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.     

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.     

The binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.     

Interaction between actin and HMM

It is shown that the interaction between actin and HMM results in a rapid precipitation of acto-HMM gel upon addition of MgATP. This is a simple demonstration of the idea that the formation of myosin filaments is not essential for mechanochemical reaction (muscle contraction) to occur and that the soluble myosin heads are competent to interact with actin to produce mechanical effect. Our findings also strongly support earlier suggestion that each head of one HMM molecule is able to bind to a different actin filament.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

The influence of substoichiometric concentrations of myosin subfragment 1 on the state of aggregation of actin under depolymerizing conditions

In 3 mM KCl, 2 mM Tris/HCl pH 7.5, 22 degrees C, 0.38 microM myosin subfragment 1 delays the depolymerization of F-actin (7.2 microM measured as monomer). The depolymerization proceeds rapidly for a few minutes and then slows down suddenly when the ratio between the monomers in the actin filaments and myosin subfragment 1 reaches the value of 11. At this time myosin subfragment 1 is substantially all bound to the actin polymers which form an irregular and discontinuous network of filaments running in doublets and in triplets, perhaps cross-linked by myosin subfragment 1. Depolymerization proceeds then for several hours, apparently ending up with the formation of the 1:1 actin-S1 heteropolymer. The ratio between the monomers in the actin filaments and myosin subfragment 1 at the end of the rapid depolymerization process is different for different protein preparations and may be as low as 5.5. In 2 mM Tris/HCl pH 7.5, 25 degrees C, 1 microM myosin subfragment 1 is able to induce the formation of undecorated actin filaments from 12 microM ATP--G-actin. These filaments probably originate by redistribution of myosin subfragment 1 between the newly formed 1/1 actin-S1 heteropolymer and G-actin in the medium, a process which allows the transient formation of undecorated actin filaments.     

The tail of myosin reduces actin filament velocity in the in vitro motility assay

It has been observed that heavy meromyosin (HMM) propels actin filaments to higher velocities than native myosin in the in vitro motility assay, yet the reason for this difference has remained unexplained. Since the major difference between these two proteins is the presence of the tail in native myosin, we tested the hypothesis that unknown interactions between actin and the tail (LMM) slow motility in native myosin. Chymotryptic HMM and LMM were mixed in a range of molar ratios (0-5 LMM/HMM) and compared to native rat skeletal myosin in the in vitro motility assay at 30 degrees C. Increasing proportions of LMM to HMM slowed actin filament velocities, becoming equivalent to native myosin at a ratio of 3 LMM/HMM. NH4+ -ATPase assays demonstrated that HMM concentrations on the surface were constant and independent of LMM concentration, arguing against a simple displacement mechanism. Relationships between velocity and the number of available heads suggested that the duty cycle of HMM was not altered by the presence of LMM. HMM prepared with a lower chymotrypsin concentration and with very short digestion times moved actin at the same high velocity. The difference between velocities of actin filament propelled by HMM and HMM/LMM decreased with increasing ionic strength, suggesting that ionic bonds between myosin tail and actin filaments may play a role in slowing filament velocity. These data suggest the high velocities of actin filaments over HMM result from the absence of drag generated by the myosin tail, and not from proteolytic nicking of the motor domain.     

Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex

Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.     

Effect of actin filament length and filament number concentration on the actin-activated ATPase activity of Acanthamoeba myosin I

The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+-ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F-actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I.     

Heavy meromyosin induces sliding movements between antiparallel actin filaments

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of the movement of leucocytes

In a study of the movement of human leucocytes it was clarified that characteristic contraction waves were observed on the cell surface during movement and an initial morphological change directly related to the appearance of the wave originated in the surface of the granuloplasm and not in the cell membrane. From these findings, together with physicochemical properties of the contractile protein from equine leucocytes, it was proposed that the wave observed in moving leucocytes might be conducted, in some way, by contraction and relaxation of the contractile protein in the cells. Myosin A and actin as constituents of the contractile protein were extracted separately from leucocytes in polymerized form, which resemble myosin aggregate and F-actin from muscle, respectively. The thick and thin filaments of about 150 and 80 Å in diameter were observed in glycerinated leucocytes with electron microscopy. When glycerinated leucocytes were incubated with heavy meromyosin (HMM) from rabbit skeletal myosin A, the thin filaments developed a structure resembling the ‘arrowhead structure’ of the HMM F-actin complex in vitro. The thick filaments seemed to correspond to myosin aggregates and the thin ones to filaments containing F-actin.     

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement.     

Filament assembly from profilin-actin

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 microM under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 microM under physiological conditions. The PAcov polymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.     

Fluorescence properties and contraction characteristics of ANM (N-(1-anilinonaphthyl-4)maleimide)-labeled rabbit psoas muscle fibers

Fluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (Vo), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though Vo decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM + MgATP or HMM + MgATP + F-actin in comparison with HMM + F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).     

Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility

The effects of crosslinking of monomeric and polymeric actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), disuccinimidyl suberate (DSS) and glutaraldehyde on the interaction with heavy meromyosin (HMM) in solution and on the sliding movement on glass-attached HMM were examined. The Vmax values of actin-activated HMM ATPase decreased in the following order: intact actin = EDC F-actin greater than DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than EDC G-actin. The affinity of actin for HMM in the presence of ATP decreased in the following order: DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than intact actin greater than EDC F-actin greater than EDC G-actin. However, sliding movement was inhibited only in the case of glutaraldehyde-crosslinked F and G-actin and EDC-crosslinked G-actin. Interestingly, after copolymerization of "non-motile" glutaraldehyde or EDC-crosslinked monomers with "motile" monomers of intact actin sliding of the copolymers was observed and its rate was independent of the type of crosslinked monomer, i.e. of the manner of their interaction with HMM. These data strongly indicate that inhibition of the sliding of actin by crosslinking cannot be explained entirely by changes in the Vmax value or affinity for myosin heads. We conclude that movement is generated by interaction of myosin with segments of F-actin containing a number of intact monomers, and the mechanism of inhibition involves an effect of the crosslinkers on the structure of F-actin itself.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.     

A synthetic peptide of the N-terminus of actin interacts with myosin

Research reported from numerous laboratories suggested that the N-terminal region of actin contained one of the binding sites between actin and myosin. A synthetic peptide corresponding to residues 1-28 of skeletal actin was prepared by solid-phase peptide methodology. The formation of a complex between this peptide and myosin subfragment 1 (S1) was demonstrated by high-performance size-exclusion chromatography (pH 6.8). The actin peptide precipitated S1 at higher pH (7.4-8.2) but remained soluble when bound to heavy meromyosin (HMM) or S1 in the presence of F-actin. The actin peptide 1-28 bound to S1 and HMM and activated the ATPase activity in a manner similar to that of F-actin. These results demonstrate that the N-terminal region of actin, residues 1-28, contains a biologically important binding site for myosin.     

Nonlinear dependence of actin-activated Mg2+-ATPase activity on the extent of phosphorylation of gizzard myosin and H-meromyosin

1. The actin-activated Mg2+-ATPase activity of gizzard HMM increased in proportion to the square of the extent of LC phosphorylation. This result indicates that the LCs of HMM are randomly phosphorylated, and the phosphorylation of both heads of HMM is required for the activation of HMM Mg2+-ATPase by F-actin. 2. In 75 mM KCl, the Mg2+-ATPase activity of gizzard myosin was activated by F-actin only slightly when a half of the total LC was phosphorylated. From 1 to 2 mol LC phosphorylation, the activity was enhanced by F-actin almost linearly. In 30 mM KCl, the activity of acto-gizzard myosin increased sigmoidally with increase in the extent of LC phosphorylation. On electron microscopy, side-by-side aggregates of myosin filaments were observed in 30 mM KCl, but not in 75 mM KCl. It was suggested that the activation of the Mg2+-ATPase activity of acto-gizzard myosin LC phosphorylation is modified by formation of myosin filaments and their aggregates. 3. The relationship between the actin-activated Mg2+-ATPase activity of HMM or myosin and the extent of LC phosphorylation was unaffected by tropomyosin.     

A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB

Acanthamoeba myosins IA and IB are single-headed, monomeric molecules consisting of one heavy chain and one light chain. Both have high actin-activated Mg2+-ATPase activity, when the heavy chain is phosphorylated, but neither seems to be able to form the bipolar filaments that are generally thought to be required for actomyosin-dependent contractility. In this paper, we show that, at fixed F-actin concentration, the actin-activated Mg2+-ATPase activities of myosins IA and IB increase about 5-fold in specific activity in a cooperative manner as the myosin concentration is increased. The myosin concentration range over which this cooperative change occurs depends on the actin concentration. More myosin I is required for the cooperative increase in activity at high concentrations of F-actin. The cooperative increase in specific activity at limiting actin concentrations is caused by a decrease in the KATPase for F-actin. The high and low KATPase states of the myosin have about the same Vmax at infinite actin concentration. Both myosins are completely bound to the F-actin long before the Vmax values are reached. Therefore, much of the actin activation must be the result of interactions between F-actin and actomyosin. These kinetic data can be explained by a model in which the cooperative shift of myosin I from the high KATPase to the low KATPase state results from the cross-linking of actin filaments by myosin I. Cross-linking might occur either through two actin-binding sites on a single molecule or by dimers or oligomers of myosin I induced to form by the interaction of myosin I monomers with the actin filaments. The ability of Acanthamoeba myosins IA and IB to cross-link actin filaments is demonstrated in the accompanying paper (Fujisaki, H., Albanesi, J.P., and Korn, E.D. (1985) J. Biol. Chem. 260, 11183-11189).