Carolyn Cohen

Paramyosin and the filaments of molluscan "catch" muscles. II. Native filaments: isolation and characterization.

Abstract A method has been developed for the partial separation of the filaments of molluscan muscles. A fraction has been obtained which consisted mainly of actin filaments many of which were attached to dense bodies. When complexed with rabbit heavy meromyosin, the actin filaments showed arrowhead structures which appeared to have opposite polarity at either end of the dense bodies. The other fraction consisted of very long and thick filaments containing essentially all of the paramyosin and the myosin in the muscle. Some actin filaments were also present in this fraction. When myosin solubilization was prevented, myosin filaments similar to those of vertebrate striated muscle were rarely found in either of the two fractions. Myosin could be selectively extracted from the thick filaments without the solubilization of paramyosin; extraction of paramyosin, however, was always accompanied by the solubilization of myosin. The removal of myosin changed the surface appearance of the thick filaments: a characteristic pattern of darkly staining nodes or gap regions in the paramyosin filament became visible in negatively stained preparations. The nodes have a roughly triangular shape defining the polarity of the structure. A number of filaments were found which showed a reversal of polarity along their length. These results lead to the conclusion that paramyosin forms a bipolar core of the thick filament which is covered by a surface layer of myosin. The specific interactions between paramyosin and myosin could be demonstrated structurally and enzymically. Molluscan myosin formed filaments resembling those of rabbit myosin when precipitated in vitro . Its assembly in the molluscan thick filament is therefore determined by the underlying paramyosin core. Paramyosin greatly inhibited the actin activated ATPase of myosin in a selective manner when these proteins were mixed in about a mole to mole ratio. The long bipolar thick filaments with myosin on the surface can account for the tension development in these molluscan muscles according to the sliding filament theory. These results also suggest that paramyosin may have a specific regulatory role in tension maintenance: in the catch mechanism a phase change in paramyosin may be coupled to the movement of cross-bridges formed between myosin and actin.

Tropomyosin: Crystal structure, polymorphism and molecular interactions☆

Abstract The α-protein tropomyosin forms a variety of ordered aggregates. True crystals with a very open lattice and at least two other net forms are produced near the isoelectric point together with fibrous aggregates. Three distinctive types of tactoids with axial periodicities about 400 A are formed with divalent cations. The electron microscope observations on the polymorphic forms have been related to X-ray diffraction measurements on the crystal lattice and the tactoids produced with magnesium. The X-ray pattern of one projection of the crystal has been interpreted at low resolution from a model composed of rod-shaped molecules arranged in accord with the electron micrographs. The tropomyosin molecules in the crystal are associated head-to-tail in polar filaments with a 400 A period. The filaments are periodically bent as a consequence of the cross connections at two sites alternatively separated by about 230 A and 170 A in the 400 A axial period. The principal conclusions of this study are: there is a specific polar end-to-end bonding of tropomyosin molecules which defines the period of about 400 A observed in the polymorphic nets and tactoids; there are two cross-connecting sites which are involved in net formation; in many of the forms, the polar filaments are arranged in oppositely directed pairs; the molecular coiled-coil is often supercoiled in the polar filaments. The periodicity in the I band of muscle can be identified with the 400 A repeat characteristic of the end-to-end association of tropomyosin. The polymorphism observed in vitro may be related to the structural and regulatory functions of tropomyosin in muscle.

A tropomyosin-like protein from human platelets.

Abstract A tropomyosin-like protein has been isolated from human platelets. This two-chain α-helical molecule has a subunit molecular weight of 30,000 and forms paracrystals with an axial period of 345 A, which is close to the length of the molecule. It is therefore smaller in size than the 400 A long tropomyosins from muscle. This protein appears to bind to platelet actin. Actomyosin from platelets shows relaxing-factor activity, and this tropomyosin-like protein may have a role in the regulation of contraction.

Tropomyosin Paracrystals Formed by Divalent Cations

Rabbit tropomyosin exhibits a polymorphism dependent on divalent cations and pH. Above a critical divalent cation concentration fibers with a period of about 400 angstroms are formed. Below this concentration, and near the isoelectric point, lattices are formed. Implications for the morphology and function of striated and smooth muscle are discussed.

Assembly of myosin

Abstract Myosin and its helical subfragments form bipolar “segment” aggregates which may be related to the bare zone of the thick filament. Two distinct modes of aggregation have now been observed: one with an overlap of 1300 A and another with an overlap of about 900 A. Both are consistent with a value of 1450 A for the length of the rod. The helical arrangement of the bridges on the thick filament has been determined by X-ray diffraction results. Placing rods of length 1450 A on this surface lattice generates the molecular overlaps found in the in vitro “segment” aggregates.

X-ray diffraction from microtubules

Abstract X-ray diffraction patterns from sperm-tail microtubules indicate that subunits with a 40 to 50 A packing diameter form filaments, alternately half-staggered, parallel to the tubule axis. A 12- or a 13-stranded structure fits best with the X-ray diagram. The strongest bonding is that between units within a longitudinal filament; the weaker lateral interactions are disrupted by drying.

Segments from myosin rods

Abstract The rod portion of myosin forms bipolar “segment” aggregates when precipitated with divalent cations. This in vitro structure establishes a minimum length for the rod, and indicates one mode of molecular packing which may be related to the bare zone of the myosin filament.

Segments from vertebrate smooth muscle myosin rods

Abstract The rod portion of myosin from gizzard muscle forms “compound” segments when precipitated with divalent ions. These compound segments can be accounted for by the interaction of two pairs of molecular arrays related by 2-fold axes. Similar but shorter compound segments are formed from subfragments obtained by tryptic and chymotryptic digestion of gizzard myosin. All the different segments display a bipolar 430 A overlap as well as a 430 A axial translation.

Paracrystalline Forms of Fibrinogen

Electron microscope and x-ray diffraction observations on highly ordered tactoids of fibrinogen show that these structures have the same axial period as fibrin. Implications of these results for the analysis of clot formation are discussed.