Sequential disassembly of vertebrate muscle thick filaments

Native thick filaments from rabbit psoas muscle have been sequentially dissolved by incremental rises in salt concentration. Three quite separate stages of depolymerization can be detected; these presumably reflect constraints imposed on the disassembly process by variations in the packing of myosin and by the presence of other thick filament proteins.     

Molecular organization of paramyosin in the core of molluscan thick filaments

An arrangement of paramyosin molecules in the polar part of molluscan thick filaments is proposed which accounts for the X-ray diffraction pattern of the smooth adductor muscle (other than the part ascribed to actin) and for the appearance of separated filaments in the electron microscope. The proposed structure is based on the PI arrangement of Cohen et al. (1971), and contains sets of parallel, equidistant molecules with successive molecules displaced along the molecular axis by 72 nm, which we call PI sheets. Every molecule belongs to two PI sheets which are nearly perpendicular. This array is not propagated throughout the filament, but is sheared periodically in the direction of the molecular (filament) axis by 2/5 X 72 nm. The shear occurs along parallel equidistant planes which are inclined to the PI sheets. The analysis of the X-ray data has been made possible by concentrating on those patterns from filaments in which the two sets of PI sheets appear to be mutually perpendicular, a condition brought about by bathing the muscle in aqueous acetone. In one set, there are four intermolecular spaces between shear planes (this appears to be true at least for the smooth adductors of Ostrea edulis, Crassostrea angulata and Mercenaria mercenaria). In the other set, the number varies with species and probably lies between eight and ten in the first two and appears to be six in the last named species. The known paracrystalline nature of paramyosin filaments suggests that this number, though dominant in one species, is not exactly constant.     

Direction and speed of actin filaments moving along thick filaments isolated from molluscan smooth muscle

The active movement of fluorescence-labeled actin filaments along thick filaments isolated from molluscan smooth muscle was observed. Along a single thick filament, actin filaments moved toward the center of the thick filament at the speed of 1.19 +/- 0.38 microns s-1 (mean +/- SD, n = 42) and detached themselves from it upon reaching the central zone. Movement of actin also occurred in the opposite direction, i.e., away from the center, albeit at a much lower velocity (0.09 +/- 0.07 microns s-1, n = 17). Thus, the thick filament shows functional bipolarity in terms of velocity but does not determine the direction of the movement.     

Polymerization of myorod, a surface protein of thick filaments of molluscan smooth muscle

The study is concerned with the polymerization of myorod, a protein from thick filaments of molluscan smooth muscles, which is an alternative product of the gene of myosin heavy chains. The dependences of the properties and polymer structure of myorod on the conditions of its formation were investigated. It was shown that myorod loses the ability to form viscous polymers after proteolytic removal of the unique sequence. It was supposed that the specificity of polymerization of myorod are determined by its unique N-terminal sequence.     

Extraction and functional reformation of thick filaments in chemically skinned molluscan catch muscle fibers.

A method for the almost complete extraction of myosin from smooth muscle fibers of the anterior byssal retractor muscle (ABRM) of Mytilus edulis was developed, and functional reformation of thick filaments in the fibers was achieved. Complete removal of myosin from the glycerol-extracted ABRM fibers with a solution containing 600 mM KCl, 5 mM MgCl2, and 5 mM ATP was difficult. However, successive treatments of the ABRM fibers with glycerol and saponin made the plasma membrane permeable to Mg-ATP and myosin. The extraction of myosin completely eliminated the tension induced by the addition of Mg-ATP. Partial recovery of tension development was observed by irrigation of myosin into fibers from which myosin had been extracted. Similar results were obtained using rabbit myosin instead of ABRM myosin. Addition of heavy meromyosin, on the other hand, had a suppressive effect on the tension development, as is the case in glycerinated rabbit psoas muscle fibers.     

N-ethylmaleimide inhibits polymerization of myorod, a contractile protein of thick filaments of molluscan smooth muscles

The effect of N-ethylmaleimide on the polymerization of myorod, a protein of molluscan smooth muscles, which is colocalized with myosin on the surface of paramyosin core of thick filaments and is a product of the alternative splicing of the gene of heavy myosin chains, was studied. It was shown that myorod modified by N-ethylmaleimide completely loses the polymerization ability but acquires the ability to aggregate in the presence of Mg2+. At the same time, treatment of molluscan myosin with N-ethylmaleimide did not affect its polymerization. It was supposed that the effect of N-ethylmaleimide on myorod polymerization is related to the modification of the myorod SH domain containing Cys722.     

Structure of Limulus telson muscle thick filaments

Computer analysis of electron micrographs of negatively stained thick filaments isolated from the telson levator muscle of the horseshoe crab (Limulus polyphemus) has shown that they have a four-stranded helical structure. The repeating units along each helix have a bent extended shape (measuring approximately 20 nm × 8 nm × 8 nm) and are inclined at an angle of about 30 ° to the helical path. At the resolution of this study, it was difficult to establish the exact size of the surface subunits, but our results are probably more consistent with each unit representing the two heads of a single myosin molecule rather than larger aggregates.     

Structure and paramyosin content of tarantula thick filaments

Muscle fibers of the tarantula femur exhibit structural and biochemical characteristics similar to those of other long-sarcomere invertebrate muscles, having long A-bands and long thick filaments. 9-12 thin filaments surround each thick filament. Tarantula muscle has a paramyosin:myosin heavy chain molecular ratio of 0.31 +/- 0.079 SD. We studied the myosin cross-bridge arrangement on the surface of tarantula thick filaments on isolated, negatively stained, and unidirectionally metal-shadowed specimens by electron microscopy and optical diffraction and filtering and found it to be similar to that previously described for the thick filaments of muscle of the closely related chelicerate arthropod, Limulus. Cross-bridges are disposed in a four-stranded right-handed helical arrangement, with 14.5-nm axial spacing between successive levels of four bridges, and a helical repeat period every 43.5 nm. The orientation of cross-bridges on the surface of tarantula filaments is also likely to be very similar to that on Limulus filaments as suggested by the similarity between filtered images of the two types of filaments and the radial distance of the centers of mass of the cross-bridges from the surfaces of both types of filaments. Tarantula filaments, however, have smaller diameters than Limulus filaments, contain less paramyosin, and display structure that probably reflects the organization of the filament backbone which is not as apparent in images of Limulus filaments. We suggest that the similarities between Limulus and tarantula thick filaments may be governed, in part, by the close evolutionary relationship of the two species.     

Structure of short thick filaments from Limulus muscle

Shortened Limulus thick filaments, isolated from stimulated muscle, are structurally similar to long filaments, isolated from unstimulated muscle, except for length. Both have 3-fold screw symmetry with a helical repeat at approximately 43 nm, axial spacing of 14.5 nm between successive crowns of crossbridges and 4-fold rotational symmetry as estimated from the Bessel argument, by analysis of optical transforms of electron micrograph negatives of negatively stained samples. Both short and long filaments also have similar radii for the location of their crossbridges, thus similar diameters. Equal numbers of subunits/helical strand are also apparent on images of metal-shadowed long and short filaments. Since these data argue against molecular reorganization during filament shortening, it is suggested that the change in length of Limulus thick filaments may occur by reversible disaggregation of constituent protein molecules.     

The WD repeat: a common architecture for diverse functions.

Our knowledge of the large family of proteins that contain the WD repeat continues to accumulate. The WD-repeat proteins are found in all eukaryotes and are implicated in a wide variety of crucial functions. The solution of the three-dimensional structure of one WD-repeat protein and the assumption that the structure will be common to all members of this family has allowed subfamilies of WD-repeat proteins to be defined on the basis of probable surface similarity. Proteins that have very similar surfaces are likely to have common binding partners and similar functions.     

Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.     

Thin filaments of molluscan catch muscles contain a calponin-like protein

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.     

The alpha/beta fold uracil DNA glycosylases: a common origin with diverse fates

Background  

Uracil DNA glycosylases (UDGs) are major repair enzymes that protect DNA from mutational damage caused by uracil incorporated as a result of a polymerase error or deamination of cytosine. Four distinct families of UDGs have been identified, which show very limited sequence similarity to each other, although two of them have been shown to possess the same structural fold. The structural and evolutionary relationships between the rest of the UDGs remain uncertain.     

Assembly and structure of calcium-induced thick vimentin filaments.

Using a viscometric assay and various electron microscopic procedures (negative staining, rotary shadowing, ultrathin sectioning) we have determined the influences of different kinds of ions and of ionic strength on the structures formed by assembly of soluble subunits of vimentin from bovine lens tissue or from Escherichia coli transformed with Xenopus vimentin cDNA. In contrast to the assembly of typical, i.e., 8 to 14-nm, intermediate-sized filaments (IFs) at elevated (e.g., 160 mM) concentrations of monovalent cations and at millimolar Mg2+ concentrations, filaments formed in the presence of Ca2+ ions (e.g., 5 mM) appeared at a lower rate, attained lower viscosity and were considerably thicker and shorter. The largest diameter measured was that for the recombinant amphibian protein: 24.2 +/- 8.5 nm in negative staining, 28.7 +/- 5.6 nm in sections. These thick Ca(2+)-induced filaments, however, revealed the same approximately 2 nm protofilament composition and approximately 20 nm cross-striation pattern as typical IFs, indicative of a similar molecular arrangement. The significance of this unusual structural IF protein assembly is discussed.     

A model for length-regulation in thick filaments of vertebrate skeletal myosin.

A mechanism for length regulation in the parallel-packed section of the thick filament is proposed. It is based on experiments done on synthetic, mini- and native filaments, and its primary purpose is to explain the physical basis of the kinetic mechanism for the assembly of synthetic thick filaments from myosin alone. Kinetically, length is regulated by a dissociation rate constant that increases exponentially as the filament grows bi-directionally from its center. Growth ceases at the point of equilibrium between invariant on and length-dependent off rates. The three subfilaments structure of the parallel-packed region of the thick filament is fundamental to the proposed scheme. The intra-subfilament bonding is strong and predominantly ionic in character, whereas the inter-subfilament bonding is relatively weak. These strong and weak interactions participate directly in the strictly sequential mechanism of assembly of dimer subunit observed in the kinetics. A third domain, independent of the sequential mechanism, consists of opposing negative charges on the subfilament surface, juxtaposed at or close to the thick filament axis. The weak and repulsive domains are additively coupled to each other through the rigidity in the subfilaments. Length regulation occurs through the repulsive component rising in intensity more rapidly with length than the initially stronger positive interactions. Growth ceases at the point where the repulsive interactions weaken the attractive interactions to the extent that equilibrium is established between head-to-tail dimer subunit and its binding sites at the tips of the arms of thick filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical aspects of the visual process. XXXVI. Calcium accumulation in cattle rod outer segments: Evidence for a calcium-sodium exchange carrier in the rod sac membrane

Accumulation of calcium has been studied in bovine rod outer segments (rods), isolated by sucrose density gradient centrifugation. Calcium-depleted rods are obtained by having ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA) present during isolation.Rods thus isolated have a leaky plasma membrane, as shown by the effects of ionophore A23187 and by their light-induced phosphorylation behaviour. The accumulation of ⁴⁵Ca, determined by incubation followed by a single fast washing-filtration procedure, thus represents translocation across the rod sac membrane.Accumulation in non-depleted rods is independent of the external calcium level and of ATP, suggesting exchange of ⁴⁰Ca by ⁴⁵Ca. In depleted rods in the presence of ATP there is net uptake, sigmoidally increasing with the external calcium concentration to the level attained in non-depleted rods. This net uptake is abolished by omission of ATP, its replacement by β,γ-methylene ATP and lowering the temperature to 0° C, suggesting involvement of enzymatic hydrolysis of ATP.Replacement of KCl by NaCl in the medium causes marked inhibition of ⁴⁵Ca uptake, both net uptake and exchange. Oligomycin, ruthenium red, lanthanum and ouabain do not inhibit accumulation.Efflux of ⁴⁵Ca from pre-loaded rods is slow in a KCl medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～30}\text{min}$ at 25° C), but is greatly accelerated by addition of NaCl or Ca²⁺ ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{10}\text{s}$ at 25°C).It is concluded that the rod sac membrane contains a carrier system, which is sensitive towards Ca²⁺ and Na⁺ and which requires ATP for net uptake of Ca²⁺ but not for exchange transport of Ca²⁺ with Ca²⁺ or Na⁺.     

Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle

Rabbit psoas muscle filaments, isolated in relaxing buffer from non-glycerinated muscle, have been applied to hydrophilic carbon films and stained with uranyl acetate. Electron micrographs were obtained under low-dose conditions to minimize specimen damage. Surrounding the filament backbone, except in the bare zone, is a fringe of clearly identifiable myosin heads. Frequently, both heads of individual myosin molecules are seen, and sometimes a section of the tail can be seen connecting the heads to the backbone. About half the expected number of heads can be counted, and they are uniformly distributed along the filament. The majority of heads appear curved. The remainder could be curved heads viewed from another aspect. Three times as many heads curve in a clockwise sense than in an anticlockwise sense, suggesting a preferential binding of one side of the head to the carbon film. The two heads of myosin molecules exhibit all the possible combinations of clockwise, anticlockwise and straight heads, and analysis of their relative frequencies suggests that the heads rotate freely and independently. The heads also adopt a wide range of angles of attachment to the tail. The lengths of heads cover a range of 14 to 26 nm, with a peak at 19 nm. The average maximum width is 6.5 nm. Both measurements are in excellent agreement with values for shadowed molecules. Since our data are from heads adsorbed to the film in relaxing conditions and the shadowed molecules were free of nucleotide, gross shape changes are not likely to be produced by nucleotide binding. The length of the link between the heads and the backbone was found to vary between 10 nm and 52 nm, with a broad peak at about 25 nm. Thus, the hinge point detected in the tail of isolated molecules was not usually the point from which the crossbridges swung out from the filament surface. The angle made by the link to the filament axis was between 20 degrees and 80 degrees, with a broad maximum around 45 degrees. These lengths and angles concur with our observation of an average limit of the crossbridges from the filament surface of 30 nm. This is sufficient to enable heads in the myofibril lattice to reach out beyond the nearest thin filament and should allow considerable flexibility for stereospecific binding to actin in active muscle.