Subfragment 1 Research Articles

Denaturation mode of carp myofibrils when affected by temperature and salt concentration

Heating temperatures of 30-40°C and KCl concentrations of 0.1-0.5 M altered the denaturation mode of carp myofibrils. In 0.1 M KCl medium, heating temperature affected the denaturation of rod more significantly than of subfragment-1 (S-1), and a slow decrease in solubility at 30°C was accompanied by a slow denaturation of rod. KCl concentration at heating altered the denaturation mode differently at 30°C and 40°C. Increased KCl concentrations for heating reduced the rod denaturation rate at 40°C, but it was increased at 30°C. At concentrations above 0.3 M KCl, the denaturation rate for rod became identical to that for S-1 at both temperatures. Upon heating of chymotryptic digest of myofibrils, S-1 denaturation was similarly detected as in intact myofibrils, whereas practically no rod denaturation was detected. Thus, it was concluded that myosin structure connecting S-1 and rod has an important role in the denaturation process.

Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1.

A new fluorescent ribose-modified ATP analogue, 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic]-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1.NBD-ADP.BeF(n) and S-1.NBD-ADP.AlF(4)(-) complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10(-2) s(-1) for BeF(n) and 1.1 x 10(-3) s(-1) for AlF(4)(-). These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1.ADP.BeF(n) or AlF(4)(-) reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.

Changes in myosin S1 orientation and force induced by a temperature increase.

Force generation in myosin-based motile systems is thought to result from an angular displacement of the myosin subfragment 1 (S1) tail domain with respect to the actin filament axis. In muscle, raised temperature increases the force generated by S1, implying a greater change in tail domain angular displacement. We used time-resolved x-ray diffraction to investigate the structural corollary of this force increase by measuring M3 meridional reflection intensity during sinusoidal length oscillations. This technique allows definition of S1 orientation with respect to the myofilament axis. M3 intensity changes were approximately sinusoid at low temperatures but became increasingly distorted as temperature was elevated, with the formation of a double intensity peak at maximum shortening. This increased distortion could be accounted for by assuming a shift in orientation of the tail domain of actin-bound S1 toward the orientation at which M3 intensity is maximal, which is consistent with a tail domain rotation model of force generation in which the tail approaches a more perpendicular projection from the thin filament axis at higher temperatures. In power stroke simulations, the angle between S1 tail mean position during oscillations and the position at maximum intensity decreased by 4.7 degrees, corresponding to a mean tail displacement toward the perpendicular of 0.73 nm for a temperature-induced force increase of 0.28 P(0) from 4 to 22 degrees C. Our findings suggest that at least 62% of crossbridge compliance is associated with the tail domain.

γ-Amido-ATP stabilizes a high-fluorescence state of myosin subfragment 1

On binding to myosin subfragment 1 (S1), the γ-amido derivative of ATP (ATPγNH 2), an isomer of adenosine 5′-[β,γ-imido]-triphosphate (AMPPNP), induces a larger increase in the intrinsic (tryptophan) fluorescence than is seen with ATP. A binding constant of 1.7×10 7 M −1 was measured for ATPγNH 2, compared to 2.1–2.4×10 7 M −1 for AMPPNP. ATPγNH 2 was hydrolyzed only very slowly by S1. ATPγNH 2 appears to stabilize the ‘closed’ conformation of S1, and does so without cleavage of the β–γ phosphate bond. The dissociation of actin-S1 by ATPγNH 2 and that of S1.ATPγNH 2 by actin are both strikingly slow.

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament.

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.

β-Myosin Heavy Chain Gene Mutations in Familial Hypertrophic Cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder arising from mutations in sarcomeric protein genes. Human genetic studies have implicated at least 9 different genes in FHC, emphasizing the enormous genetic and allelic heterogeneity associated with FHC.1 β-Myosin heavy chain (βMyHC, MYH7 ) is the most commonly mutated gene in FHC, and at least 60 different MYH7 gene mutations have been described in human FHC subjects.2 The vast majority of these are single base pair mutations that produce missense amino acid substitutions. Myosin is a hexamer composed of 2 heavy chains and 2 each of the regulatory and essential light chains (see Figure). Myosin is a highly asymmetric protein with a long rod domain and 2 globular heads. The globular domain of myosin, heavy meromyosin (HMM), can be proteolyzed into subfragment 1 (S1) and subfragment 2 (S2). The S1 head is the enzymatic “business” end of the molecule in that it possesses actin-activated ATPase activity and is capable of directing the sliding of actin filaments in vitro. The crystal structure of S1 has been determined, shedding light on the potential conformational changes that occur in response to ATP hydrolysis and that ultimately are responsible for myocyte shortening, skeletal muscle movement, and the beating of our hearts.3 Myosin was one of the first proteins to be purified and proteolytically separated into distinct functional domains contributing to the domain theory of proteins. Similarly for human genetic studies, the domain structure of myosin has proved useful because all the reported missense mutations in βMyHC map to the head and neck region, a region of the rod most proximal to the globular head. Within the S1 head, FHC mutations cluster to …

Stabilization of squid myosin subfragment-1 and myofibrils by ATP-related compounds

: Adenosine triphosphatase (ATPase) inactivation of squid myosin subfragment-1 (S-1) was strongly suppressed upon addition of adenylimidophosphate (AMPPNP), an analogous compound of adenosine triphosphate (ATP); whereas suppression by adenosine diphosphate (ADP) and pyrophosphate (PPi) was not as effective. Adenylimidophosphate did not stabilize myofibrils in which myosin is stabilized by F-actin. Thus, the stabilization effect by F-actin and AMPPNP is not additive. Pyrophosphate markedly accelerated the inactivation of myofibrils through the loss of protection by F-actin. The suppressive effect of F-actin on myosin ATPase inactivation was much greater than that by AMPPNP. The stabilization effects of AMPPNP and F-actin on S-1 were almost completely lost when the medium contained approximately 1 M KCl.

A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1.

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 [Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP (2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate), is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of 20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.

Dynamic docking of myosin and actin observed with resonance energy transfer.

Atomic models of myosin subfragment-1 (S1) and the actin filament are docked together using resonance energy-transfer data from both pre- and postpowerstroke conditions. The quality of the resulting best fits discriminated between neck-region orientations of the S1 for a given set of experimental conditions. For measurements of the postpowerstroke states in the presence of ADP, resonance energy-transfer data alone are sufficient to dock the atomic models and provide evidence that S1 exists with at least two neck-region orientations under these conditions. To dock the prepowerstroke state, resonance energy-transfer data were used in combination with previous chemical cross-linking data to determine that a neck-region orientation similar to that of a proposed prepowerstroke state best fit the data. The resulting models determined independently from electron microscopy compare favorably with micrographs from the recent literature. The docking models by resonance energy transfer suggest that the larger movements in the light-chain binding domain are accompanied by twisting and rotating movements of the catalytic domain, causing a tilt of approximately 30 degrees during the weak-to-strong transition. This transition provides the displacement necessary to support motility and force generation.

The alanine-scanning mutagenesis of Dictyostelium myosin II at the ionic interface with actin.

Cyclic interaction of actin and myosin coupled with ATP hydrolysis is essential for generation of force by the actin-myosin system. The major force maintaining the actin-myosin contact during ATP hydrolysis, actin-myosin sliding and force generation seems to be ionic interactions, since these functions are highly sensitive to the ionic strength of the solvent. In the absence of ATP, i.e., under rigor conditions, however, hydrophobic interactions become dominant in maintaining the actin-myosin association. Based on the three-dimensional reconstruction of electron microscopic images of the rigor complex of actin and myosin subfragment 1 (S1), these ionic and hydrophobic interaction sites have been tentatively assigned to several locations of actin and myosin (Schroder et al. 1993; Milligan 1996).KeywordsActin FilamentMalachite GreenBasic ResidueHeavy Chain GeneDictyostelium CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Actin in Pumpkin Phloem Exudate

When analyzing cytoskeletal proteins in Cucurbita pepo phloem exudate by immunoblotting, we detected actin in an amount comparable to that in some plant tissues and a small amount of α-tubulin. Electron-microscopic examination of the exudate permitted us to observe filaments that were capable of interacting with the myosin subfragment S1 from rabbit skeletal muscle and with phalloidin conjugated with colloidal gold. The addition of 0.5 mM phalloidin to the exudate in the medium containing 20 mM dithiothreitol (DTT) resulted in an increased number of filaments. Since high DTT concentrations induce a breakdown of filaments of the phloem protein PP1, it seems likely that the produced filaments were composed of actin. The addition of 50 mM MgCl2 to the exudate resulted in the formation of dense bundles and paracrystals, which resembled those produced by muscle actin under similar conditions. Our results demonstrated that actin in phloem sap was capable of polymerization with filament formation.

A small-molecule inhibitor of skeletal muscle myosin II.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

Role of neck region in the thermal aggregation of myosin.

Carp dorsal myosin formed oligomers that retained ATPase activity upon heating. Cleavage of the oligomeric myosin at subfragment-1 (S-1)/rod junction released monomeric S-1 and rod, indicating that ATPase retaining myosin associated near the S-1/rod junction. The digest also contained rod oligomers. Heating a mixture of S-1 and rod generated neither ATPase retaining S-1 oligomers nor rod oligomers. Electron microscopic observation of the heated myosin revealed that some oligomers were formed by associating at the S-1/rod joining region, exhibiting a recognized double head, probably ATPase retaining oligomers. No myosin oligomers associated at the tail region were observed, thus, rod aggregation would be formed at its very restricted region near the S-1/rod junction. Based on the findings, we proposed that the neck structure is important in the thermal oligomerization process of myosin.

The interdomain motions in myosin subfragment 1

The interdomain motions in myosin subfragment 1 (S1) were studied by steady-state and time-resolved fluorescence of tryptophan residues and N-(iodoacetyl)- N′-(5-sulfo-1-naphtyl)ethylenediamine (AEDANS) attached to Cys178 of alkali light chain 1 (A1) exchanged into S1. The efficiency of fluorescence resonance energy transfer (FRET) from tryptophan residues of motor domain to AEDANS at A1 decreased dramatically after addition of ATP to S1A1-AEDANS. The efficiency of FRET calculated from the crystal structure of chicken S1 corresponded to the experimental one measured in the presence of ATP. The results showed that AEDANS at Cys178 of A1 became more mobile and distant from the motor domain of S1 upon ATP binding. These findings led to the suggestion that a release of the products of ATP hydrolysis and power stroke might be associated with movement of light chain-binding domain towards the N-terminal domain of S1.

Effect of deuterium oxide on actomyosin motility in vitro

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D 2O) and water (H 2O) to examine the effect of D 2O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D 2O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H 2O assay solution, but the maximum velocity (4.1±0.5 μm/s, n=11) at pD 8.5 in D 2O was about 60% of that (7.1±1.1 μm/s, n=11) at pH 8.5 in H 2O. The K m values of 95 and 80 μM and V max values of 3.2 and 5.1 μm/s for the D 2O and H 2O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis–Menten equation. The K m value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 μM [actin] in H 2O to 33 μM [actin] in D 2O without any significant changes in V max (9.4 s −1 in D 2O and 9.3 s −1 in H 2O). The rate constants of ADP release from the acto-S1–ADP complex measured by the stopped flow method were 361±26 s −1 ( n=27) in D 2O and 512±39 s −1 ( n=27) in H 2O at 6°C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D 2O results from a slowing of the release of ADP from the actomyosin–ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D 2O.

Monitoring Myosin Degradation During Conditioning in Chicken Meat Using an Immunological Method

ABSTRACT: Changes of chicken breast myosin during storage at 2°C and 37°C were monitored immunochemically. Anti‐myosin subfragment‐1 (S‐1) monoclonal antibody, which recognized epitopes within the 27 kDa fragment of S‐1, and the anti‐myosin rod polyclonal antiserum, were prepared. Myosin degradation products were not detected in muscle extracts stored for 3 weeks at 2°C. In contrast, storage at 37°C brought about the degradation of myosin heavy chain to immunologically detectable small fragments. While, myosin rod produced during the conditioning period was not decomposed into any small filaments. Namely, storage of muscle at 37°C resulted in minor amounts of myosin heavy chain degradation, with initial conversion to rod and S‐1 fragments, and subsequent breakdown occurred in the S‐1 region only. Immunoblot assay also suggested that the pattern of changes in myosin heavy chain in muscle incubated at 37°C was similar to that produced by in vitro digestion with cathepsin D.

Ca 2+-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca 2+-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)- N′-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4′-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin–tropomyosin (Tn–Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn–Tm complex with DABmal-labeled actin, in the presence and absence of Ca 2+, resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn–Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 Å to 44.1 Å when Ca 2+ bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn–Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca 2+. The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of ∼0.1 in the presence of Ca 2+, and ∼0.3 in the absence of Ca 2+, were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by ∼7 Å in the presence of Ca 2+ and by ∼2 Å in the absence of Ca 2+ when S1 bound to actin. Our results suggest that TnI’s interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.

Dimethyl sulphoxide enhances the effects of Pi in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before Pi release. DMSO (5–30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto–S1MgATPase activities in the same proportions, without altering acto–S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin–myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca2+ and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca2+ sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for Pi is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP·Pi or ATP bound to them.

Dimethyl sulphoxide enhances the effects of P(i) in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins.

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before P(i) release. DMSO (5-30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto-S1 MgATPase activities in the same proportions, without altering acto-S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin-myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca(2+) and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca(2+) sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for P(i) is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP.P(i) or ATP bound to them.

Phosphorylation-dependent regulation is absent in a nonmuscle heavy meromyosin construct with one complete head and one head lacking the motor domain.

To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase measurements showed that phosphorylation did not alter the affinity for actin by more than a factor of 2 for any construct. Phosphorylation increased V(max) by a factor of 10 for construct 1 and 1.5-3 for construct 2 but had no effect for construct 3. Single turnover measurements, a better measure of slow rates inherent to unphosphorylated regulated myosins, showed that the single-headed construct 2, like construct 3 retains less than 1% of the regulatory properties of the double-headed construct 1 (300-fold activation). Therefore, a complete head cannot be down-regulated by a regulatory domain (without the motor domain) on the partner head. Two motor domains are required for regulation. This result is predicted by a structural model (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390) showing interaction between the motor domains for unphosphorylated smooth muscle myosin, if motor-motor interaction is the basis for down-regulation.