In vitro at low ionic strength (pm = 0.02 M) and 50C, myosin subfragment-I shows significant binding to regulated actin in the presence of ATP, independent of the concentration of free Ca'. Under the same conditions, single skinned rabbit psoas muscle fibers develop force only in the presence of Ca2+ and are relaxed in its absence. However, the stiffness, measured with very rapid stretches (0.5% ofmuscle length in 0.1Ims), is high even when the fibers are relaxed. This "rapid stiffness" of the resting muscle is sensitive to ionic strength, becoming small at normal ionic strength (p = 0.17 M). At low ionic strength, the rapid stiffness is approximately proportional to the overlap between the actin and myosin filaments. At zero overlap (sarcomere length = 3.8 pim), the stiffness is less than 20% ofthe value measured at full overlap. This remaining 20% is relatively insensitive to ionic strength, like the passive resting tension, and it may in fact be due to the structures responsible for the~resting tension. Thus, both in vitro binding and the effect of overlap on rapid stiffness measurements in fibers suggest that cross-bridges are attached to actin in relaxed muscle at low' ionic strength. (8). The troponintropomyosin complex was prepared according to Eisenberg and Kielley (9). Regulated actin was prepared by mixing troponintropomyosin with actin in a molar ratio of 1.5:7. The binding of S-1 to actin-troponin-tropomyosin (regulated actin) was determined by using a Beckman air-driven ultracentrifuge as described earlier (5) except that the centrifugation was at 5YC and for 30 min.Fiber Preparation. Single fibers were prepared from bundles cut from the lateral margin of rabbit psoas muscles. They were kept for up to several hours in skinning solution (10, 11); before the experiments they were soaked for 30 min in 1:3 (vol/ vol) glycerol/skinning solution to ensure permeability of the sarcolemma to larger molecules such as creatine kinase. Low ionic strength relaxing solution contained 1 mM EGTA, 3 mM MgCl2, 1 mM Na2ATP, and 10 mM imidazole, pH 7.0. Contracting solution, pH 7.0, contained 1 mM CaEGTA, 3 mM MgCl2, 10 mM imidazole, 1 mM Na2ATP, 10 mM phosphocreatine, and creatine kinase at 300 Sigma units/m-l. All solutions also contained 1 mM dithiothreitol. Ionic strength was increased, where appropriate, with KC1.Mechanical Apparatus. The mechanical setup is described elsewhere (11). The moving coil was modified to increase its stiffness in order-to reduce possible oscillations when using a feedback system to control the muscle length. The time required to complete a stepwise change in length was about 0.3 ms. The stiffness measurements described below were complete in about 100 pus. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Remodelling of the contractile apparatus within smooth muscle cells is an essential process that allows effective contractile activity over a wide range of cell lengths. The thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. The structure of this folded molecule has been controversial. Using negative stain electron microscopy of individual folded molecules from turkey gizzard we show they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Smooth muscle heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin whose features are identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2-D crystals on lipid.

Myosin subfragment 1 (S-1) with its two reactive cysteine groups crosslinked by N,N'-p-phenylenedimaleimide (pPDM), is shown to be a stable analogue of S-IFATP and S-I'ADP-P;, the predominant complexes present during the steadystate hydrolysis of ATP by S-1. pPDM-S-1 binds to actin with about twice the affinity of S-IATP or S-1ADP-P;, whereas its affinity is 1/100th of that of S-15'-adenylyl imidodiphosphate and 1/1,000th of that of S-1FADP. pPDM-S-i is also similar to S-IFATP and SIlADP Pi in that its binding to actin is not inhibited by troponintropomyosin. In contrast, the binding of S-1, S-FADP, and S-1F5'-adenylyl imidodiphosphate to actin is markedly inhibited by troponin-tropomyosin in the absence of Ca2" when actin is in large excess over S-1. This suggests that modifying S-1 with pPDM stabilizes a conformation which mimics that induced by the binding of ATP.ping a molecule of ADP (10). Two observations suggest that pPDM-S-1 may be similar to S-I ATP. The circular dichroism spectrum of pPDM-S-1 resembles that of S-1 in the presence of ATP and, like S-1 in the presence of ATP, pPDM-S-1 does not bind to actin at high ionic strength (11). Under the same high ionic strength conditions, S-1 having both sulfhydryl groups alkylated but not crosslinked is able to bind to actin. On the other hand, the intrinsic fluorescence of pPDM-S-1 resembles that of S-IPADP rather than S-1FATP (11,12). To determine whether pPDM-S-1 is an analogue of S-IFATP or S-1FADP we have thoroughly studied the binding of pPDM-S-1 to actin in both the presence and the absence of troponin-tropomyosin. Our results suggest that pPDM-S-1 is an excellent analogue of S-1ATP.Force generation in vertebrate skeletal muscle is thought to occur as a result of a cyclic interaction of myosin cross-bridges with actin (1). Various cross-bridge models of muscle contraction suggest that the globular head region of myosin undergoes a rotation or a conformational change while attached to actin, causing movement of the thin actin filaments past the thick myosin filaments (2-4). After this step of shortening, the myosin molecule detaches, and then reattaches to a different actin monomer to repeat the process. Because this cyclic process is coupled to the hydrolysis of ATP, it is thought that myosin must exist in two or more conformational states as ATP is hydrolyzed. In the absence of ATP (rigor) the cross-bridge seems to be bound to actin at a 450 angle (5, 6), whereas in the relaxed state this angle may be closer to 900 (5). Eisenberg and Greene have suggested that the 900 or relaxed conformation may be associated with states containing bound ATP or ADP-Pi, whereas the 450 or rigor conformation may be associated with states containing bound ADP or no nucleotide (7). Accordingly, it is of interest to be able to dissect and study the individual states. It is possible to study the structure of acto S-1 (S-1, myosin subfragment 1) in the "450" state, but it is difficult to study the structure of the "90°" ATP-bound state because of the rapidity with wh...

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Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.electron microscopy ͉ molluscan muscle ͉ regulation ͉ smooth muscle ͉ image processing

Recent theoretical work on the cooperative equilibrium binding of myosin subfragment-1-ADP to regulated actin, as influenced by Ca2+, is extended here to the cooperative steady-state ATPase activity of myosin subfragment-1 on regulated actin. Exact solution of the general steady-state problem will require Monte Carlo calculations. Three interrelated special cases are discussed in some detail and sample computer (not Monte Carlo) solutions are given. The eventual objective is to apply these considerations to in vitro experimental data and to in vivo muscle models.