle. Only this portion of the exchange proceeds at a speed comparable with superprecipitation . The rest of t he exchange , though consist ing of similar cycles, is slower becau se of t he time required to complet e t he pr eviou s cycles and t o bring successive sites of actin t o approximate myosin sites. It is imp ortant that the cyclic structura l and chemica l rea cti ons cont inue as long as ATP is present in t he medium, long afte r t he macroscopic cha nges associated wit h superprecipitatio n and eontra ct ion ha ve been completed . Such a reaction seque nce is in good agreement with t he mechanism proposed by H an son & Huxley (1955) in t heir original paper describing the sliding model of contraction, and is not dissimil ar from the description of Asakura et al. (1963a) of the role of acti n in contract ion . There is no evidence at present as to what the local cha nge in actin structure may be, except that locally and for a short period actin behave s as if it were G-actin. It may represent a local br eak in the actin filament. It may also be a local cyclic conversion of the helical actin struct ure into a linear polymer, the F )- f transition of Oosawa & Kasai (1962). The exchange rea cti on in our experiments stopped at a point at which half of the a ctin-bound nucleotides have reacted. This behavior may be due to peculiarities of act in structure or to some limitations imposed by in teraction betw een act in and myosin . The la tter possibility is more likely , since essentially all the bound ADP in an ac to myos in can excha nge pro vided the superprecipitate d actomy osin is dissolved , reprecipitated and re-treated with ATP several t imes. The following exper imental evidence suggests that the chemical and st ruct ural cha nges are of a cycl ic nature. For the exchange of bound ADP, the presence of ATP (or other nucleotide t riphosphate) in the medium is req uired . Th e ATP participating in the excha nge is rapidly deph osphorylaterl and recovered as bound ADP. ADP

536

A. G. SZENT-GY()RGYI AND G. PRIOR

alone does not lead to an exchange reaction. The alteration in actin or in actin filament structure allowing exchange is only transitory and the newly incorporated ADP is protected by the structure. The same sites can undergo reaction more than once, and previous reactions do not influence the reactivity of the majority ofthe sites. The cycle measured by exchange would consist at least of the following steps: (1) formation of actin and myosin bond in the presence of ATP; (2) a myosin-induced localized change in actin; (3) release of bound ADP; (4) incorporation of ATP; (5) dephosphorylation of bound ATP; (6) repair of the actin structure; (7) rupture of the actin-myosin bond. The exchange itself, represented by steps (3) and (4), may not be necessarily an obligatory part of the cycle. Alternative routes are possible, but for all of these the reversible change taking place at sites of actin and myosin interaction would be required. There is the possibility that actin is repaired without participation of ATP. Hayashi & Tsuboi (1960) and also Grubhofer & Weber (1961) showed that G-actin containing ADP can polymerize under certain conditions. If that were the case, the findings described here would have no importance for the energetics of muscle contraction. Another alternative for steps (3) and (4) would be a direct rephosphorylation of the actin-bound ADP as proposed by Lorand (1953), by Weber (1960) and particularly by Carlson & Siger (1960). Whether exchange or rephosphorylation takes place will depend on the time the actin sites are open and new actin and myosin sites are brought to interaction, possibly influenced by the regularity of the organization of actin and myosin filaments with respect to each other. It will also depend on the presence of appropriate transphosphorylating enzymes and relative speeds of direct rephosphorylation and of exchange. Rephosphorylation and exchange both could be a part of mechano-chemical coupling. It is noteworthy that whereas exchange can take place extensively in myofibrils, it occurs more slowly than with actomyosin, even though contraction is faster in myofibrils. It is possible that in myofibrils, exchange is an accident and represents only a relatively small fraction of the cycles taking place, rephosphorylation dominating the reaction. Martonosi et al. (1960b) reported that equilibration of actin-bound ADP with pool ATP in vivo takes about a day and is not dependent on exercise. Although this equilibration is slow, it is still much faster than the turnover rate of actin, as pointed out by Biro & Miihlrad (1960). The slow equilibration may be explained by assuming that the exchange reaction route is not the predominant one, besides the fact that only a fraction of the total sites may participate in physiological contraction. The studies reported here suggest that a change in actin is part of the contraction cycle in myofibrils and in actomyosin. Since techniques used here cannot detect any conformational changes on myosin, such changes are not excluded. Further information is required to demonstrate cyclic alterations in myosin, if any, and to elucidate the sequence and the importance of the steps proposed here. We acknowledge the helpful discussions with Dr Lafayette H. Noda, Department of Biochemistry, Dartmouth Medical School. This investigation was conducted during the tenure of a United States Public Health Service Research Career Award, CA-K6-14218 to one of us (A. G. S-G.) and supported by grants from the National Science Foundation (National Science Foundation, G22004) and U.S. Public Health Service grant GMO-9808.

EXCHANGE OF ADP ON ACTIN IN CONTRACTION

537

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