Control of filament length by the regulatory light chains in skeletal and cardiac myosins

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.     

Unidirectional sliding of myosin filaments along the bundle of F-actin filaments spontaneously formed during superprecipitation

I reported previously (Higashi-Fujime, S., 1982, Cold Spring Harbor Symp. Quant. Biol., 46:69-75) that active movements of fibrils composed of F-actin and myosin filaments occurred after superprecipitation in the presence of ATP at low ionic strengths. When the concentration of MgCl2 in the medium used in the above experiment was raised to 20-26 mM, bundles of F-actin filaments, in addition to large precipitates, were formed spontaneously both during and after superprecipitation. Along these bundles, many myosin filaments were observed to slide unidirectionally and successively through the bundle, from one end to the other. The sliding of myosin filaments continued for approximately 1 h at room temperature at a mean rate of 6.0 micron/s, as long as ATP remained in the medium. By electron microscopy, it was found that most F-actin filaments decorated with heavy meromyosin pointed to the same direction in the bundle. Myosin filaments moved actively not only along the F-actin bundle but also in the medium. Such movement probably occurred along F-actin filaments that did not form the bundle but were dispersed in the medium, although dispersed F-actin filaments were not visible under the microscope. In this case, myosin filament could have moved in a reverse direction, changing from one F-actin filament to the other. These results suggested that the direction of movement of myosin filament, which has a bipolar structure and the potentiality to move in both directions, was determined by the polarity of F-actin filament in action.     

Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.     

Effect of the length and effective diameter of F-actin on the filament orientation in liquid crystalline sols measured by x-ray fiber diffraction.

We examined factors that affect the filament orientation in F-actin sols to prepare highly well-oriented liquid crystalline sols suitable for x-ray fiber diffraction structure analysis. Filamentous particles such as F-actin spontaneously align with one another when concentrated above a certain threshold concentration. This alignment is attributed to the excluded volume effect of the particles. In trying to improve the orientation of F-actin sols, we focused on the excluded volume to see how it affects the alignment. The achievable orientation was sensitive to the ionic strength of the solvent; the filaments were better oriented at lower ionic strengths, where the effective diameter of the filament is relatively large. Sols of longer filaments were better oriented than those of shorter filaments at the same concentration, but the best achievable orientation was limited, probably because of the filament flexibility. The best strategy for making well-oriented F-actin sols is therefore to concentrate F-actin filaments of relatively short length (<1 micrometer) by slow centrifugation in a low-ionic-strength solvent (<30 mM).     

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G- actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G- actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.     

Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments

Striking effects of F-actin and the reconstituted thin filament of muscle on the catalytic activity of rabbit muscle phosphofructokinase are demonstrated through direct measurements of enzymatic activity by using the pH stat. The addition of F-actin to solutions of phosphofructokinase at low ionic strength (10 mM KCl and 5 mM MgCl2) partially reverses the inhibition of the enzyme seen at high ATP concentrations and increases the apparent affinity of the enzyme for fructose 6-phosphate with slight effect on Vmax. F-Actin augments the activation of the enzyme obtained with AMP and partially counters the inhibition obtained with citrate. The maximum effect in the reversal of ATP inhibition is about the same for combinations of either F-actin or the thin filament with AMP as it is for AMP alone. In general, the effect of F-actin on the catalytic activity of phosphofructokinase is larger than that of the thin filament. The activation of phosphofructokinase by F-actin persists at physiological ionic strength.     

Ionic strength-dependent interaction of rat liver casein kinase I with plasma membrane

Casein kinase I binding to rat liver plasma membrane was rapidly released from membrane by increasing the ionic strength above physiological level. The released activities at 250-300 mM NaCl were 3-4-fold higher than those obtained under lower ionic strength below 100 mM NaCl. This reaction occurred nonenzymatically and was reversible. By lowering the ionic strength from 250 mM to 50 mM NaCl by dilution at least 50% of the released enzyme was rebound to plasma membrane. By gel filtration analysis, most of the released enzyme activity under higher NaCl concentration was recovered around the molecular mass of 35,000 which corresponded to that of casein kinase I. This enzyme showed the tendency to aggregate under lower ionic strength (50 mM NaCl), but existed as monomer under higher ionic strength (250 mM NaCl). These results suggest that the release of casein kinase I from plasma membrane and the rebinding to membrane induced by the alteration of ionic strength seem to be an important regulatory mechanism in determining the subcellular distribution of this enzyme.     

The Pathway of Assembly of Intermediate Filaments from Recombinant α-Internexin

The pathway of filament assembly from the neuronal intermediate filament α-intermexin was investigated. Optimal assembly occurred in solutions of pH 6.5 to 7 and moderate ionic strength at 37°C. Short filaments formed upon dialysis at 24°C, which elongated further when incubated at 37°C. Soluble forms of α-internexin were characterized by analytical ultracentrifugation and electron microscopy. In 10 mM Tris, pH 8, conditions that favor formation of tetramers and other small oligomers for other intermediate filament proteins, α-internexin formed 10.5 S particles, apparently unit-length half-filaments in the form of rods 10.6 nm in diameter and 68 nm long. Dialysis vs the same buffer with added 10 mM NaCl yielded 16 S rods, probably unit-length filaments, of the same length but 13.0 nm in diameter. At 50 mM NaCl, rods about 13 nm in diameter and heterogeneous in length were observed in electron micrographs, apparently formed from longitudinal annealing of unit-length rods. The results favor a model of assembly in which coiled coil dimers aggregate laterally to form first “unit-length half-filaments” (Herrmann, H., and Aebi, U. (1998)Curr. Opin. Struct. Biol.8, 177–185) and then “unit-length filaments,” which subsequently elongate by annealing.     

Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii

Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed.     

Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network

Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. Kitanishi-Yumura. 1990. Cell Struct. Funct. 15:355-364). Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCl2, which stabilized myosin II filaments, myosin II remained at the foci. Ultrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin II filaments remained in the foci in the presence of 10 mM MgCl2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form. Using [gamma 32P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin II ATPase) and/or ATP gamma S (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the foci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm.     

Activation of casein kinase II by sphingosine

Sphingosine activates casein kinase II in the presence of endogenous substrates as well as a synthetic peptide substrate. The activation response occurred between 12 and 25 micrograms/ml sphingosine and exhibited positive cooperativity with a Hill coefficient of 3.0. Sphingosine not only increased the Vmax of casein kinase II but decreased the Km(app) for the peptide substrate from 0.5 to 0.08 mM. In contrast, the Km(app) for MgCl2 was increased from 0.12 to 0.7 mM. Consequently, sphingosine altered significantly several parameters which determine casein kinase II activity. The effect of sphingosine was relatively specific, inasmuch as related lipids were less potent activators or largely ineffective in stimulating casein kinase II. On the other hand, the effect of sphingosine itself could be potentiated or inhibited by other lipids. Ceramide and sphingosylphosphorylcholine augmented the sphingosine effect. Phospholipids alone did not alter the activity of casein kinase II significantly, but abolished enzyme activation by sphingosine with different potencies (phosphatidylserine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine). Moreover, the sphingosine effect could be abrogated by KCI and NaCl, which alone are known to induce enzyme activation and dissociation of aggregated casein kinase II protein; LiCl and NH4Cl also inhibited the sphingosine effect. Polyamines, known activators of casein kinase II, partially mimicked the effect of sphingosine on endogenous polypeptide phosphorylation but failed to do so with the peptide substrate. These observations demonstrate that sphingosine is a potent activator of casein kinase II. The potential pharmacological and physiological modulation of casein kinase II by sphingoid bases is discussed.     

X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths

Equatorial x-ray diffraction patterns from single skinned rabbit psoas fibers were studied at various ionic strengths to obtain structural information regarding cross-bridge formation in relaxed muscle fibers. At ionic strengths between 20 and 50 mM, the intensity of the 11 reflection, I11, of the relaxed state was close to that of the rigor state, whereas the intensity of the 10 reflection, I10, was approximately twice that of rigor reflection. Calculations by two-dimensional Fourier synthesis indicated that substantial extra mass was associated with the thin filaments under these conditions. With increasing ionic strength between 20 and 100 mM, I10 increased and I11 decreased in an approximately linear way, indicating net transfer of mass away from the thin filaments towards the thick filaments. These results provided evidence that cross-bridges were formed in a relaxed fiber at low ionic strengths, and that the number of cross-bridges decreased as ionic strength was raised. Above mu = 100 mM, I10 and I11 both decreased, indicating the onset of increasing disorder within the filament lattice.     

Characterization of distinct early assembly units of different intermediate filament proteins

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.     

Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect.

Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle.     

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.     

Rate of binding of tropomyosin to actin filaments

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.     

Cytochalasin B slows but does not prevent monomer addition at the barbed end of the actin filament

We used Limulus sperm acrosomal actin bundles to examine the effect of 2 microM cytochalasin B (CB) on elongation from both the barbed and pointed ends of the actin filament. In this paper we report that 2 microM CB does not prevent monomer addition onto the barbed ends of the acrosomal actin filaments. Barbed end assembly occurred over a range of actin monomer concentrations (0.2-6 microM) in solutions containing 75 mM KCl, 5 mM MgCl2, 10 mM Imidazole, pH 7.2, and 2 microM CB. However, the elongation rates were reduced such that the rates at the barbed end were approximately the same as those at the pointed end. The association and dissociation rate constants were 8- to 10-fold smaller at the barbed end in the presence of CB along with an accompanying twofold increase in critical concentration at that end. Over the time course of experimentation there was little evidence for potentiation by CB of the nucleation step of assembly. CB did not sever actin filaments; instead its presence increased the susceptibility of actin filaments to breakage from the gentle shear forces incurred during sample preparation. Under these experimental conditions, the assembly rate constants and critical concentration at the pointed end were the same in both the presence and the absence of CB.     

Mechanical properties of actin

We used a cone and plate rheometer to evaluate the mechanical properties of actin over a wide range of oscillation frequencies and shear rates. Remarkably, both filamentous and nonfilamentous actin behaved as viscoelastic solids in both oscillatory and shear type experiments, providing that they were given ample time to equilibrate. Actin was purified by gel filtration from rabbit skeletal muscle and Acanthamoeba. Nonfilamentous actin in 2 different buffers had similar properties. In a low ionic strength buffer the absence of filaments was confirmed by electron microscopy, ultracentrifugation, and the fluorescence of pyrene-labeled actin. In 0.6 M KI, actin was monomeric by gel filtration. Filamentous actin had similar properties in 2 mM MgCl2 with either 50 mM KC1 or 500 mM KC1. Under all 4 of these conditions, actin required about 1000 min at 25 degrees C for the rheological properties to equilibrate. Under conditions where the oscillation of the rheometer did not affect the mechanical properties, all of the actin preparations had dynamic viscosities that were inverse functions of the frequency and dynamic elasticites that leveled off at low frequencies as expected for viscoelastic solids. For filamentous actin, the values of these parameters were about 2 times higher than for nonfilamentous actin. In shear experiments, both filamentous and nonfilamentous actin exhibited shear rate-dependent yield stresses. When filamentous and nonfilamentous actin structures were disrupted by transient shearing, the dynamic elasticity recovered to 90% in 30 min. Ovalbumin in the low ionic strength buffer also behaved as a viscoelastic material with elasticity and viscosity about 10 times lower than nonfilamentous actin, while cytochrome c behaved as a Newtonian fluid with a viscosity of 0.02 poise.     

Ionic effects on the rotational dynamics of cross-bridges in myosin filaments, measured by triplet absorption anisotropy

We have measured the rotational motion of myosin heads in synthetic thick filaments at 4 degrees C in the time range from 10(-7) to 10(-4) seconds, by measuring transient absorption anisotropy of an eosin probe attached to a reactive sulfhydryl on the myosin head. Under conditions that result in monomeric myosin (500 mM ionic strength), the anisotropy decay is independent of pH in the range from 7.0 to 8.2 and [Mg2+] in the range from 0.1 to 10 mM; the anisotropy decays bi-exponentially with correlation times of 0.4 and 2 microseconds to a constant value of 0.016. Under more physiological conditions (115 mM ionic strength), resulting in filament formation, the anisotropy decay is sensitive to both pH and [Mg2+]. The anisotropy at pH 8.2 and 0.1 mM-Mg2+ decays with correlation times of 0.5 and 3.8 microseconds to a constant limiting anisotropy of 0.038. When the [Mg2+] is increased to 10 mM, the correlation times are 0.6 and 5.7 microseconds and the limiting anisotropy value is 0.055. Identical changes in the anisotropy decay are caused by an increase in [H+] to pH 7.0, in the presence of 0.1 mM-Mg2+. Increasing the total ionic strength to 187 mM decreases the amplitude of the cation effects. These results provide direct evidence that the rotational dynamics of myosin heads in thick filaments are influenced by physiological concentrations of cations. The results are qualitatively consistent with the proposal that these and other ionic conditions regulate transitions between "spread" and "compact" cross-bridge conformations, but the quantitative results indicate that cross-bridges undergo large-amplitude microsecond rotations even under conditions where the compact state should predominate.