J. Biochem. 112, 568-572 (1992)

Muscle Actin Cleaved by Proteinase K: Its Polymerization and In Vitro Motility1 Sugie Higashi-Fujime,* Masami Suzuki," Koiti Titani," and Tetsu Hozumi*" 'Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464; "Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-11; and "'Depertment of Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya, Aichi 467 Received for publication, May 25, 1992

Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 36 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-Factin: Vmax=0.24 s"1, and X e p p =2.8/iM, while V m u = 7 . 6 s 1 , and KtPP = 13pM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 ptm/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.

Muscle contraction is based on the sliding between myosin and actin filaments, consuming energy liberated by ATP hydrolysis in myosin ATPase activated by F-actin. Little is known about how chemical energy is transduced into mechanical energy for sliding. During the interaction between actin and myosin, both myosin and actin may undergo dynamic changes in their molecular structures to produce force for the mutual sliding of filaments. Biochemical investigations have revealed that the binding sites of actin with the myosin head are located in the N-terminal region (1, 2), particularly in loop regions consisting of residues 1-5, 22-29, 92-100 which reside in the subdomain 1 of the small domain, as revealed by X-ray diffraction studies (3-5). Although no enzyme has been found to cut directly the binding sites of the myosin head, enzymatic (6-11) and nonenzymatic (12) cleavages of actin have been performed to elucidate its functional structure. Most proteolytic enzymes examined so far cut a loop region at residues 40-50 from the amino terminus to produce a 35 kDa polypeptide fragment or the stable "core" of 33 kDa. Cleavage of actin by trypsin yields a 33 kDa fragment which can bind ATP but does not polymerize into F-actin (6, 7), even in the presence of phalloidin (8). Chymotryptic cleavage yields a 35 kDa fragment which is ' This study was supported by a Grants-in-Aid for Scientific Research on Priority Areas (Nos. 03223207 to S.H.-F. and No. 03223214 to T.H.) from the Ministry of Education, Science and Culture of Japan. Abbreviations: DTT, dithiothreitol; HMM, heavy meromyosin; X,pp, the apparent binding constant of heavy meromyosin with F-actin in the presence of ATP; kDa, kilodalton; PMSF, phenylmethylsulfonyl fluoride; proK-actin, actin cleaved by proteinase K; proK-F-actin, proK-actin polymerized into F-actin; PVDF, polyvinylidene difluoride; S-l, myosin subfragment 1; Tris, tris-(hydroxymethyl)aminomethane.

subsequently cleaved to a 33 kDa fragment (9, 10). A mixture of intact actin and cleaved actin containing the 35 kDa fragment are polymerizable but actin containing the 33 kDa fragment is not (10, 12). When G-actin is cleaved by subtilisin, a 35 kDa fragment is specifically produced (21). This cleaved actin can polymerize into F-actin on addition of salt and activates HMM or S-l ATPase at the same rate as intact F-actin at "infinite" concentration (11, 14), but its sliding movement in vitro is impaired (14). Thus, cleaved actin containing 35 kDa fragment but no actin containing 33 kDa fragment can polymerize, as far as is known. We report here that cleavage of G-actin by proteinase K produces a fairly stable 35 kDa C-terminal fragment, which is polymerizable into F-actin (proK-F-actin) only in the presence of phalloidin, and the very weak activation of HMM ATPase by proK-Factin (Vrmax = 0.24 s"1) correlates with very slow sliding in vitro of proK-F-actin on HMM (0.47 //m/s). MATERIALS AND METHODS Preparation of Muscle Proteins—Myosin and actin were prepared from rabbit skeletal muscle by the method of Perry (15) and Ebashi et aL (16), respectively, with slight modification (17). Heavy meromyosin (HMM) was prepared according to Weeds and Pope (18) with some modification (19). Cleavage of Actin with Proteinase K—G-Actin (2 mg/ ml) was digested by proteinase K at a concentration of 0.01 mg/ml at 25'C for 90 min in a solution containing 2 mM Tris-HCl, 0.2 mM ATP, 0.1 mM CaCl,, and 0.1 mM NaN3 at pH 8.0. The reaction was terminated by addition of 2 mM PMSF. Under these conditions, G-actin was cleaved

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Proteinase K-Cleaved Actin mainly into the 35 kDa polypeptide fragment, but small amounts of fragments with molecular mass of about 25 and 15 kDa were also generated (Fig. 1). The amounts of these minor fragments and remaining intact actin slightly differed from preparation to preparation. Mechanochemical properties of the different preparations of cleaved actin were very similar. Measurement of Actin-Activated ATPase Activity of HMM—The ATPase activity was asssayed by measuring the amount of inorganic phosphate liberated by the method of LeBel et aL (20). Actin-activated ATPase was assayed in a solution containing 30 mM KC1, 1 mM MgCl2( 0.5 mM ATP, 0.2 mM DTT, 30 mM imidazole buffer pH 7.6 at 25'C. The concentration of HMM was 0.05 mg/ml, and that of actin ranged from 0.1 to 1.0 mg/ml. In Vitro Motility Assay—Intact actin or cleaved actin filaments were labeled with rhodamine-phalloidin at a molar ratio of 1 to 1 (21). Motility was assayed at 23'C under the same medium conditions as for ATPase assay except that 40 mM KC1 was used instead of 30 mM, at 23"C. Twenty millimolar DTT, 0.22 mg/ml glucose oxidase, 0.036 mg/ml catalase, and 4.5 mg/ml glucose were added to prevent rhodamine from bleaching (22). The sliding movement of F- actin filaments on HMM or on myosin attached to a coverslip coated with collodion film (23-25) was observed with a fluorescence microscope, and was recorded on video tape by an SIT camera attached to the microscope (19). The velocity was measured by monitoring the sliding movement recorded on the video tape by using a program (19). SDS-PAGE—Gel electrophoresis was carried out as described previously (26), using a 7-18% polyacrylamide gradient gel. The molecular mass of a cleaved fragment was estimated from the mobility of molecular mass markers: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21 kDa), lysozyme (14 kDa), and cytochrome c (12 kDa), and also insulin (5.8 kDa) which was eluted in the absence of 2-mercaptoethanol. Electron Microscopy—F-Actin at a concentration of about 0.1 mg/ml was mounted on a carbon-coated grid and stained negatively with uranyl acetate. The structures were observed with an electron microscope (JEOL100C) operated at 80 kV. Protein Sequencing—Protein sequence analysis was carried out by the method of Matsudaira (27). Fragments separated by SDS-PAGE was electroblotted onto an Immobilon (PVDF) transfer membrane (Nihon Millipore, Tokyo) and stained with Coomassie Brilliant Blue. Protein bands were cut off and subjected to arnino-terminal sequence analysis on a model 470A Protein Sequencer equipped with a model 120A on-line PTH analyzer (Applied Biosystems, Foster City, CA). Chemicals—Glucose oxidase, catalase,

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velocity Fig. 4. Velocity distributions of sliding movements of intact F-actin and proK-F-actin filaments on HMM. About a half of proK-F-actin filaments were immobile and only filaments sliding were counted in a defined area of a microscopic field for about 2 min. Shaded bars, proK-F-actin; open bars, intact F-actin. The arrow and double arrow point to the average velocity of proK-F-actin 0.4 s (n = 86), and intact F-actin, 5.70^m/s (n = 81), respectively.

was about 30 times lower than that activated by intact F-actin. In Vitro Motility Assay—The sliding of fluorescently labeled F-actin on HMM in vitro can be observed by fluorescence microscopy. Figure 4 shows histograms of J. Biochem.

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Proteinase K- Cleaved Actin sliding velocity of intact F-actin and of proK-F-actin on HMM. Intact F-actin slid at an average velocity of 5.70± 0.77 /*m/s (n—81) and almost all filaments were sliding, whereas proK-F-actin slid at 0.47±0.21//m/s (n=82). This was the average velocity of moving filaments. The fraction of moving filaments ranged from 10 to 60%, depending on the condition of the surface to which HMM was attached, probably on the depth of the collodion film. To compare the motile activity of intact F-actin with that of proK-F-actin, we avoided the dependence of motile activity on the individual surface as follows. First, we selected a good surface on which intact F-actin slid at about 5//m/s and recorded the movement, then the assay medium containing 0.15 M KC1 without F-actin was perfused to wash away the F-actin filaments sliding on and attached to the surface (18). Subsequently, proK-F-actin in the same assay medium was perfused in, and the movement on the same surface was recorded without displacing the microscopic stage. On a surface where intact F-actin slid faster, more filaments of proK-F-actin moved and they slid faster. The movement of proK-F-actin shown in Fig. 4 illustrates the behavior on a good surface. The sliding velocity of proK-F-actin on myosin was very similar to that on HMM: 0.40±0.21 ^ m / s (n = 23), on the same surface where intact F-actin slid at the velocity of 5.48 ±0.92 fun/ s (n=22). In a specimen of proK-F-actin, one or two filaments were occasionally found to slide at the velocity of about 4 //m/s, even when proK-F-actin was initially applied onto the HMM-coated surface. This indicates that a small amount of intact actin, remaining undigested in such samples, polymerized with itself without copolymerizing with cleaved actin. The slow velocity of sliding of proK-F-actin correlates with its low activation of HMM ATPase (Fig. 3). DISCUSSION Proteinase K-cleaved actin can polymerize into F-actin on adding phalloidin, suggesting that it retains the global molecular structure without dissociation of subfragments. Yet, the rate of polymerization is very low, even in the presence of phalloidin. The overall structure of proK-Factin and the arrowhead structure of its rigor complex with HMM do not appear defective, as judged by electron microscopy. This proK-F-actin, however, activates HMM ATPase very weakly. The maximum rate of ATPase, Vmai, is 0.24 8"1, about 1/30 that of F-actin, and the sliding velocity of proK-F-actin is 0.47 //m/s, about 1/12 that of intact F-actin. Subtilisin cleaves actin into 35 and 9 kDa fragments and cuts at the same site as proteinase K {11). Although cleavage is at the same site, subtilisin-cleaved actin can polymerize on adding salt, and activates HMM ATPase at the same rate as does intact F-actin (14). Since proteinase K is a nonspecific protease, it may cut other site(s) in the N-terminal 9 kDa region where actin-actin binding sites and myosin-binding sites reside (3, 4). In fact, the 9 kDa fragment was not found on the SDS-PAGE of proK-actin (Fig. 1). Although the N-terminal fragment with an apparent molecular mass of 9 kDa was reported to be stained poorly by Coomassie Blue (11, 13)', we failed to find a band with a molecular mass of 9 kDa or 5.5 kDa, which was calculated from the amino acid sequence of the N-terminal Vol. 112, No. 4, 1992

fragment (29), even when the protein was overloaded. But faint and smeared staining without any distinct bands was observed below the molecular mass of 12 kDa (data not shown). Therefore, the N-terminal fragment seemed to be fragmented into shorter peptides and parts of the molecular structure of the actin would be damaged. This may explain the requirement of phalloidin for polymerization, and its weak activation of HMM ATPase. Interestingly, phalloidin binds to the residues 117 and 355 (30) and is thought to stabilize the genetic helix through its contacts between the residues 110-112 and residues 195-197 (4), both of which reside in the large 35 kDa C-terminal fragment. Nevertheless, phalloidin was a potent activator of polymerization of proK-actin, indicating again that cleavages in the N-terminal region including the 40-50 loop, had damaged the molecular structure of G-actin. Site-directed mutation in the charge cluster at the N-terminus, which is thought to play an important role for myosin binding in the presence of ATP (1), impaired both the actin-activated myosin ATPase activity and the sliding movement in vitro (31). Cleavage in or near the charge cluster region of proK-actin has not been demonstrated yet, but cleavage in the 9 kDa N-terminal region could crucially influence the biochemical and motile activity of actin. The sliding velocity of proK-F-actin is strongly rliminished, but about a half of the population of proK-F-actin filaments slides continuously. As described in "RESULTS,* however, a few filaments were observed to slide as fast as intact F-actin filaments, suggesting that remaining uncleaved actin would polymerize exclusively into F-actin without copolymerizing with cleaved actin. Therefore, this slow sliding of proK-F-actin can be attributed to motility of proteinase K-cleaved actin. Assuming the nonmotile fraction of proK-F-actin (about 50%) does not participate at all in ATPase activation, the ATPase activity activated by proK-F-actin correlated roughly with the sliding velocity. ProK-F-actin has a low KaPP value, 2.8 ^M, and apparently binds to HMM more tightly than does intact F-actin in the presence of ATP. Therefore, the weak activation of HMM ATPase by proK-F-actin is not due to lack of binding, but is due to some unfavorable structural change that directly impairs the catalytic process. The strongly inhibited sliding movement and ATPase activation of proK-Factin suggest that perhaps cleavage in the N-terminal region impairs the transfer of information to and from the myosin head for the catalytic and force-generating processes. We thank to Prof. M.F. Morales for his critical reading of this manuscript and valuable suggestions. REFERENCES 1. Sutoh, K. (1982) Biochemistry 21, 3654-3661 2. Bertrand, R., Chaussepied, P., & Kassab, R. (1988) Biochemistry 27, 5728-5736 3. Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes, K.C. (1990) Nature 347, 37-44 4. Holmes, K.C, Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49 5. Flaherty, K.M., McKay, D.B., Kabsch, W., & Holmes, K.C. (1991) Proc NaU. Acad. Sd. USA 88, 5041-5045 6. Muazbek, L. & Laki, K. (1974) Proc. NaU. Acad. Scl USA 71, 2208-2211

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S. Higashi-Fujime et al. (1990) J. Biochem. 107, 671-679 20. LeBel, D., Poirier, G.G., & Beauboinl, A.R. (1978) AnaL Biochem. 86, 86-89 21. Yanagida, T., Nakase, M., Nishiyama, K., & Oosawa, F. (1984) Nature 307, 58-60 22. Kishino, A. & Yanagida, T. (1988) Nature 334, 74-76 23. Toyoshima, Y.Y., Kron, S.J., McNally, E.M., Niebling, K.R., Toyoshima, C, & Spudich, J.A. (1987) Nature 328, 536-539 24. Higashi-Fujime S. (1991) Int. Rev. Cytol. 126, 95-138 25. Okagaki, T., Higashi-Fujime, S., Ishikawa, R., Takano-Ohmuro, H., & Kohama, K. (1991) J. Biochem. 109, 858-866 26. Hozumi, T. (1983) Biochemistry 22, 799-804 27. Matsudaira, P. (1987) J. BioL Chem. 262, 10035-10038 28. Knecht, D., Cohen, S.M., Loomis, W.F., & Lodish, H.F. (1986) Mol Cell Biol. 6, 3973-3983 29. Elzinga, M.( Collins, J.H., Kuehl, W.M., & Adekrtein, R.S. (1973) Proc. Nad. Acad. Sci. USA 70, 2687-2691 30. Vandekerckhove, J., Deboben, A., Nassal, M., & Wieland, T. (1985) EMBO J. 4, 2815-2818 31. Sutoh, K., Ando, M., Sutoh, K., & Toyoshima, Y.Y. (1991) Proc. NatL Acad. Sci. USA 88, 7711-7714

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