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219 Smooth and Skeletal Muscle Actin Are Mechanically Indistinguishable in the In Vitro Motility Assay David E. Harris and David M. Warshaw Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 Smooth muscle produces as much stress as skeletal muscle with less myosin. To determine if the actin isoforms specific to smooth muscle contribute to the enhanced force generation, the motility of actin filaments from smooth and skeletal muscle were compared in an in vitro assay in which single fluorescently labeled actin filaments slide over a myosin-coated coverslip. No difference was observed between the velocity of smooth versus skeletal muscle actin filaments over either smooth or skeletal muscle myosin over a large range of assay conditions (changes in pH, ionic strength, and [ATP]). Similarly, no difference was observed between the two actins when the filaments moved under load over mixtures of phosphorylated smooth and skeletal muscle myosin. Thus, it appears that the actin isoforms of smooth and skeletal muscle are mechanically indistinguishable in the motility assay and that smooth muscle's enhanced force generation may originate within the myosin molecule specific to smooth muscle. (Circulation Research 1993;72:219-224) KEY WoRDs * actin * smooth muscle * motility * protein isoform T he basic mechanism of smooth muscle contraction' and the biochemical steps of the smooth muscle actomyosin ATPase cycle2 are thought to be qualitatively similar to those of skeletal muscle. Nevertheless, smooth muscle's mechanical properties distinguish it from skeletal muscle. One obvious difference is smooth muscle's slower maximum velocity of shortening. More interestingly, both vascular and visceral smooth muscle generate as much force per crosssectional area as skeletal muscle with only 20% as much myosin.3-5 This apparent enhancement in smooth muscle myosin's force production may simply reflect greater numbers of crossbridges working effectively in parallel. Such a mechanical advantage could result from longer (i.e., 2.2-gm) myosin filaments6 or a more parallel arrangement of contractile units within the smooth muscle cell.1 At the actomyosin level, however, differences in kinetics of the smooth muscle crossbridge cycle could result in crossbridges spending a higher fraction of their cycle in the active force-generating state (i.e., higher duty cycle) under isometric conditions.1 The distinct mechanical properties of smooth and skeletal muscle have been primarily explained on the basis of differences in the myosin molecule, with little attention given to the role of actin. Interest in actin's contribution to the force-generating event has gained momentum now that the primary structure of actin can From the Department of Physiology and Biophysics, University of Vermont, Burlington. Supported by National Institutes of Health grants AR-34872 and HL-45161 (D.M.W.). D.M.W. is an Established Investigator of the American Heart Association. Address for correspondence: Dr. D.E. Harris, Department of Physiology and Biophysics, University of Vermont, Burlington, VT 05405. Received August 3, 1992; accepted October 6, 1992. be correlated to its tertiary structure, derived from actin:DNAse I cocrystals.7 G-actin is a bilobed structure consisting of large and small domains, with the myosin binding site contained within the small domain. Given that a single genetically engineered amino acid change to actin, at its putative myosin binding site, can result in altered crossbridge kinetics in Drosophila flight muscle,8 actin's contribution to force generation must be considered. This is important, since smooth and skeletal muscle express different actin isoforms, which, in part, may account for smooth muscle's enhanced force-generating ability. There are two smooth muscle actin isoforms (asmooth muscle actin and y-smooth muscle actin), one or both of which are found in every smooth muscle tissue studied.9-11 The a-smooth muscle actin isoform predominates in vascular tissue, whereas visceral smooth muscle tissue expresses y-smooth muscle actin. Extensive amino acid homology exists between the smooth and skeletal muscle actins, with only six differences between chicken gizzard (y-smooth muscle) and chicken pectoralis (a-skeletal muscle) actin.i2 However, two of these changes occur within the cluster of acidic residues at the N-terminal end of actin (residues 1 and 3), believed to be part of the myosin binding site.13 In vitro motility can be altered in actins with reduced acidity in this cluster, including wild-type yeast actin14 and mutated Dictyostelium actin.15 Thus, it is possible that the naturally occurring amino acid differences between smooth and skeletal muscle actin, which include the absence of the initial aspartic acid found in skeletal muscle actin, may contribute to smooth muscle's unique mechanical properties. The goal of this study was to determine if, in an in vitro motility assay in which single fluorescently labeled 220 Circulation Research Vol 72, No 1 January 1993 actin filaments were observed sliding over a myosincoated coverslip,16 the characteristics of actin motility were dependent on actin isoform. Specifically, we wished to determine if the motility of smooth muscle actin differed from that of skeletal muscle actin over either phosphorylated smooth or skeletal muscle myosin over a wide range of assay conditions. The assay conditions studied were chosen so that direct comparisons could be made to the extensive body of literature on skeletal muscle actin motility in vitro. Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 Materials and Methods Contractile Protein Isolation and Preparation Chicken skeletal pectoralis muscle myosin and turkey gizzard smooth muscle myosin were prepared as previously described.17 Smooth muscle myosin was 100% thiophosphorylated by the addition of myosin light chain kinase, calmodulin, CaCI2, and ATP-y-S. Skeletal muscle actin was isolated from chicken pectoralis acetone powder.18 Smooth muscle actin was purified from chicken gizzard acetone powder as described,19 except that, before the final polymerization, size fractionation was carried out with a Sephacryl 300 column (Pharmacia). The purity of all protein-containing fractions was assessed on a 12% polyacrylamide gel, and the purest fraction was retained for polymerization and use. Both smooth and skeletal muscle actin were stored in filamentous form at 4°C and fluorescently labeled with tetramethylrhodamine phalloidin (Sigma Chemical Co., St. Louis, Mo.).17 Actin purity and composition were determined by one- and two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), as previously described.20 One-dimensional SDS-PAGE of both smooth and skeletal muscle actin showed a single major protein band with minor contaminants visible in both preparations only when the actin was overloaded. Twodimensional electrophoresis revealed that the smooth muscle actin focused overwhelmingly as a single species (data not shown). This is in agreement with previous determinations that chicken gizzard actin is 80% y-smooth muscle actin with minor contributions from the nonmuscle actin species.10 In Vitro Motility Assay The in vitro motility assay was carried out essentially as described previously.172' Briefly, flow-through chambers were constructed from a nitrocellulose-coated coverslip and a glass microscope slide. Then the following solutions were introduced into the chamber: 30 gl myosin (250 ,ug/ml) in a 300 mM KCl buffer, which allowed the myosin to adhere to the nitrocellulose without forming filaments (i.e., in monomeric form); 60 gl bovine serum albumin (0.5 mg/ml) in the same buffer to wash out unbound myosin and block any uncovered nitrocellulose; 60 ,ul fluorescently labeled actin (0.5 ,ug/ml) in ATP-free assay buffer; 60 ,l ATP-free assay buffer to remove unbound actin; and 90 gl assay buffer to initiate motility. Under standard conditions, the assay buffer contained 25 mM KCI, 25 mM imidazole, 1 mM EGTA, 4 mM MgCl2, 1 mM dithiothreitol, 0.5% methylcellulose, and 1 mM ATP, pH 7.4, with an enzymatic oxygen scavenger system (0.1 mg/ml glucose oxidase, 0.018 mg/ml catalase, and 2.3 mg/ml glucose) added to retard photobleaching. The content of this buffer was adjusted depending on the variable being examined (e.g., changes in [KCl], pH, and [ATP]). Where required, smooth and skeletal muscle myosin were mixed before introduction into the flow-through chamber. Temperature was controlled at 30°C by an objective heater (Vermont Technologies, Burlington, Vt.). Fluorescently labeled actin filaments were viewed through an inverted microscope equipped for epifluorescence.'7 Intensified video images were digitized and analyzed for actin filament velocity by computer.22 Statistics All velocity measurements were obtained from at least 10 actin filaments and are presented as mean+SD. Since the velocities that contribute to each mean value were all obtained from the actin population in a single flow-through chamber, mean velocities were considered an n of one for subsequent statistical analysis. The effect of actin filament type as a function of assay condition (Figures 2-5) was determined by fitting velocity data for each filament type by polynomial regression (order of polynomial determined by analysis of variance [ANOVA]) and then comparing the quality of the regressions by F test. To allow comparison to previously published data in which no difference (p>0.05) was found between actin filament types, data were combined and fit to the most appropriate polynomial as determined by ANOVA. Curve fitting was performed with the TABLECURVE and SIGMAPLOT software packages (Jandel Scientific, Corte Madera, Calif.). Results Smooth and Skeletal Muscle Actin Filament Motility With Changes in Assay Condition Under standard assay conditions (see "Materials and Methods"), skeletal and smooth muscle actin filament velocities over monomeric skeletal muscle myosin were not significantly different (p>0.05), as was the case with monomeric smooth muscle myosin (Figure 1). As previously reported for filamentous myosin,17 the velocity of actin filament motion over monomeric skeletal muscle myosin was approximately an order of magnitude faster than over monomeric phosphorylated smooth muscle myosin. To more rigorously probe for differences between myosin's interaction with smooth and skeletal muscle actin, the motilities of the two actin filament types were compared over a wide range of assay conditions. Starting from standard assay conditions, each of the following parameters was varied independently: pH, [ATP], and [KCl]. These comparisons were performed over both smooth and skeletal muscle monomeric myosin. When ionic strength was varied by changing [KCI], both smooth and skeletal muscle actin filament velocities increased with increasing ionic strength ([KCI], 12.5-75 mM) when observed over either phosphorylated smooth muscle (Figure 2A) or skeletal muscle (Figure 2B) myosin. No significant difference (p>0.05) was observed between the ionic strength dependence of skeletal versus smooth muscle actin motility over either smooth or skeletal muscle myosin. Harris and Warshaw Effect of Actin Isoform on Motility Smooth Muscle Myosin 221 Skeletal Muscle Myosin 0.8 0 0iW1 FIGURE 1. Bar graphs showing the velocity of smooth muscle versus skeletal muscle actin filaments with smooth muscle myosin (left panel) and skeletal muscle myosin (right panel). Data are mean + SD of six independent experiments. The comparison between actins was made by t test. 0 -J uJ Skeletal Actin Skeletal Actin Smooth Actin Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 Smooth and skeletal muscle actin motility reacted similarly to changes in pH when measured over either smooth or skeletal muscle myosin. Actin filament velocity on both phosphorylated smooth (Figure 3A) and skeletal (Figure 3B) muscle myosin reached a broad peak between pH 7.0 and pH 8.0. As [ATP] was reduced, actin filament velocity slowed for both skeletal and phosphorylated smooth muscle myosin (Figure 4). Since no difference was observed A Smooth Actin between the velocity of skeletal versus smooth muscle actin over either smooth or skeletal muscle myosin (p>0.05), actin filament types were combined for subsequent analysis. Double reciprocal (Lineweaver-Burk) analysis showed that the ATP-dependent Km of actin filament velocity on smooth muscle myosin (46 ,uM) was not significantly different from that on skeletal muscle myosin (53 ,uM). The ionic strength, pH, and [ATP] dependence of actin filament velocity over both phosphorylated A 1.5 r 0.9 F 1.0 [ S hi 0 E 0.6 F -j m 0 0 _i W 0 _3 W 0.3 h 0.5 F Smooth Muscle Myosin 0.0 0 25 B 50 75 (mM) [KCI] A l 0.0 6.0 100 t 6.5 7.0 osi 8.0 7.5 pH B 9r y Smooth Muscle Myosin 9r 0 E 6 E6 C.) 0 _1 W 0 -J3 W A 3F Skeletal Muscle Myosin Skeletal Muscle Myosin Dv 0 0 25 50 75 [KCII (mM) 100 FIGURE 2. Graphs showing smooth muscle actin filament (A) and skeletal muscle actin filament (o) velocity as a function of [KCl] with monomeric phosphorylated smooth muscle myosin (panel A) and skeletal muscle myosin (panel B) at pH 7.4, 1 mMATP, and 300C. Data are mean +SD. Solid lines are polynomialfits to data from both actin filament types: for panel A, y= -0.00014x2+0.02x+0.27 (r2=0.92); for panel B, y=0. 02x+5.08 (r2=0. 94). 6.0 6.5 7.5 7.0 8.0 8.5 pH FIGURE 3. Graphs showing smooth muscle actin filament (A) and skeletal muscle actin filament (o) velocity as a function of pH with monomeric phosphorylated smooth muscle myosin (panel A) and skeletal muscle myosin (panel B) at 25 mMKCl, 1 mMATP, and 300C. Data are mean ±SD. The solid lines are polynomial fits to data from both actin filament types: forpanel A, y=-0.33x2+5.0x-18.03 (r2=0.94); for panel B, y=-2.55x2+38.89x-141.63 (r2=0.86). Circulation Research Vol 72, No 1 January 1993 222 6r 0) E SMOOTH PHOSPHORYLATED MYOSIN (%) / Smooth Muscle / Myosin /// / / 4 100 0 w 60 40 20 0 20 40 60 80 100 / // a/// 0.75 W / 0 80 1.00 Er: / ¢ 0.50 2 /^/ / ,/ A W 0.25 /=:==-' / / -50 Skeletal Muscle Myosin 0 50 100 1/[ATP] (l/mM) Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 FIGURE 4. Double reciprocal plot showing the effect of [ATP] on actin filament velocity for smooth muscle actin (A) and skeletal muscle actin (o) at 25 mM KCI, pH 7.4, and 30°C with both monomeric phosphorylated smooth and skeletal muscle myosin. Solid lines are best-fit linear regression with 95% confidence limits as dashed lines. The comparison of the [A TP] needed for half-maximum actin filament velocity (ATP-dependent Km) for smooth versus skeletal myosin was made by noting that the 95% confidence limits of the linear regressions for the Lineweaver-Burk plots of the two myosins overlap at the x axis. The linear fits are as follows: for smooth muscle myosin, y=0.055x+1. 188 (r2=0.98); for skeletal muscle myosin, y=O.009x+0. 17 (r2=0. 96). smooth and skeletal muscle myosin monomers reported here (Figures 2-4) are in agreement with previous results from the motility assay.16 '7,23,24 Smooth and Skeletal Muscle Actin Filament Motility With Monomeric Myosin Mixtures Actin filament velocity over mixtures of myosins having different cycling rates may be determined by mechanical interactions between myosins.l7,2526 If so, comparison of smooth and skeletal muscle actin filament motility over mixtures of monomeric phosphorylated smooth and skeletal muscle myosins may reveal differences between the two actins under loaded conditions. When increasing amounts of monomeric phosphory- lated smooth muscle myosin were mixed with skeletal muscle myosin, the velocity of both smooth and skeletal muscle actin filament velocity dropped precipitously (Figure 5). By increasing the proportion of smooth muscle myosin to 32%, actin filament velocity was reduced by 50% of the velocity for skeletal muscle myosin alone. No significant difference (p>0.05) was observed between smooth and skeletal muscle actin velocities in this experiment. Discussion Actin's role in muscle contraction has been viewed generally as both a cofactor to enhance myosin ATPase activity and a passive mechanical structure that transmits force generated by myosin to the ends of the contractile unit (i.e., sarcomere in striated muscle). However, a series of recent studies on nonmammalian actin isoforms'4 and actins altered by enzymatic cleav- 0.00 L 0 SKELETAL MYOSIN (%) FIGURE 5. Smooth muscle actin filament (A) and skeletal muscle actin filament (o) velocities (V) plotted against monomeric myosin mixtures of phosphorylated smooth and skeletal muscle myosin at 25 mM KCl, pH 7. 4, 1 mM A TP, and 30°C. Velocities are mean values normalized to maximum value (Vmax) for each data set. Error bars are omitted for clarity. Solid line is fit to data sets from both actins. Vmax is 7.21 um/sec for smooth muscle actin and 7.43 gm/sec for skeletal muscle actin. The equation of the line is as follows: y=O.00009X2-O. 00057x+0.095 (r 2=O. 96). age,27 chemical cross-linking,28 and genetic engineering8s15 suggests that actin may play a significant role in the chemomechanical transduction process (see Morel and Mereh29 for review). In fact, models in which actomyosin force generation originates within the actin filament itself have been proposed.30 Is it possible that smooth muscle's enhanced force-generating capacity is related in part to the smooth muscle-specific actin isoform? Previous biochemical studies, designed to address this question, have shown that both smooth and skeletal muscle actin are equally capable of activating skeletal myosin ATPase activity.3' However, actomyosin ATPase measurements in solution merely reflect the enzymatic properties rather than the mechanical characteristics of the actomyosin interaction. Therefore, the in vitro motility assay provides a unique opportunity to assess the mechanical consequences of actin isoform on the actomyosin interaction through alterations in actin filament motility. At least three studies using the in vitro motility assay have attempted to characterize whether the velocity of actin motility is related to either the actin or the myosin isoform.i624,32 Kron and Spudich'6 demonstrated that Dictyostelium and rabbit skeletal muscle actin filament velocity are similar over both Dictyostelium and rabbit skeletal muscle myosin. Similarly, Umemoto and Sellers24 and Okagaki et a132 found no difference between the velocity of gizzard and skeletal muscle actin over smooth muscle myosin. These investigators concluded that actin filament motility was independent of actin isoform and that'the velocity of movement was governed by the myosin isoform. However, this conclusion was drawn in each case from a very limited experimental protocol performed at only one ionic strength, pH, and Harris and Warshaw Effect of Actin Isoform on Motility Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 [ATP]. Given that both muscle fiber mechanics and in vitro actin filament motility are exquisitely sensitive to the assay conditions, we believe that a comparison of smooth versus skeletal muscle actin motility over a far wider range of assay conditions was necessary before a definitive conclusion could be drawn about the effect of actin isoform on the actomyosin interaction in vitro. The results of the present study suggest that, over the wide ranges of pH and [KCl] investigated in which actin filaments are presumed to be under no load, the y-smooth muscle and a-skeletal muscle actin isoforms are mechanically indistinguishable over both smooth and skeletal muscle myosin. Even though the two actin isoforms reacted similarly to changes in assay conditions, actin isoform may still affect actin filament motility when actin filaments experience a load. We approached this problem by studying the motility of actin filaments with mixtures of smooth and skeletal muscle myosin. Mechanical interactions between myosin species occur in the motility assay whenever two myosin populations having different cycling rates attach to the same actin filament.172526 The resultant actin filament velocity is dependent on the force-generating capacity of the faster cycling myosin and the magnitude of the load created by the slower cycling species as it is negatively strained by the faster cycling crossbridges.17,21 Therefore, actin filaments moving over a myosin mixture must be experiencing the opposing forces created by the mechanical interactions between the two myosin species. Our laboratory has previously characterized the dependence of actin filament velocity on the proportion of phosphorylated smooth and skeletal muscle myosin.17 In these earlier studies in which skeletal muscle actin was used, actin filament velocity for an equal mixture of smooth and skeletal muscle myosin was more similar to the velocity over smooth muscle myosin alone. If actin isoform is crucial to force production, then one would predict that the shape of the relation between actin filament velocity and myosin mixture content might shift if the smooth muscle actin were used. However, no difference was observed between smooth versus skeletal muscle actin filament velocity over mixtures of smooth and skeletal muscle myosin (Figure 5). Once again, it appears that actin isoforms do not alter or modulate the force produced by the actomyosin interaction. This conclusion is supported by the finding that no difference was observed between the velocities of smooth and skeletal muscle actin filaments with either smooth or skeletal muscle myosin at low [ATP] (Figure 4), where slowing is presumably caused by the load imposed by rigor bridges. Therefore, differences in myosin isoforms between smooth and skeletal muscle may be the predominate cause for smooth muscle's enhanced stress production. These results argue against models proposing that force generation originates in the actin filament.30 The fact that actin isoforms are mechanically indistinguishable in the motility assay may relate to the conserved nature of actin. In fact, the naturally occurring amino acid substitutions that do exist between chicken a-skeletal and chicken y-smooth muscle actin are quite conservative with respect to amino acid charge and polarity.12 Therefore, the present results may not be too surprising. It still remains to be determined whether, under truly isometric conditions, changes in 223 actin isoform can modulate actomyosin force production. Future studies that include isometric force measurements on single actin filaments may reveal a mechanical difference between smooth and skeletal muscle actin.33 Alternatively, differences between actin isoforms may only be revealed when a more native smooth muscle thin filament, containing tropomyosin and possibly caldesmon and calponin, is reconstituted. Acknowledgments We would like to thank Steven Work for the computer programs used to track actin filament motion, Janet Woodcock-Mitchell and John Mitchell for running the SDS-PAGE, Gary Badger and Joe Carpenter for assistance with the statistical analysis, and Kathy Trybus for providing many of the proteins and for her comments and contributions to an earlier version of this manuscript. References 1. Fay FS, Rees DD, Warshaw DM: The contractile mechanism in smooth muscle, in Bitter EE (ed): Membrane Structure and Function. New York, John Wiley & Sons, Inc, 1981, vol 4, pp 79-130 2. Marston SB, Taylor EW: Comparisons of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles. J Mol Biol 1980;139:573-600 3. Murphy RA, Herlihy JT, Megerman J: Force-generating capacity and contractile protein content of arterial smooth muscle. J Gen Physiol 1974;64:691-705 4. Hellstrand P, Paul RJ: Vascular smooth muscle: Relations between energy metabolism and mechanics, in Crass MF, Barnes CD (ed): Vascular Smooth Muscle: Metabolic, Ionic, and Contractile Mechanisms. New York, Academic Press, Inc, 1982, pp 1-35 5. Haeberle JR, Hathaway DR, Smith CL: Caldesmon content of mammalian smooth muscles. J Muscle Res Cell Motil 1992;13:81-89 6. Ashton FT, Somlyo AV, Somlyo AP: The contractile apparatus of vascular smooth muscle: Intermediate high voltage stereo electron microscopy. J Mol Biol 1975;98:17-29 7. Kabsch W, Mannherz HG, Suck D, Pai EF, Holmes KC: Atomic structure of the actin: DNase 1 complex. Nature 1990;347:37-43 8. Drummond DR, Peckham M, Sparrow JC, White DCS: Alteration in crossbridge kinetics caused by mutations in actin. Nature 1990; 348:440-442 9. Vandekerckhove J, Weber K: Actin typing on total cellular extracts. Eur J Biochem 1981;113:595-603 10. Small JV, Sobieszek A: Contractile and structural proteins of smooth muscle, in Stephens NL (ed): Biochemistry of Smooth Muscle. Boca Raton, Fla, CRC Press, 1983, vol 1, pp 85-140 11. Fatagati V, Murphy RA: Actin and tropomyosin variants in smooth muscle. J Biol Chem 1984;259:14383-14388 12. Vandekerckhove J, Weber K: The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. Differentiation 1979;14: 123-133 13. Sutoh K: Identification of myosin-binding sites on the actin sequence. Biochemistry 1982;21:3654-3661 14. Kron SJ, Drubin DG, Botstein D, Spudich JA: Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin. Proc Natl Acad Sci U S A 1992;89:4466-4470 15. Sutoh K, Ando M, Sutoh K, Toyoshima YY: Site-directed mutations of Dictyostelium actin: Disruption of a negative charge cluster at the N terminus. Proc Natl Acad Sci U S A 1991;88:7711-7714 16. Kron SJ, Spudich JA: Fluorescent actin filaments move on myosin fixed to a glass surface. Proc Natl Acad Sci U S A 1986;83: 6272-6276 17. Warshaw DM, Desrosiers JM, Work SS, Trybus KM: Smooth muscle cross-bridge interactions modulate actin filament sliding velocity in vitro. J Cell Biol 1990;111:453-463 18. Pardee JD, Spudich JA: Purification of muscle actin. Methods Enzymol 1982;85:164-181 19. Strzelecka-Golaszewska H, Prochniewicz E, Nowak E, Smorzynski S, Drabikowski W: Chicken-gizzard actin: Polymerization and stability. Eur J Biochem 1980;104:41-52 20. Mitchell JJ, Low RB, Woodcock-Mitchell JL: Cytomatrix synthesis in MDK epithelial cells. J Cell Physiol 1990;143:501-511 224 Circulation Research Vol 72, No 1 January 1993 21. Warshaw DM, Desrosiers JM, Work SS, Trybus KM: Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin. J Biol Chem 1991;266:24339-24343 22. Work SS, Warshaw DM: Computer-assisted tracking of actin filament motility. Anal Biochem 1992;202:275-285 23. Harada Y, Noguchi A, Kishino A, Yanagida T: Sliding movement of single actin filaments on one-headed myosin filaments. Nature 1987;326:805-808 24. Umemoto S, Sellers JR: Characterization of in vitro motility assays using smooth muscle and cytoplasmic myosins. J Biol Chem 1990; 265:14864-14869 25. Sellers JR, Spudich JA, Sheetz MP: Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J Cell Biol 1985;101:1897-1902 26. Tawada K, Sekimoto K: A physical model of ATP-induced actin- myosin movement in vitro. Biophys J 1991;59:343-356 27. Schwyter DH, Krin SJ, Toyoshima YY, Spudich JA, Reisler E: Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin. J Cell Biol 1992;111:465 -470 28. Prochiewicz E, Yanagida T: Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility. J Mol Biol 1990;111:465-470 29. Morel JE, Mereh Z: Muscle contraction and in vitro movement: Role of actin? J Muscle Res Cell Motil 1992;13:5-6 30. Schutt CE, Lindberg U: Actin as the generator of tension during muscle contraction. Proc Natl Acad Sci U S A 1992;89:319-323 31. Hartshorne DJ: Biochemistry of the contractile process in smooth muscle, in Johnson LR (ed): Physiology of the Gastrointestinal Tract. New York, Raven Press, Publishers, 1981, pp 243-267 32. Okagaki T, Higashi-Fujime S, Ishikawa R, Takano-Ohmuro H, Kohama K: In vitro movement of actin filaments on gizzard smooth muscle myosin: Requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon. J Biochem 1991; 109:858-866 33. Kishino A, Yanagida T: Force measurements by micromanipulation of a single actin filament by glass needles. Nature 1988;334: 74-76 Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 Smooth and skeletal muscle actin are mechanically indistinguishable in the in vitro motility assay. D E Harris and D M Warshaw Downloaded from http://circres.ahajournals.org/ by guest on April 3, 2017 Circ Res. 1993;72:219-224 doi: 10.1161/01.RES.72.1.219 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1993 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. 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