调节轻链在平滑肌肌球蛋白分子上的定位

 应用凝胶电泳覆盖技术和放射自显影法研究了32~P-标记的平滑肌肌球蛋白调节轻链在肌球蛋白分子上的定位。实验结果表明调节轻链(LC_(20))可重新结合于平滑肌肌球蛋白重链(200kD),重酶解肌球蛋白(130kD)及其62kD和26kD肽段上。这提示调节轻链的结合点位于平滑肌肌球蛋白亚段-1羧基端的26kD肽段上。     

Acrylamide fluorescence quenching studies on the actin-induced change in protein dynamics in the subfragment-1/subfragment-2 link region of cardiac myosin

In order to obtain information about the actin-induced conformational change around the subfragment-1/subfragment-2 link region of myosin, measurements of the fluorescence quenching by acrylamide were made on cardiac myosin and its heavy meromyosin, in which the reactive lysyl residue located in the link region was labeled with an extrinsic fluorophore, the N-methyl-2-anilino-6-naphthalenesulfonyl group. The results with the model compound indicated the involvement of a collisional quenching mechanism for the fluorophore. The quenching rate constant calculated from measured quenching constants using available lifetime data was extremely low for the labeled myosin (0.59 X 10(8) M-1 . S-1), suggesting that the fluorophore bound to myosin is surrounded by segments of proteins. This value was independent of the solvent viscosity, indicating that the quenching reaction is limited by fluctuations in the protein matrix, which produce the inward movement of acrylamide. Chymotryptic digestion of the labeled myosin, which yielded the light chain-2-deficient heavy meromyosin, made the bound fluorophore slightly exposed. Addition of F-actin resulted in about 40% reduction in the quenching rate constants for the labeled myosin and heavy meromyosin. The actin effect was reversed by adding ATP. These results suggest that the binding of actin to myosin makes the protein matrix around the subfragment-1/subfragment-2 link region less mobile.     

Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

Regulation of smooth muscle contractile proteins by calmodulin and cyclic AMP

The various protein components of a reversible phosphorylating system regulating smooth muscle actomyosin Mg-ATPase activity have been purified. The enzyme catalyzing phosphorylation of smooth muscle myosin, myosin-kinase, requires Ca2+ and the Ca2+-binding protein calmodulin for activity and binds calmodulin in a ratio of 1 mol calmodulin to 1 mol of myosin kinase. Myosin kinase can be phosphorylated by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, and phosphorylation of myosin kinase that does not have calmodulin bound results in a marked decrease in the affinity of this enzyme for Ca2+-calmodulin. This effect is reversed when myosin kinase is dephosphorylated by a phosphatase purified from smooth muscle. When the various components of the smooth muscle myosin phosphorylating-dephosphorylating system are reconstituted, a positive correlation is found between the state of myosin phosphorylation and the actin-activated Mg-ATPase activity of myosin. Unphosphorylated and dephosphorylated myosin cannot be activated by actin, but the phosphorylated and rephosphorylated myosin can be activated by actin. The same relationship between phosphorylation and enzymatic activity was found for a chymotryptic peptide of myosin, smooth muscle heavy meromyosin. The findings reported here suggest one mechanism by which Ca2+ and calmodulin may act to regulate smooth muscle contraction and how cAMP may modulate smooth muscle contractile activity.     

Localization of a light-chain binding site on smooth muscle myosin revealed by light-chain overlay of sodium dodecyl sulfate-polyacrylamide electrophoretic gels

The interactions of smooth muscle myosin and its light chains have been examined by incubating sodium dodecyl sulfate-polyacrylamide gels of myosin with radioactively labeled regulatory or essential light chains. The technique involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fixation with methanol and acetic acid followed by an extensive series of washes. The gel is incubated overnight with labeled light chains in the presence of bovine serum albumin and then washed extensively to remove unbound protein. Following staining and destaining, the gel is autoradiographed to reveal which protein bands have bound light chain. The myosin heavy chain was able to rebind labeled regulatory or essential light chains despite the harsh procedure described above. By fragmenting the myosin heavy chain proteolytically, we were able to determine the binding site for both types of light chains to be within the 26,000-Da COOH-terminal segment of smooth muscle subfragment 1 (S-1) or the 20,000-Da COOH-terminal segment of skeletal muscle S-1. The extent of binding was 0.1-0.4 mol of light chain/mol of S-1 heavy chain. No binding was observed to portions of the myosin molecule which do not contain this segment such as myosin rod, light meromyosin, S-2, or the NH2-terminal 75,000-Da segment of S-1.     

Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis

Monoclonal antibodies were used to identify and localize by immunoelectron microscopy epitopes on myosin isozymes. An antibody that reacts with an amino-terminal fragment of the myosin heavy chain maps on the myosin head 140 A distal to the head-rod junction. It identifies an epitope that is shared on adult and embryonic myosin, and detects two transitions in myosin expression during avian pectoralis myogenesis. Another antibody maps to the carboxyl terminus of the myosin rod. It is specific for an adult fast myosin epitope that is not detected in early developing pectoralis muscle. In contrast, an epitope that is present throughout development is identified by an antibody that reacts with a myosin light chain. This light chain epitope is localized at the head-rod junction. These results demonstrate structural changes in widely separated regions of the myosin molecule accompanying the sequential expression of developmental myosin isozymes.     

Light chain dependent effects of actin binding on the S-1/S-2 swivel in myosin

The S-1/S-2 swivel in myosin provides a flexible link between the head and tail portions of the molecule. We have investigated the properties of the swivel by employing limited proteolysis methods. Our results indicate that the binding of actin to heavy meromyosin inhibits both the chymotryptic and papain cleavage of the S-1/S-2 swivel, and that this effect is dependent on the presence of intact LC-2 light chains. Actin did not slow digestions carried out using heavy meromyosin previously treated with proteases to nick the LC-2 chains to 17,000 or 14,000 Mr fragments. Although the integrity of the LC-2 light chain appears to be required to transmit the effects of actin binding from the myosin head to the S-1/S-2 swivel, the binding of Ca2+ to the 17,000 Mr LC-2 fragment can still affect the chemical reactivity of SH1 thiol groups. Both chymotryptic and papain digestions of heavy meromyosin containing intact or fragmented LC-2 light chain show substantial temperature sensitivity between 5 degrees C and 35 degrees C. Calculated apparent activation energies for this process indicate that the S-1/S-2 swivel in myosin can undergo temperature-dependent structural changes independently of the state of the LC-2 light chain. Thus, both actin binding and temperature variations can induce structural transitions in the S-1/S-2 swivel.     

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-actin filaments and myosin heads attached to them have been measured by saturation transfer electron paramagnetic resonance spectroscopy using spin-labels rigidly bound to actin, or to the myosin head region in intact myosin molecules, heavy meromyosin, and subfragment-1. The spin-label attached to F-actin undergoes rotational motion having an effective correlation time of the order of 10^?4 seconds. This cannot be interpreted as rotation of the entire F-actin filament or local rotation of the spin-label, but must represent an internal rotational mode of F-actin, possibly a bending or flexing motion, or a rotation of an actin monomer or a segment of it. The rate of this rotational motion is reduced approximately fourfold by myosin, HMM or S-1; HMM and S-1 are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S-1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S-1 prepared with papain in the presence of Mg²⁺, the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the actin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-actin is controlled by a mode of rotation within the F-actin filaments.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.     

Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.     

Immobilization of actin and myosin.

Using glutaric dialdehyde, the muscle proteins myosin, actin, actomyosin and heavy meromyosin subfragment-1 (S-1) have been immobilized on capron fibers. The ATPase activity of myosin and its capability to interact with actin have been preserved whereas the ATPase activity of its subfragment decreased significnatly. Immobilization on capron fibers changes the pH dependence of the ATPase activity of myosin and of S-1 shifting the maximum towards the acid zone (pH 5.5) and increases the thermal stability of the enzyme. Calcium ions produce a stimulatory effect on ATPase; Mg2+ions yield no effect on myosin and S-1 but enhance the activity in the case of immobilized actomyosin though to a lesser degree than the ions of Ca2+. Immobilized actin retains its ability to form actomyosin complex.     

Preparation and characterization of heavy meromyosin and subfragment 1 from vertebrate cytoplasmic myosins

The soluble fragments of myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) have been instrumental in elucidating the kinetic mechanisms of the actin-activated MgATPase activity of both skeletal and smooth muscle myosin. To date, relatively little has been published on these fragments from vertebrate cytoplasmic myosins. We now describe the preparation and steady-state kinetic characterization of S-1 and HMM from human platelet and avian intestinal epithelial brush border myosin. The HMM prepared from each of these tissues was similar both in their SDS-polyacrylamide gel pattern and in their steady-state kinetic properties. The Vmax of the actin-activated MgATPase activity varied between 0.8 and 2.5 s-1, and the KATPase (the apparent dissociation constant derived from a double-reciprocal plot of the MgATPase activity) was about 1-2 microM. This low value for the apparent dissociation constant was similar to the dissociation constant of HMM for actin directly measured under similar conditions and is about 40 times lower than that determined with avian smooth muscle HMM. The KATPase of the cytoplasmic HMM was only slightly increased when the ionic strength was raised from 12 to 112 mM.     

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

Antibody against 25,000 dalton tryptic fragment of subfragment-1 from chicken skeletal muscle myosin: functional implication of the 25,000 fragment region in subfragment-1

Antibody was prepared against the 25,000-dalton tryptic fragment of subfragment-1 from skeletal muscle myosin. The antibody was found to inhibit the Mg2+-ATPase activity and the initial P1-burst of the ATPase. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. The acto-S-1 ATPase activity was also inhibited by the antibody. These results suggest that there is a site in the 25K fragment region responsible for the transition of the myosin-ATP complex to another high energy complex.     

Incorporation of the Ca2+ -binding component (g2) into heavy meromyosin and subfragment-1 of pig cardiac myosin.

A new protein component was found in heavy meromyosin and in subfragment-1 (S-1) prepared by chymotrypsin digestion of pig cardiac myosin in the presence of Ca2+. The molecular weight of this protein was estimated as 15,000 dalton. It was able to bind Ca2+ and showed a similar UV absorption spectrum to that of the g2 light chain. Heavy meromyosin and subfragment-1 which contained the 15,000 dalton component incorporated exogenous g2 and the 15,000 dalton component disappeared after such treatment. We concluded that the 15,000 dalton component was produced from g2 by limitted proteolysis. The subfragment-1 was separated into two protein fractions in equal yield by recycling the gel filtration. One contained the 15,000 dalton component and was able to bind Ca2+ while the other did not contain the component and was unable to bind Ca2+. According to analysis by SDS gel electrophoresis, the large polypeptide chain (the f component) of the first S-1 was approximately 5,000 dalton larger than the f component of the second S-1. The polypeptide corresponding to 5,000 dalton was designated polypeptide-C, because it was released from the C terminal of the f component. It seems to be essential for the attachment of the Ca2+-binding light chain g2. The location of g2 in myosin may thus be at the polypeptide-C which links the head to the tail of myosin.     

Mode of caldesmon binding to smooth muscle thin filament: possible projection of the amino-terminal of caldesmon from native thin filament.

The structure of smooth muscle thin filament was examined by various electron microscopy techniques, with special attention to the mode of caldesmon binding. Chemical cross-linking was positively used to avoid the dissociation of accessory proteins upon dilution. Caldesmon in reconstituted thin filament was observed as fine filamentous projections from thin filament. Native thin filament isolated from smooth muscle showed similarly numerous fine whisker-like projections by all the techniques employed here. Antibody against the amino-terminus of caldesmon labeled the end of such projections indicating the possibility that the amino-terminal myosin binding moiety might stick out from the shaft of the thin filament. Such whiskers are often projected out as a cluster to the same side of native thin filament. Further, we could visualize the assembly of dephosphorylated heavy meromyosin (HMM) with native or reconstituted thin filament forming "nonproductive" complex in the presence of ATP. The association of HMM to the shaft of thin filament was through subfragment-2 moiety, in accordance with biochemical studies. Some HMM particles bound closer to the thin filament shaft, possibly suggesting the presence of the second myosin-binding site on caldesmon. Occasionally two kinds of HMM association as such coexisted at a single site on this filament in tandem. Thus, we constructed a structural model of thin filament. The proposed molecular arrangement is not only compatible with all the biochemical results but also provides additional support for our recent findings (E. Katayoma, G. C. Scott-Woo, and M. Ikebe (1995) J. Biol. Chem. 270, 3919-3925) regarding the capability of caldesmon to induce dephosphorylated myosin filament, which explains the existence of thick filaments in relaxed smooth muscle cells.     

Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards

A phosphoprotein phosphatase that dephosphorylates smooth muscle myosin has been purified to apparent homogeneity from turkey gizzards. Smooth muscle phosphatase (SMP) IV has a molecular weight of 150,000 as determined by gel filtration on a Sephadex G-200 column and is composed of two subunits (Mr = 58,000 and 40,000). Although it is active toward a number of proteins, its activities toward the contractile proteins, intact myosin, heavy meromyosin, and isolated myosin light chains are higher than its activities toward phosphorylase alpha, histone IIA, and phosphorylase kinase. SMP-IV preferentially dephosphorylates the beta-subunit of phosphorylase kinase. The properties of the enzyme have been studied using heavy meromyosin, a soluble chymotryptic fragment of myosin, and isolated myosin light chains as substrates. SMP-IV has high affinity for both substrates and is optimally active at neutral pH. Divalent cations, Ca2+ and Mg2+, activate the dephosphorylation of heavy meromyosin but inhibit the activity toward myosin light chains. Low concentrations of ATP (1-5 mM) activate SMP-IV but concentrations higher than 5 mM are inhibitory. Inhibition of 50% of the activity of the enzyme by NaF and PPi requires concentrations higher than 10 mM. Rabbit skeletal muscle heat stable inhibitor-2 has no effect on the activity of SMP-IV toward heavy meromyosin, myosin light chains, and phosphorylase alpha.     

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin

Antibodies with epitopes near the heavy meromyosin/light meromyosin junction distinguish the folded from the extended conformational states of smooth muscle myosin. Antibody 10S.1 has 100-fold higher avidity for folded than for extended myosin, while antibody S2.2 binds preferentially to the extended state. The properties of these antibodies provide direct evidence that the conformation of the rod is different in the folded than the extended monomeric state, and suggest that this perturbation may extend into the subfragment 2 region of the rod. Two antihead antibodies with epitopes on the heavy chain map at or near the head/rod junction. Magnesium greatly enhances the binding of these antibodies to myosin, showing that the conformation of the heavy chain in the neck region changes upon divalent cation binding to the regulatory light chain. Myosin assembly is also altered by antibody binding. Antibodies that bind to the central region of the rod block disassembly of filaments upon MgATP addition. Antibodies with epitopes near the COOH terminus of the rod, in contrast, promote filament depolymerization, suggesting that this region of the tail is important for assembly. The monoclonal antibodies described here are therefore useful both for detecting and altering conformational states of smooth muscle myosin.