Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.     

Subunit exchange between smooth muscle myosin filaments

Filaments formed from phosphorylated smooth muscle myosin are stable in the presence of MgATP, whereas dephosphorylated filaments are disassembled to a mixture of folded monomers and dimers. The stability of copolymers of phosphorylated and dephosphorylated myosin was, however, unknown. Gel filtration, sedimentation velocity, and pelleting assays were used to show that MgATP could dissociate dephosphorylated myosin from copolymers containing either rod and myosin or dephosphorylated and phosphorylated myosin. Copolymers were typically formed by dialyzing monomeric mixtures into filament-forming buffer but, unexpectedly, could also be formed within minutes of mixing preformed rod and myosin minifilaments. This result suggested that molecules can rapidly and extensively exchange between filaments, presumably via the monomeric pool of myosin in equilibrium with polymer. An exchange of molecules between filaments was demonstrated directly by electron microscopy using gold-labeled streptavidin or antibody to detect the exchanged species. By this approach it was shown that smooth muscle myosin filaments, like other macromolecular assemblies, are dynamic structures that can readily alter their composition in response to changing solvent conditions. Moreover, because folded monomeric myosin is unable to polymerize, these experiments suggest a mechanism for the disassembly of the filament by MgATP.     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

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.     

Role of magnesium binding to myosin in controlling the state of cross-bridges in skeletal rabbit muscle

The effect of Mg2+ on the disposition of myosin cross-bridges was studied on myofibrils and synthetic myosin and rod filaments by employing chymotryptic digestion and chemical cross-linking methods. In the presence of low Mg2+ concentrations (0.1 mM), the proteolytic susceptibility at the heavy meromyosin/light meromyosin (HMM/LMM) junction in these three systems sharply increases over the pH range from 7.0 to 8.2. Such a change has been previously associated with the release of myosin cross-bridges from the filament surface [Ueno, H., & Harrington, W.F. (1981) J. Mol. Biol. 149, 619-640]. Millimolar concentrations of Mg2+ block or reverse this charge-dependent transition. Rod filaments show the same behavior as myosin filaments, indicating that the low-affinity binding sites for Mg2+ are located on the rod portion of myosin. The interpretation of these results in terms of Mg2+-mediated binding of cross-bridges to the filament backbone is supported by cross-linking experiments. The normalized rate of S-2 cross-linking in rod filaments at pH 8.0, kS-2/kLMM, increases upon addition of Mg2+ from 0.30 to 0.65 and approaches the cross-linking rate measured at pH 7.0 (0.75), when the cross-bridges are close to the filament surface. In rod filaments prepared from oxidized rod particles, chymotryptic digestion proceeds both at the S-2/LMM junction and at a new cleavage site located in the N-terminal portion of the molecule. Kinetic analysis of digestion rates at these two sites reveals that binding of Mg2+ to oxidized myosin rods has a similar effect at both sites over the pH range from 7.0 to 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformations of vertebrate striated muscle myosin monomers in equilibrium with filaments.

Porcine cardiac myosin monomers in equilibrium with filaments under physiological conditions were observed to have two conformations, extended and folded forms, upon electron microscopy and gel filtration HPLC. The conformational state was independent of ATP and the phosphorylation of regulatory light chain. The folded monomers of cardiac myosin were mainly in an open conformation with only one bend in the tail, and may not trap the hydrolysis products of ATP, as assessed by single turnover experiments. These properties are similar to those of the folded monomers of rabbit skeletal myosin [Katoh, T., Konishi, K., and Yazawa, M. (1998) J. Biol. Chem. 273, 11436-11439]. The conformational states of skeletal and cardiac myosin monomers were not affected by pH between 7.0 and 8.5. Although significant disassembly of filaments and thus an increase in the monomer concentration were observed with an increase in pH. The results indicate that the pH-dependent change in filament assembly is due to a shift of equilibrium between the filaments and extended monomers toward filament disassembly. The Mg2+-ATPase activity of these myosin monomers decreased with a decrease in the salt concentration below approximately 0.1 M, suggestive of the formation of a closed conformation similar to the conformation of 10S smooth myosin. The results suggest that the conformational change from the extended to the folded form is a common property of various myosin IIs.     

Modulation of myosin filament conformation by physiological levels of divalent cation

The effect of divalent cation, in particular Mg²⁺, on the properties of synthetic myosin filaments has been studied; and substantial changes in sedimentation and light scattering demonstrated to occur in the physiological range of free Mg²⁺. A pre-requisite for these studies has been the definition of a modified method for the preparation of myosin in highly monodisperse filament form, rigorously free from thin filament proteins. The sedimentation coefficient at infinite dilution shows a large increase (169 S to 193 S) in the range 0.2 mm to 3 mm in Mg²⁺. The anomalous frictional increment found for these filaments is thus substantially reduced. The concentration dependence (k_s), however, shows a substantial decrease (470 ml/g to 334 ml/g) in the same range of Mg²⁺, and the calculated filament molecular weight is virtually unchanged. A change in the filament conformation is thus indicated. This is confirmed by an analysis of the turbidity of the filaments in the centrifuge cell, which shows a similar increase in response to the addition of Mg²⁺. These effects have been found to be independent of ionic strength (0.07 to 0.11), pH (7.0 to 7.6), the presence of MgATP or the presence of low levels of Ca²⁺ (~100 μm). These effects studied indicate the action of Mg²⁺ through a low-affinity binding site (K_d ~ 1.5 × 10^?3m). We consider that a significant change in crossbridge conformation can adequately explain these changes in physical and enzymic properties. A provisional model is proposed, in which the effect of Mg²⁺ is to bring the crossbridges into closer proximity to the filament shaft.     

Effects of C-protein on synthetic myosin filament structure.

In the absence of C-protein, synthetic filaments prepared from column-purified myosin exhibit the following features: individual filament diameters are uniform over a long length, but a wide distribution of diameters is apparent over the population; approximately 25% of the filaments have a frayed appearance and take up stain poorly, whereas the remaining 75% are well-stained; optical diffraction of well-stained filaments reveals a 14.3-nm subunit period and a 43-nm axial period (Koretz, 1978; Koretz, 1979). Addition of C-protein to myosin before filament formation affects all of these features in a manner related to C-protein concentration. At the physiological ratio of C-protein to myosin in the banded region of the natural thick filament, synthetic aggregates are uniform in diameter over the population and show less than 10% frays. Whereas the subunit period remains unchanged, the axial period has increased to 114.4 nm, or eight times the subunit repeat. Above and below the physiological ratio, disorder of a specific nature is apparent. Addition of C-protein after filament formation appears to coat the aggregates so that elements of backbone ultrastructure are obscured, and some evidence of axial period change is visible in diffraction patterns. A model is presented for the binding of C-protein to myosin, and its observed effects on filament structure are explained in terms of this model.     

Effects of ionic strength on calcium binding to rabbit skeletal myofibrils,thin filaments and myosin

Calcium binding by rabbit skeletal myosin, thin filaments and myofibrils was measured in solutions with and without 2 mM MgATP and with ionic strengths adjusted with KCl to 0.05, 0.10 and 0.14 M. Free Mg²⁺ was held constant at 1 mM, pH at 7.0 and temperature at 25 °C. In the presence of MgATP, the relation between free Ca²⁺ and myofibrillar bound calcium shifted to the left as ionic strength was decreased from 0.14 to 0.05 M. In the absence of MgATP, myofibrillar calcium binding was enhanced over a wide range of free Ca²⁺ concentration, but calcium binding was no longer a function of ionic strength. Similarly, calcium binding by thin filaments and myosin was unaffected by changes in ionic strength from 0.05 to 0.14 M. In view of evidence that cross-bridge connections between thick and thin filaments increase as ionic strength decreases, our results suggest that these connections enhance myofibrillar calcium binding. These results thus confirm previous data of Bremel and Weber (Bremel, R. D. and Weber, A. (1972) Nature New Biol. 238, 97–101) who first showed that nucleotide-free cross-bridge connections enhance thin filament calcium binding.     

The structure of thick filaments on longitudinal sections of rabbit psoas muscle

By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different.     

The regulatory light chain is required for folding of smooth muscle myosin

Light chain phosphorylation causes the folded monomeric form of myosin to extend and assemble into filaments. This observation established the involvement of the 20-kDa regulatory light chain (LC20) in conformational transitions of smooth muscle myosin. To further assess the role of this subunit in the intramolecular folding of myosin, LC20 was removed from turkey gizzard myosin at elevated temperatures in the presence of EDTA through the use of an antibody affinity column. Metal-shadowed images showed that LC20-deficient myosin had a tendency to aggregate through the neck region. When MgATP was added to filaments formed from this myosin, less than 10% of the myosin was solubilized, indicating that myosin could not fold in the absence of light chain. Readdition of native regulatory light chain restored the myosin to its original solubility properties, thus establishing reversibility. Addition of foreign light chains from skeletal muscle myosin or a chymotryptic-cleaved gizzard light chain produced the same amount of monomeric myosin in high salt that was obtained by recombination with the homologous light chain. However, the ability of the hybrid myosins to assume the folded conformation was impaired, and only a partially folded species was obtained. Single-headed myosin, like rod and light chain-deficient myosin, remained filamentous in the presence of MgATP. These results are consistent with the hypothesis that the regulatory light chain in the neck region of myosin contributes to a binding site for the myosin tail.     

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin

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

Self-association of a high molecular weight subfragment-2 of myosin induced by divalent metal ions

The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form ordered, high molecular weight aggregates whereas short S-2 does not associate. The association process is co-operative and results from binding two to four divalent cations within the light meromyosin-heavy meromyosin (LMM-HMM) hinge region of long S-2. Optical diffraction of electron micrographs of the long S-2 aggregates revealed several periodicities including reflections near 143 A. High molecular weight HMM showed a similar divalent metal induced self-association. Chymotryptic digestion studies of rod filaments reveal that cleavage within the LMM-HMM hinge is also strongly dependent on the presence of divalent cations. At pH 8, in the absence of divalent cations, the S-2 region appears to be displaced away from the filament backbone resulting in rapid proteolysis in the hinge domain. At high cation concentrations (greater than 10 mM) proteolytic cleavage is suppressed. A similar depression of the (substantially lower) hinge cleavage rate was also observed at neutral pH following addition of these divalent metal ions. Results suggest that binding of Mg2+ within the hinge domain under physiological conditions may act to lock the cross-bridge onto the thick filament surface in its resting-state orientation.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.     

Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin- dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.     

Actin-myosin interaction: The role of myosin in determining the actin pattern in self-assembled ‘hybrid’ contractile units

Self-assembly of actin-myosin filamentous complexes was assayed by polymerizing rabbit G-ADP actin on formed filaments of lobster myosin. The resulting contractile units indicate a 12-member actin orbital rather than the six-member orbital obtained previously using rabbit myosin and actin. Furthermore, the pattern of actin distribution surrounding the myosin filament is similar to that of the lobster tonic muscle sarcomere rather than the trigonal actin position characteristic of vertebrate muscle. The results show that the pattern and mode of actin complexing is determined by the specific myosin and the arrangement of the cross-bridges on the organized filament.     

Effect of adenosine triphosphate, inorganic phosphate and divalent cations on the size and structure of synthetic myosin filaments. An electron microscope study

We have studied the influence of ATP, inorganic monophosphate, MgCl₂ and CaCl₂, alone or in combination, on the formation of synthetic myosin filaments by means of electron microscopy. Both crude and column-purified rat skeletal myosin were studied systematically, and in some instances parallel experiments were carried out using crude rabbit skeletal myosin.The behaviour of filaments formed by a standard polymerization procedure at pH 6.8, in the absence of ATP or inorganic phosphate, is not influenced by MgCl₂, CaCl₂ or ethylenediaminetetraacetic acid at concentrations up to 5 mm. Such filaments are homogeneously 30 to 50 nm wide and 5 to 15 μm. long, i.e. larger than physiological size. They do not systematically display tapered ends. Filaments are built up from 2 to 3-nm wide threadlike subunits, arranged roughly parallel to the long axes. Sometimes filamentous projections up to 60 nm long, not associated with accidentical filament bending, are seen. In heavily contrasted preparations these projections are replaced by irregular globular structures. This transformation of the projections is accompanied by a slight decrease in the diameter of the filament shaft.When polymerization is carried out in the presence of millimolar amounts of ATP or inorganic phosphate, but in the absence of divalent cations, regular filaments are not formed. Instead long branched structures, or small twig-like filaments are obtained, which are 10 to 15 nm wide and 0.2 to 0.4 μm long. The addition of ATP or inorganic phosphate to preformed regular filaments does not bring about this structural disorder.The presence of millimolar amounts of MgCl₂ or CaCl₂ in the polymerization medium efficiently counteracts the disorganizing effect of ATP and inorganic phosphate. The size of filaments formed under these circumstances critically depends upon the nature of the divalent cation. CaCl₂ induces the formation of filaments similar in every respect to those already described. In the presence of MgCl₂ distinctly thinner ones, with close to physiological diameters (15 to 17 nm), are obtained. In both cases the lengths are still 5 to 15 μm.The filaments with physiological diameters consistently display tapered ends. They are built up from the same threadlike subunits as wider ones, and most display filamentous projections up to 60 nm long, systematically pointing in the direction of the filament ends. Central zones of polarity reversal are easily identified. In heavily contrasted preparations these filamentous projections are again replaced by irregular globular structures and the apparent diameter of the shaft is slightly diminished.In no case did we notice any significant influence of the origin, or degree of purity, of the myosin on filament behaviour.     

Effect of H-protein on the formation of myosin filaments and light meromyosin paracrystals

H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.     

The aggregation characteristics of column-purified rabbit skeletal myosin in the presence and absence of C-protein at pH 7.0.

The aggregation properties of column-purified rabbit skeletal myosin at pH 7.0 were investigated as functions of ionic strength, protein concentration, and time. Filaments prepared by dialysis exhibited the same average length and population distribution at 0.10 and 0.15 M KCl at protein concentrations greater than 0.10 mg/ml; similar results were obtained at .0.20 M KCl, although average filament length was approximately 0.5 micrometer shorter. Once formed, these length distributions remained virtually unchanged over an 8-d period. At and below 0.10 mg/ml, average filament length decreased as a function of protein concentration; filaments prepared from an initial concentration of 0.02 mg/ml were half the length of those prepared at 0.2 mg/ml. Filaments prepared by dilution exhibited a sharp increase in average length as the time-course increased up to 40 s, then altered only slightly over a further period of 4 min. Addition of C-protein in a molar ratio of 1-3.3 myosin molecules affected most of these results. Average filament length was affected neither by ionic strength nor by initial protein concentration down to 0.04 mg/ml or over an 8-d period. Filaments formed by dilution in the presence of C-protein exhibited a constant average length and hypersharp length distribution over variable time courses up to 7 min. It is possible that C-protein acts to stabilize the antiparallel intermediate during filamentogenesis, and may also affect subunit addition to this nucleus.     

Hydrostatic pressure studies of native and synthetic thick filaments: in vitro myosin aggregates at pH 7.0 with and without C-protein

Column-purified myosin at pH 7.0 will reproducibly aggregate into filaments of known average length and structure when dialyzed against a low ionic strength medium under controlled conditions. When exposed to increased hydrostatic pressure, followed by quick return to atmospheric pressure, the original filaments shorten linearly with increasing pressure; in addition, a second population of filaments is seen, presumably the result of reaggregation of myosin after release of pressure. This second population is about 0.5 microns long, bipolar, and about half the diameter of the original filaments. The number of these filaments, but not their physical characteristics, is a function of the shortening of the original filament population. Both the remnants of the original population and the new aggregates, once formed, are stable over time and at room temperature. The addition of C-protein to myosin solutions before filament preparation results in a filament population of slightly shorter length. When these filaments are exposed to increased hydrostatic pressure, they are more resistant to disaggregation than myosin filaments without C-protein. However, like the filaments prepared in the absence of C-protein, a second population of shorter, thinner filaments is visible after exposure to pressure.