Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis.

The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.

Conformational dynamics of loop 262-274 in G- and F-actin.

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.

Activation dependence of stretch activation in mouse skinned myocardium: implications for ventricular function.

Recent evidence suggests that ventricular ejection is partly powered by a delayed development of force, i.e., stretch activation, in regions of the ventricular wall due to stretch resulting from torsional twist of the ventricle around the apex-to-base axis. Given the potential importance of stretch activation in cardiac function, we characterized the stretch activation response and its Ca2+ dependence in murine skinned myocardium at 22 degrees C in solutions of varying Ca2+ concentrations. Stretch activation was induced by suddenly imposing a stretch of 0.5-2.5% of initial length to the isometrically contracting muscle and then holding the muscle at the new length. The force response to stretch was multiphasic: force initially increased in proportion to the amount of stretch, reached a peak, and then declined to a minimum before redeveloping to a new steady level. This last phase of the response is the delayed force characteristic of myocardial stretch activation and is presumably due to increased attachment of cross-bridges as a consequence of stretch. The amplitude and rate of stretch activation varied with Ca2+ concentration and more specifically with the level of isometric force prior to the stretch. Since myocardial force is regulated both by Ca2+ binding to troponin-C and cross-bridge binding to thin filaments, we explored the role of cross-bridge binding in the stretch activation response using NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1. NEM-S1 treatment at submaximal Ca2+-activated isometric forces significantly accelerated the rate of the stretch activation response and reduced its amplitude. These data show that the rate and amplitude of myocardial stretch activation vary with the level of activation and that stretch activation involves cooperative binding of cross-bridges to the thin filament. Such a mechanism would contribute to increased systolic ejection in response to increased delivery of activator Ca2+ during excitation-contraction coupling.

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium.

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton.

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.

Strong binding of myosin increases shortening velocity of rabbit skinned skeletal muscle fibres at low levels of Ca(2+).

1. At low levels of activation, unloaded shortening of skinned skeletal muscle fibres takes place in two phases: an initial phase of high-velocity shortening followed by a phase of low-velocity shortening. The basis for Ca(2+) dependence of unloaded shortening velocity (V(o)) in the low-velocity phase was investigated by varying the level of thin filament activation with Ca(2+) and N-ethyl-maleimide myosin subfragment-1 (NEM-S1), a non-tension-generating, strong binding derivative of subfragment-1. V(o) was measured with the slack-test method. 2. Treatment of skinned fibres with 5 microM NEM-S1 eliminated the low-velocity phase of shortening but had no effect on the high-velocity phase of shortening during submaximal activation with Ca(2+), or on V(o) during maximal activation with Ca(2+). 3. Extensive washout of NEM-S1 from the treated fibres restored the low-velocity phase of shortening and returned low-velocity V(o) to pre-treatment values. 4. The effect of NEM-S1 to increase low-velocity V(o) can be explained in terms of a model in which strong binding myosin cross-bridges activate the thin filament to a state in which the rate of ADP release from the actin-myosin-ADP complex and the rate of cross-bridge detachment from actin are accelerated during unloaded shortening.

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers.

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.

Strongly binding myosin crossbridges regulate loaded shortening and power output in cardiac myocytes.

This study investigated the possible roles of strongly binding myosin crossbridges in determining loaded shortening and power output in cardiac myocytes. Single skinned cardiac myocytes were attached between a force transducer and position motor, and shortening velocities were measured over a range of loads during varying levels of Ca(2+) activation. Lowering the [Ca(2+)] slowed shortening velocities, decreased relative power output, and increased the curvature of length traces. We tested the hypothesis that Ca(2+) activation dependence of loaded shortening is determined primarily by strongly binding crossbridges or by [Ca(2+)] per se, which was done by measuring loaded shortening before and after addition of N-ethylmaleimide-conjugated myosin subfragment-1 (NEM-S1), a strongly binding myosin analogue that cooperatively enhances thin filament activation. At fixed [Ca(2+)], NEM-S1 reduced the curvature of length traces and sped loaded shortening velocities. Even when [Ca(2+)] was adjusted so that force was equal with and without NEM-S1, myocyte shortening was faster and exhibited less curvature with NEM-S1. In the presence of NEM-S1, peak relative power output was also significantly greater during activations either at the same [Ca(2+)] or when [Ca(2+)] was adjusted to achieve the same force. Consequently, NEM-S1 eliminated any Ca(2+) dependence of relative power output that is normally observed in cardiac myocytes. These results indicate that strongly binding crossbridges play a significant role in determining loaded shortening and power output and suggest that previously observed Ca(2+) dependence of power output is mediated by alterations in numbers of crossbridges bound to the thin filament.

The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state.

Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons 1a or 1b and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to tropomyosins with an exon 1a-encoded N-terminus, found in long isoforms, the exon 1b-encoded sequence, expressed in 247-residue nonmuscle tropomyosins, increases actin affinity in tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a > acetylated 1a9a > 1a9d >> 1a9a > or = 1a9c congruent with 1b9c. Myosin S1 greatly increases the affinity of all tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state; generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with tropomyosin: 1b9d >1a9d> 1b9a > or = acetylated 1a9a > 1a9a > 1a9c congruent with 1b9c.

Effects of two familial hypertrophic cardiomyopathy-causing mutations on alpha-tropomyosin structure and function.

Missense mutations in alpha-tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled tropomyosins differed in their response to myosin S1 binding to the actin-tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.

A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using microgram quantities of protein.

The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is <<1 microM at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a Kd below 0.1 microM. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for Kd's as low as 10 nM and we demonstrate that the Kd's can be estimated using only microgram quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.

Myosin drives retrograde F-actin flow in neuronal growth cones.

Actin filaments assembled at the leading edge of neuronal growth cones are centripetally transported via retrograde F-actin flow, a process fundamental to growth cone guidance and other forms of directed cell motility. Here we investigated the role of myosins in retrograde flow, using two distinct modes of myosin inhibition: microinjection of NEM inactivated myosin S1 fragments, or treatment with 2,3-butanedione-2-monoxime, and inhibitor of myosin ATPase. Both treatments resulted in dose-dependent attenuation of retrograde F-actin flow and growth of filopodia. Growth was cytochalasin sensitive and directly proportional to the degree of myosin inhibition, suggesting that retrograde flow results from superimposition of two independent processes: actin assembly and myosin-based filament retraction. These results provide the first direct evidence for myosin involvement in neuronal growth cone function.

Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop.

The protease subtilisin has been reported to cleave skeletal muscle G-actin between Met 47 and Gly 48 generating a core fragment of 33 kDa and a small N-terminal peptide, which remains attached to the core fragment [Schwyter, D. Phillips, M., & Reisler, E. (1989) Biochemistry 28, 5889-5895]. However, amino acid sequencing and mass spectroscopy of subtilisin cleaved-actin revealed two cleavage sites, one between Met 47 and Gly 48 and a second between Gly 42 and Val 43, generating an actin core of 37 kDa and a nicked 4.4 kDa N-terminal peptide. Here we describe a procedure for purifying the actin core fragment and the attached N-terminal peptide from the linking pentapeptide comprising amino acid residues 43-47 under native conditions by anion exchange chromatography. After removal of the pentapeptide, the salt-induced polymerization of actin was abolished. However, the purified fragments could be polymerized by addition of salt plus myosin subfragment 1 or salt plus phalloidin as shown by sedimentation and fluorescence increase using N-(1-pyrenyl)iodoacetamide labeled actin. These results confirm earlier reports proposing that cleavage in the DNase I binding loop is affecting the ion induced polymerization of actin [Higashi-Fujime, S., et al. (1992) J. Biochem. (Tokyo) 112, 568-572; and Khaitlina, S., et al. (1993) Eur. J. Biochem. 218, 911-920]. Monomeric and filamentous subactin exhibited reduced abilities to inhibit deoxyribonuclease I (DNase I) and to stimulate the myosin subfragment 1 ATPase activity. Direct binding of subactin to DNase I was verified by gel filtration and to myosin subfragment 1 by affinity chromatography, chemical cross-linking, and electron microscopy.

Flexation of caldesmon: effect of conformation on the properties of caldesmon.

The contribution of the extended and bent forms of caldesmon to its function was investigated by examining chemically modified forms of this protein. The bent 'hairpin' form of caldesmon was enhanced between pH 6.0 and 8.0 and at low ionic strengths, as reported by an increase in excimer fluorescence of pyrene-labelled caldesmon under these conditions. The presence of nucleotides also produced significant conformational changes in caldesmon, as detected by fluorescence measurements and protease digestions. Titrations of pyrene caldesmon with actin, heavy meromyosin, and calmodulin resulted in a decrease in excimer fluorescence. The function of the bent form of caldesmon was investigated by using intramolecular 1-ethyl-3-(3-dimethylamino propyl) carbodiimide-crosslinked caldesmon. The inhibition of acto-S-1 ATPase activity by crosslinked caldesmon was less efficient compared with that by pyrene modified and control caldesmons. Caldesmon's ability to switch from an activator to an inhibitor of actin-activated ATPase of myosin was also affected by the folding. Cosedimentation experiments revealed normal binding of crosslinked caldesmon to smooth muscle myosin. These results indicate the importance of caldesmon's transition from extended to folded forms and suggest possible functional roles for these different forms of caldesmon.

Myosin subfragment 1 activates ATP hydrolysis on Mg(2+)-G-actin.

The interaction of myosin subfragment 1 isoenzyme A2 (S1A2) with Mg(2+)-G-actin was studied. Polarization titrations of 1,5-IAEDANS-Mg(2+)-G-actin and of epsilon ATP-Mg(2+)-G-actin with S1A2 provided evidence that, similar to Ca(2+)-G-actin, the proteins form a tight binary complex. Significant amounts of oligomeric forms of actin in the presence and absence of S1 were not detected. The effect of S1A2 on the rates of nucleotide and metal dissociation and hydrolysis from Mg(2+)-actin was measured. The hydrolysis rate for [gamma-32P]ATP-actin in the G-acto-S1A2 complex (k- = 0.016 s-1) was faster than the rate of 32P liberation from the complex (k- = 0.004 s-1), obtained by measuring the liberation of [32P]orthophosphate from [alpha-32P]ATP-actin in the presence of a large excess of alkaline phosphatase. This indicates that most of actin's ATP was hydrolyzed before it was released to solution and that the dissociating nucleotide was ADP, for which the dissociation rate is higher than that for ATP. In agreement with this mechanism, S1A2 accelerated the dissociation of epsilon ATP but inhibited the dissociation of epsilon ADP from the complex. The activation of actin's ATPase is specific for Mg(2+)-G-actin and does not occur in Ca(2+)-G-actin. The effect of deoxyribonuclease I on the rates of nucleotide dissociation and hydrolysis was examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of the polymerization of actin from the actin:thymosin beta 4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin.

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin beta 4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked+ends. Free, slowly growing-ends were unable to induce polymerization of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin beta 4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin beta 4. This Kd was found to be 0.4 microM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin beta 4 complex; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.

Polymerization of actin from the thymosin beta 4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1.

Thymosin beta 4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin-thymosin beta 4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin beta 4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non-muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin-stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.

Both isoforms of skeletal muscle subfragment 1 (S1A1 and S1A2) can induce actin polymerization with equal speed in the absence of ATP.

The ability of myosin subfragment 1 to induce actin polymerization was reinvestigated using the DNase I inhibition assay, by electron microscopy after negative staining, cosedimentation, measurement of viscosity and the fluorescence increase of pyrenyl-labeled actin. Using these techniques we demonstrate that rabbit skeletal muscle myosin subfragment 1 containing either the alkali light chain 1 (S1A1) or the alkali light chain 2 (S1A2) is able to promote actin polymerization even in the absence of divalent cations or salt. In the presence of ATP the rate of induction of actin polymerization by S1A2 is slower than by A1A1. In contrast, in the absence of free ATP, both subfragment 1 variants exhibit equal ability to induce actin polymerization. Evidence is given that the slower rate of induction of actin polymerization by S1A2 in the presence of free ATP is due to a slower rate of ATP-hydrolysis by S1A2 and thus to a slower rate of ATP depletion. We therefore assume that the formation of rigor type complexes involving the subfragment 1 heavy chain is necessary for the induction of actin polymerization. The ability of subfragment 1 to induce actin polymerization is retarded by a synthetic heavy chain mimetic peptide which inhibits its actin binding or after proteolytic cleavage of the subfragment 1 heavy chain by trypsin.

The effect of Ca2+ on the conformation of tropomyosin and actin in regulated actin filaments with or without bound myosin subfragment 1.

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.

The myosin molecule--charge response to nucleotide binding.

A decrease in the net fixed electric charge in the A-bands of cross-striated muscle was observed by Bartels and Elliott [2,10] when the muscle went from the rigor to the relaxed condition. The current work localises the source of the charge decrease by following the net charge on myosin (in the form of concentrated gels) and also myosin rod and light meromyosin gels when the gels are exposed to different concentrations of ATP. The work includes a study of muscle A-bands when the muscle is exposed to the same variations in ATP concentrations as the protein gels. The work shows that (i) Only 100-200 microns ATP is needed to initiate the charge decrease between the rigor and relaxed conditions; (ii) the effect of ATP is seen in the muscle A-band and the myosin and myosin rod gels, but not in LMM gels; (iii) pyrophosphate (PPi) shows a similar charge effect to ATP. ADP does not affect the charge on myosin gels, on the other hand. The results suggest that the charge decrease caused by ATP or PPi is due to ligand interaction with one or more sites on the myosin molecule. This interaction causes a disseminated effect in the protein, and a consequent loss in net negative charge either by a decrease in the absorption, of anions to Saroff sites on the protein, or, less probably, by an increase in the absorption of cations at those sites.