Selective Removal of the Carboxyl-terminal Tail End of the Dictyostelium Myosin I1 Heavy Chain by Chymotrypsin*

Dictyostelium myosin I1 is a conventional myosin consisting of two heavy chains of 243,000 Da and two pairs of light chains of 16,000 and 18,000 Da. In this paper, we show that the heavy chain of myosin I1 can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular weights of 195,000 and 38,000 Da as estimated from sodium dodecyl sulfate-polyacrylamide gels. Chymotryptic cleavage at this site occurs most readily in the absence of salt and is greatly inhibited as the salt concentration is increased from 0 to 60 mM. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 of the myosin I1 heavy chain. If the fragment extends to the carboxyl terminus of the myosin I1 heavy chain, it would have a molecular weight of 33,700. Upon digestion of myosin I1 which has been phosphorylated with a high molecular weight Dictyostelium myosin heavy chain kinase (Cijte, G. P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072), all of the phosphate is recovered on the 33,700-Da tail-end fragment. Chy-motrypsin-cleaved myosin I1 is shown to be capable of forming filaments at salt concentrations between 20 and 100 mM as judged by its ability to be sedimented by centrifugation. Only the large fragment of myosin I1 hydrolysis


Selective Removal of the Carboxyl-terminal Tail End of the
Dictyostelium myosin I1 is a conventional myosin consisting of two heavy chains of 243,000 Da and two pairs of light chains of 16,000 and 18,000 Da. In this paper, we show that the heavy chain of myosin I1 can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular weights of 195,000 and 38,000 Da as estimated from sodium dodecyl sulfate-polyacrylamide gels. Chymotryptic cleavage at this site occurs most readily in the absence of salt and is greatly inhibited as the salt concentration is increased from 0 to 60 mM. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 of the myosin I1 heavy chain. If the fragment extends to the carboxyl terminus of the myosin I1 heavy chain, it would have a molecular weight of 33,700. Upon digestion of myosin I1 which has been phosphorylated with a high molecular weight Dictyostelium myosin heavy chain kinase (Cijte, G . P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072), all of the phosphate is recovered on the 33,700-Da tail-end fragment. Chymotrypsin-cleaved myosin I1 is shown to be capable of forming filaments at salt concentrations between 20 and 100 m M as judged by its ability to be sedimented by centrifugation. Only the large fragment of myosin I1 is found in the pellet; the 33,700-dalton fragment remains soluble. Chymotrypsin-cleaved myosin I1 can bind to actin and displays a high Ca'+-activated ATPase activity but has very low actin-activated ATPase activity.
The lower eukaryote Dictyostelium discoideum contains at least two distinct enzymes with myosin-like enzymatic and actin-binding properties. The smaller of these has a native molecular weight of about 150,000 (1) and seems to be similar to the single-headed myosin I enzymes first identified in Acanthamoebu (2,3). The role played by this small myosin in cell motility is not yet elucidated. The larger enzyme (myosin 11) is structurally similar to other muscle and nonmuscle myosins and consists of two heavy chains, each of 243 kDa (4), and two pairs of light chains, of 16 and 18 kDa, arranged in a highly asymmetric molecule possessing two globular heads and a 180-nm-long a-helical coiled-coil tail (5, 6). I n vivo, Dictyostelium myosin I1 is phosphorylated both on the heavy chain and the 18-kDa light chain (7). The levels of phosphate on both the heavy and light chains transiently increase when Dictyostelium amoeba are stimulated by the *This work was supported by the Medical Research Council of Canada The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Medical Research Council of Canada Scholarship.
chemoattractant cyclic AMP (8)(9)(10). The time course of the increase in myosin phosphorylation correlates with a shape change in the amoeba (9) and with a rapid, reversible reorganization of the myosin I1 filaments within the Dictyostelium (11). It is suggested then that chemotactic stimulation of Dictyostelium alters the activity of specific myosin I1 kinases that in turn regulate the filament-forming properties and contractile activity of myosin I1 (reviewed in Ref. 12).
To date, most studies have concentrated on the role of heavy chain phosphorylation in regulating the properties of Dictyostelium myosin 11. Peptide mapping seems to indicate that multiple sites on the Dictyostelium myosin I1 heavy chain are phosphorylated (lo), and indeed several forms of Dictyostelium myosin I1 heavy kinases have been isolated. Kuczmarski (13) has identified a myosin I1 heavy chain kinase from Dictyostelium which phosphorylates serine and threonine residues, while Maruta et al. (14) have partially purified two kinases, both with a molecular weight of 70,000 as estimated by gel filtration, which phosphorylate only threonine residues. We have purified to near homogeneity a Dictyostelium myosin heavy chain kinase with a molecular weight of 130,000 as estimated by SDSI-polyacrylamide gel electrophoresis, but of greater than 700,000 as determined by gel filtration, which seems to phosphorylate a single threonine residue on each of the myosin I1 heavy chains (15). All of the kinases phosphorylate the tail region of myosin I1 and inhibit the actinactivated ATPase activity of the myosin (13)(14)(15)(16)(17).
To better understand the means by which heavy chain phosphorylation regulates the properties of Dictyostelium myosin 11, it will be necessary to first define more exactly the number and location of the phosphorylation sites on the heavy chain and then to gain some information on conformational or structural changes brought about in the myosin heavy chain by phosphorylation. In this paper, we demonstrate that a specific site toward the carboxyl terminus of the Dictyostelium myosin I1 heavy chain is extremely susceptible to chymotryptic digestion at low salt concentrations. Selective removal of the end of the myosin I1 tail, which is shown to contain the threonine residue phosphorylated by the 700-kDa myosin 11 heavy chain kinase, does not alter the Ca2+-ATPase activity of myosin I1 but does inhibit actin-activated ATPase activity. The chymotrypsin-cleaved myosin 11 retains the ability to bind to actin and form filaments.

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Type Culture Collection) was grown and harvested as described previously (1). Dictyostelium myosin I1 and the myosin I1 heavy chain kinase were purified as described (15) with the following changes.
The myosin I1 fractions recovered from the Sepharose CL-4B column were not passed over a Dowex 1-X2 column but were dialyzed immediately against 0.1 M KC1, 20% sucrose, 10 mM imidazole, 1 mM dithiothreitol, pH 7.0 buffer. Elimination of the Dowex column step had no detectable effect on the properties of the myosin. The purification of the myosin I1 heavy chain kinase involved the steps up to and including the hydroxylapatite column. The kinase was stored on ice in 0.1 M KCl, 60% sucrose, 10 mM imidazole, 2 mM dithiothreitol, pH 7.0, and used in this form. Phosphorylation of Myosin ZZ-Dictyostelium myosin 11, dialyzed into 30 mM KCl, 10 mM imidazole, 1 mM M&12,1 mM dithiothreitol, pH 7.0, was pelleted by centrifugation in a Ti-50 rotor (40,000 rpm for 20 min) and resuspended in 10 mM imidazole, 2 mM MgC12,l mM dithiothreitol, and 0.5 mM [y-32P]ATP (50 Ci/mol). One ml of the myosin solution (1.3 mg/ml) was phosphorylated at 25 "C by the addition, at 0 and 10 min, of 50-pl aliquots of myosin heavy chain kinase (0.2 mg/ml). The reaction was stopped after 20 min by the addition of 0.6 M KC1 and 5 mM EDTA, and the myosin was then separated from the kinase and ATP by passage over a 1 X 30-cm Sepharose CL-4B column equilibrated in 0.6 M KCl, 10 mM imidazole, and 1 mM dithiothreitol. The myosin, containing 2 mol of phosphate/ mol, was dialyzed overnight against 10 mM imidazole, 1 mM dithiothreitol and used within 1 week.
Chymotryptic Digestion of Myosin ZZ-Dictyostelium myosin I1 was dialyzed into 10 mM imidazole, 1 mM dithiothreitol, pH 7.5 buffer, and then diluted with this buffer to a protein concentration of 0.2-0.3 mg/ml. For experiments at higher ionic strengths, the diluting buffer contained the appropriate concentration of NaCl. Unless stated otherwise, a chymotrypsin to myosin ratio (w/w) of 1:lOO was used for digestions at 25 "C, and a ratio of 1:40 was used at 0 "C. Digestions were stopped by the addition of 0.1 M phenylmethylsulfonyl fluoride in isopropyl alcohol to give a final concentration of 0.5 mM.
ATPase Assays-The MgZ+-ATPase assay buffer contained 10 mM M&12, 30 mM KCl, 1.0 mM [y3'P]ATP (1 mCi/mmol), 10 mM imidazole, 1 mM dithiothreitol, pH 7.0. Actin, prepared from rabbit skeletal muscle by the procedure of Spudich and Watt (18), was stored as G-actin. Before use, the actin was polymerized, pelleted by centrifugation, and then resuspended in actin buffer (10 mM imidazole, 5 mM MgC12, and 1 mM dithiothreitol, pH 7.0). Assays contained 360 pl of Mg++-ATPase assay buffer, 40 pl of actin and/or actin buffer and were initiated by the addition of 60 p1 of myosin in 0.1 M KCl, 10 mM imidazole, 1 mM dithiothreitol, pH 7.5. After 20 min at 30 'C, release of 32Pi was measured by the method of Pollard and Korn (19). The rate of ATP hydrolysis was linear over this time period and proportional to the myosin concentration. Ca2+-ATPase assays were performed as described previously (15).
Amino Acid Sequence Analysis-Automated Edman sequence analyses were performed with an Applied Biosystems gas phase sequencer 470A. Phenylthiohydantoin amino acids were identified by high pressure liquid chromatography with a Beckman liquid chromatograph and an Applied Biosystems PTH-C18 (4.6 mm, inner diameter) column.
Miscellaneous Methods-Discontinuous SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (20), and gels were stained according to Fairbanks et al. (21). For autoradiography of 32P-labeled proteins, polyacrylamide gels were dried down and exposed to x-ray film (Kodak X-Omat AR-2) at room temperature without an intensifying screen. Myosin I1 and actin concentrations were determined by the method of Bradford (22) using bovine serum albumin as a standard. Chymotrypsin concentrations were determined using an Em value of 21.6.

RESULTS
Chymotryptic Digestion of Dictyostelium Myosin 11-The 243-kDa heavy chain of Dictyostelium myosin I1 can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular masses, as estimated from SDS-polyacrylamide gels, of 195 and 38 kDa (Fig. 1). When the digestion is carried out at 25 "C, using a chymotrypsin to myosin ratio (w/w) of 1:100, cleavage of the myosin heavy chain is complete by 10 min; however, loss of the 38-kDa fragment into lower molecular mass peptides is observed (Fig. 1, lanes A-D). The cleavage of the myosin heavy chain into the 195-and 38-kDa fragments occurs rapidly even when carried out on ice (Fig.  1, lanes A-D). At a chymotrypsin to myosin ratio (w/w) of 1:40, digestion of the myosin heavy chain is virtually complete by 0.5 min, and little further degradation of either of the cleavage products occurs for several minutes.
Dictyostelium myosin I1 isolated as described under "EXperimental Procedures'' has previously been shown to contain little covalently bound heavy chain phosphate, and we will refer to this myosin as "unphosphorylated myosin 11" (15). A 700-kDa Dictyostelium kinase that phosphorylates only threonine residues (15) was used to incorporate 1 mol of phosphate into each of the Dictyostelium myosin I1 heavy chains. Phosphorylation of the myosin does not seem to significantly alter the rate at which chymotrypsin cleaves the myosin heavy chain, and the digestion products, once produced, remain stable for several minutes (Fig. 2). However, in addition to the 195-and 38-kDa fragments, a minor cleavage product of 40 kDa is also evident. The 40-kDa fragment is observed when unphosphorylated myosin I1 is digested at 25 "C but does not seem to be produced when the digest is performed at 0 "C ( Fig. 1). An autoradiogram of the SDS-polyacrylamide gel demonstrates that all of the phosphate incorporated into the myosin heavy chain ends up in the 38-and 40-kDa fragments, and none of it remains associated with the large 195-kDa fragment (Fig. 2).
Optimal Conditions for Selective Cleavage of the Myosin 1 1 Heavy Chin-The specific hydrolysis of the Dictyostelium myosin I1 heavy chain into the 195-and 38-kDa fragments by chymotrypsin occurs only under certain conditions. The susceptibility of this site to chymotrypsin was found not to be dependent on the myosin concentration (0.15-1.5 mg/ml) or the sucrose concentration (0-20%) in the digestion mixture (data not shown), but was greatly reduced as the ionic strength increased (Fig. 3). Under conditions where almost complete cleavage of the myosin I1 heavy chain occurs in the absence of NaC1, virtually no cleavage is observed in 0.1 M NaCl (Fig.  3). Chymotryptic cleavage of the heavy chain remains inhibited when the NaCl concentration is increased to 0. amide gels such as those in Fig. 3 indicates that the digestion of both phosphorylated and unphosphorylated myosin I1 is very sensitive to small changes in the ionic strength (Fig. 4).
These experiments were performed at a chymotrypsin to myosin ratio of 1230 (w/w), so that after 2 min at 0 "C only about 80% of the myosin heavy chain was digested in the absence of NaC1. For unphosphorylated myosin 11, an increase in the salt concentration to 50 mM is sufficient to decrease by 80% the amount of 38-kDa fragment produced. The digestion of phosphorylated myosin I1 is affected only slightly by NaCl up to 20 mM, but then is rapidly inhibited as the NaCl concentration increases to 50 mM (Fig. 4). Myosin  There does not seem to be a correlation between the range of ionic strengths over which chymotryptic cleavage at the 195-38-kDa junction is inhibited and the ionic strengths required for myosin I1 to aggregate into filaments. In the absence of NaC1, unphosphorylated myosin I1 is soluble (perhaps due to charge repulsion between the myosin tails); however, only 10 mM NaCl is required for nearly all of the myosin to be pelleted as filaments (Fig. 5). Unphosphorylated Dictyosteliurn myosin I1 then remains in the form of filaments, as judged by the ability of the myosin to be sedimented, until the salt concentration approaches 0.15 M. Phosphorylated myosin I1 remains soluble following centrifugation at all of the salt conditions tested (Fig. 5), although chymotryptic cleavage of phosphorylated myosin I1 is strongly inhibited by increasing ionic strength (Fig. 4).
Properties of the Chymotrypsin-cleaved Myosin-Sedimentation studies were also performed on Dictyostelium myosin I1 following chymotryptic cleavage. At NaCl concentrations between 20 and 80 mM, the majority of the 195-kDa fragment is insoluble and is recovered in the pellet (Figs. 5 and 6). The 38-kDa fragment remains soluble at all salt concentrations, even at those where the 195-kDa fragment pellets (Fig. 6), indicating that the two chymotryptic fragments do not interact strongly with each other. Preliminary results show little difference between the solubility properties of the phosphorylated and unphosphorylated forms of the 38-kDa fragment.
Dictyostelium myosin I1 that has been digested by chymotrypsin into the 195-and 38-kDa fragments displays a Ca2'-ATPase activity of 1.0-1.2 pmol/min/mg, identical to that of intact myosin 11, and also retains the ability to bind in an ATP-dependent manner to filaments of actin. Studies carried out in 0.2 M NaCl demonstrate that in the presence of ATP and actin the majority of the 195-kDa fragment is recovered in the supernatant following centrifugation, but in the absence of ATP the 195-kDa fragment is recovered in the actin pellet (data not shown). In all cases, the 38-kDa fragment remains soluble.
Although both the Ca2+-ATPase activity and the actinbinding properties of Dictyostelium myosin I1 are retained following chymotryptic cleavage, the majority of the myosin I1 actin-activated ATPase activity is lost (Fig. 7). The actinactivated ATPase activity of the chymotrypsin-cleaved myosin remained low even when the actin concentration was increased to 1 mg/ml. After subtracting the Me-ATPase activity of intact and digested myosin I1 in the absence of  The 38-kDa fragment, purified over a Sepharose CL-4B column, was subjected to automated Edman degradation as described under "Experimental Procedures." Twenty-eight residues were identified (lines under the residues) which correspond to residues 1826-1853 of the Dictyostelium myosin I1 heavy chain sequence (4). The 10 residues before Lys-1826 are also shown. Boxed residues indicate the repeating pattern of nonpolar amino acids that form the hydrophobic core of the coiled-coil myosin rod. actin (0.01 prnol/min/mg), the data in Fig. 7 were analyzed using the Hanes-Woolf double-reciprocal plot. Values for V , , of 0.143 and 0.029 prnol/min/mg were obtained for intact and digested myosin 11, respectively. Both forms of myosin I1 had similar KATpase values for actin of 0.24 p~.
Amino Acid Sequence Analysis of the 38-kDa Fragment-The 38-kDa chymotryptic fragment can be purified to near homogeneity in two steps: centrifugation in 50 mM KC1 to remove the majority of the 195-kDa fragments (Fig. 6) and chromatography over a Sepharose CL-4B column. The 38-kDa fragment, which elutes from the column with an apparent molecular weight of around 160,000 (data not shown), was subjected to amino acid sequence analysis. A single sequence, extending 28 residues, was obtained (Fig. 8). Comparison of this sequence to the complete sequence of the Dictyostelium myosin I1 heavy chain (5) demonstrates that the 38-kDa fragment begins at lysine 1826 and therefore is derived from the carboxyl-terminal tail end of the myosin I1 heavy chain.

DISCUSSION
In this paper, conditions are described that restrict the cleavage of Dictyostelium myosin I1 by chymotrypsin to a small region of the heavy chains, producing two fragments with molecular weights of 195,000 and 38,000 as estimated by SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 in the complete sequence of the Dictyostelium myosin I1 heavy chain (4). If the chymotryptic fragment extends from residue 1826 to the end of the myosin heavy chain (residue 2116), it would consist of 291 amino acids and have a molecular weight, as calculated from the amino acid composition, of 33,700. The elution of this fragment from gel filtration columns with an apparent molecular weight of 160,000 suggests that under native conditions this fragment forms an asymmetric a-helical coiledcoil dimer. The large chymotryptic fragment must then have a molecular weight of close to 209,000 and consist of the globular head regions of myosin I1 along with the remainder of the tail. This fragment retains both the ATPase and actinbinding sites of the myosin I1 molecule.
The extreme susceptibility of the peptide bond between residues 1825 and 1826 of the myosin tail to hydrolysis by chymotrypsin must be due in part to the presence of Tyr-1825 in one of the exposed outer positions of the coiled-coil (Fig.  8). The only other two aromatic amino acids in this section of the myosin I1 sequence, Phe-1795 and Tyr-1816, are both buried within the hydrophobic core of the coiled-coil (4) and so may not be accessible to chymotrypsin. The a-helical coiled-coil structure can be destabilized by the presence of charged or polar residues in hydrophobic core positions (23,24). A threonine residue occupies the core position immediately preceding Tyr-1825 while, just after Tyr-1825, glutamic acid, lysine, and threonine residues fill three consecutive core positions (Fig. 8). However, charged and polar residues occur relatively frequently in core positions throughout the Dictyostelium myosin I1 tail ( 9 , and a more extensive analysis is required before it can be concluded that the section of the coiled-coil around Tyr-1825 is significantly less stable than other regions of the tail. Chymotryptic digestion at Tyr-1825 is strongly inhibited as the salt concentration is increased from 10 to 60 mM (Figs. 3  and 4). Over this range of ionic strengths, unphosphorylated myosin I1 remains filamentous. The inhibition of digestion could therefore be due to some alteration in the packing of myosin I1 molecules within the bipolar filament, resulting in the region of the tail around Tyr-1825 become inaccessible to chymotrypsin. However, chymotryptic digestion of unphosphorylated myosin I1 remains inhibited even at 0.2 M NaC1, a salt concentration at which unphosphorylated myosin I1 is monomeric. In addition, chymotryptic digestion of phosphorylated myosin I1 is inhibited by increasing salt concentrations, although the phosphorylated myosin is soluble at all ionic strengths. Therefore, the inhibition of chymotryptic digestion is not dependent on the presence of filaments of myosin 11, but is probably due to a local conformational change in the region of the myosin tail around Tyr-1825 as the ionic strength increases. Further studies will be required to determine whether this local conformation change is reflected in the structure of myosin I1 filaments or perhaps in an alteration in actin-activated ATPase activity.
Conformational changes near the end of the tail of Dictyostelium myosin I1 are clearly of physiological importance, because phosphorylation within this region (Fig. 2) results in a dramatic decrease in the ability of the myosin to form filaments (Fig. 4). Whereas greater than 90% of unphosphorylated myosin I1 sedimented at NaCl concentrations of 10-100 mM, less than 20% of the phosphorylated myosin I1 (1 mol of phosphate/mol of heavy chain) sedimented. Earlier studies indicated that about 50% of phosphorylated Dictyostelium myosin I1 sedimented at these salt concentrations (7); however, the myosin I1 used in these studies contained only 0.6 mol of phosphate/mol of heavy chain, with an unknown amount of the phosphate present on serine residues. Along with previous results (15), the studies in this paper demonstrate that phosphorylation of a threonine residue near the end of the tail of Dictyostelium myosin I1 with the 700-kDa Dictyostelium heavy chain kinase is sufficient both to inhibit the actin-activated ATPase activity of myosin 11 and its ability to polymerize.
In addition to the 700-kDa kinase, which phosphorylates only threonine residues, a lower molecular weight Dictyostelium kinase that phosphorylates both serine and threonine residues within the tail region of myosin I1 has been identified (13). Also, Maruta et al. (14) have isolated two threoninespecific kinases that elute from gel filtration columns with molecular weights of 70,000. The threonine(s) phosphorylated by one of the 70-kDa kinases has been located, by using monoclonal antibodies specific for different sections of the Dictyostelium myosin I1 molecule, to a region of the myosin tail about 32 kDa from the carboxyl terminus of the myosin (16).
Thus, despite considerable differences in their properties, all of the Dictyostelium myosin I1 heavy chain kinases so far studied phosphorylate sites on the myosin I1 tail, whereas both the high and low molecular weight threonine-specific kinases are now known to phosphorylate sites near the end of the tail. The chymotryptic digest described in this paper provides a simple means by which to obtain large amounts of the phosphorylated tail-end fragment of Dictyostelium myosin 11. Further proteolysis of this fragment should allow the eventual identification of the phosphorylated amino acids within the myosin I1 heavy chain sequence. It will be important to determine whether the same threonine residue is phosphorylated by each of the Dictyostelium myosin I1 heavy chain kinases so far isolated (in which case the possibility must be considered that the kinases represent different forms of a single enzyme) or whether each kinase phosphorylates a different site on the myosin I1 heavy chain, perhaps with different effects on the contractile activity of the myosin.
Two other conventional myosins, from Acanthamoeba (25,26) and rabbit macrophages (27), have previously been shown to contain heavy chain phosphorylation sites located very close to the carboxyl-terminal tail end of the molecule. In both cases, this regulatory region could be specifically removed by proteolytic digestion. Acanthamoeba myosin I1 lacking the regulatory region displays neither actin-activated ATPase activity nor the ability to form filaments (28). In contrast, removal of the tail end of Dictyostelium myosin I1 does not prevent the formation of filaments, although the filaments formed by the chymotrypsin-cleaved myosin are less stable to salt than filaments formed by intact unphosphorylated myosin I1 (Fig. 4). These results are in good agreement with studies employing monoclonal antibodies which have shown that two sections of the Dictyostelium myosin 11 tail are important for filament formation (29,30). Monopolar and bipolar filament formation is completely inhibited by antibodies that bind to the tail 50-80% of the distance from the heads, whereas bipolar (but not monopolar) filament assembly is abolished by an antibody that binds to the tip of the tail (30). It is surprising that the carboxylterminal end of the tail is not absolutely required for filament formation, yet it contains a site which when phosphorylated is able to prevent filament formation by the whole myosin I1 molecule.
The V,,,,, for the actin-activated ATPase of Dictyostelium myosin I1 is inhibited 80% by the removal of the tail end of the molecule (Fig. 7). Because the filament-forming assays and ATPase assays are performed under different conditions, we cannot be certain that the chymotrypsin-cleaved myosin is filamentous in the ATPase assay. However, it has been shown that a soluble S-1 fragment prepared from Dictyostelium myosin I1 has a KA*paee only 12 times greater than that of filamentous myosin (17). Therefore, even if the chymotrypsin-cleaved myosin were soluble in the ATPase assay, the actin concentrations we have tested (up to 1 mg/ml actin) should have been sufficient to reach Vmax. The results suggest that inhibition of actin activation is not due solely to the weakened ability of the digested myosin to form filaments.