Rod Portion Research Articles

Isoform specificity of monoclonal hybridoma antibodies to quail skeletal muscle myosin subunits

Monoclonal antibodies to adult quail breast muscle myosin (QBM) have been prepared and characterized using a solid phase enzyme linked immunosorbent assay and immunoblot procedures. One antibody (QBM-1) is directed against an epitope in the rod portion of the myosin heavy chain while another (QBM-2) binds exclusively to a conserved portion of the two alkali light chains of fast muscle myosin. Both of these antibodies cross-react with myosin from myotubes cultured in vitro but do not recognize non-muscle myosin. The application of these antibodies to the study of myogenesis is discussed.

Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle.

The amino acid sequence of the rod portion of nematode myosin, deduced for the sequence of the unc-54 heavy chain gene of Caenorhabditis elegans, is highly repetitive and has the characteristics of an alpha-helical coiled coil. The molecular surface contains alternate clusters of positive and negative charge. Interactions between charge clusters on adjacent molecules could account for the observed spacing of the myosin cross-bridges in muscle. Calculations also suggest that the N-terminal third of the rod is only loosely associated with the thick filament backbone. Bending of the rod near the end of this region could allow the N-terminal section to act as a hinged arm during muscle contraction.

Structurally damped bolt

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Living macrophages phosphorylate the 20,000 dalton light chains and heavy chains of myosin

Brief incubation of rabbit alveolar macrophages in medium containing 32Pi results in the incorporation of radioactivity into the 20 KD light chains and into the 220 KD heavy chains of myosin. Phosphorylation of the heavy chain is mediated by a kinase that is probably not myosin light chain kinase. Limited proteolysis of the phosphorylated myosin shows that radioactivity is associated with the rod portion of the heavy chain.

EPR and fluorescence depolarization studies on bovine cardiac myosin.

To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in steric restraints around the SH1 thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M.ADP and M.ADP.Pi. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kd (ADP) = 7 microM, n = 1.6. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins.

Fluorescence studies on the interaction of muscle M-line proteins, creatine kinase and the 165,000 dalton component, with each other and with myosin and myosin subfragments

1. 1. The binding of 8-anilino-l-naphthalene sulfonate (ANS) by M-line creatine kinase (CPK) and the 165,000 dalton protein component was studied by fluorescence. 2. 2. One mole of ANS binds to a mole of each M-protein and the binding site on these two M-proteins is hydrophobic in nature. 3. 3. M-line proteins labeled with ANS were used to demonstrate their interaction with myosin and myosin subfragments 1 and 2. 4. 4. A unique finding of this study, that labeled M-line CPK binds to subfragment 2 of myosin, is of significance from a structural view point, since only the rod portions of myosin molecules are exposed at the M-line and therefore able to interact with CPK. 5. 5. The use of a sulfhydryl fluorescent probe, N-(2-iodoacetyl)- N-(5-sulfo-l-napthyl) ethylene diamine, (1,5-AEDANS), has shown that the essential sulfhydryl group on CPK is very important for its interaction with both myosin and the 165,000 dalton M-line protein component.

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.

Topography of the myosin molecule as visualized by an improved negative staining method.

An improved negative staining method has been used to visualize monomeric myosin molecules. Each lobe (subfragment 1) of the molecule looked like an elongated pear, and the widths of the thick and thin portions were about 95 and 55 A, respectively. The length of each lobe was about 210 A. It appeared capable of moving azimuthally and altitudinally, utilizing its juncture with the rod portion as the base. The rod portion of the molecule was about 1400 A long and 30 A wide. It also appeared to possess a considerably flexible region at a point about 680 A from the tail-end.

Hydrolytically induced allosteric change in the heavy chain of intact myosin involving nonessential thiol groups

The two globular head portions, each bearing an active site, contain an uncleaved heavy chain when isolated by chymotrypsin from intact myosin. By specific labeling with radioactive N-ethylmaleimide the essential thiol 1 and thiol 2 groups were found to reside in this heavy chain. In intact myosin nonessential thiol 3 groups become the most reactive during ATP hydrolysis above 15 degrees C. These thiol 3 groups are located in a portion of the myosin heavy chain which appears as a fragment with an apparent molecular weight of 11 000 during proteolysis. The facts that this fragment is produced in an almost 1: 1 molar ratio with the head heavy chain and that it bears unblocked N-terminal amino groups whereas the heavy chain does not and is not contained in the rod portion of the myosin molecule indicate that it may orginate from the heavy chains in the neck region where the heads are joined to the rod. Since this fragment is removed by ion-exchange chromatography, it is not part of the functioning head and hence not involved in the active site. As its nonessential thiol 3 groups are rendered the most reactive of all thiol groups in the enzyme-product complex M**ADP.Pi, the hydrolytic step induces an allosteric conformational change in the neck region of intact myosin.

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 quantity 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 the 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 μm 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 ε-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.

Heat-induced denaturation of myosin total rod.

The myosin total rod, which consists of smaller segments of light meromyosin and heavy meromyosin subfragment-2 (HMM S–2), prepared by limited papain digestion of rabbit myosin, was purified by Sepharose-2B column chromatography. The purified total rod was more homogeneous than any previously reported, and the sodium dodecyl sulfate (SDS) gel electrophoretic method yielded a molecular weight of 22−23 × 104 (11−11.5 × 104 × 2).Transition temperatures of this purified myosin total rod obtained from the melting profile during heating were 47.5 and 55°C. The results of ORD and CD measurements showed almost full reversibility upon cooling after thermal treatment. However, the results obtained from difference spectra and fluorescence spectra showed incomplete reversibility with hysteresis.This ostensible dichotomy concerning the structural thermostability of the rod portion of myosin molecule may mean that although ORD and CD studies show almost full reversibility of the helix-coil transition, local irreversibl...

Infrared spectra of light and heavy meromyosin

1. 1. Infrared spectra of films of both light and heavy meromyosin are very similar to those ofmyosin. As with myosin the precipitating conditions determine the observed conformations. 2. 2. The conformation of films prepared at pH 7 appears to be a mixture of α-helical and random conformations. 3. 3. However β-structure is observed in addition to the α-helical secondary structure if the films are precipitated at low pH or with a 50-50 mixture by volume of CHCl 3-Ch 3OH. 4. 4. The occurrence of β-structure in both light and heavy meromyosin suggests that the β-structure occurs in the rod portion of the myosin molecule.

Location of the creatine phosphokinase binding site of myosin

The interaction of myosin and its subfragments with creatine phosphokinase (CPK) was studied by the method of steady state fluorescence depolarization. Perrin plots showed that CPK labeled with a sulfhydryl specific dye binds to the rod portion of the myosin molecule, and that this interaction is specific for CPK and myosin.

General model of myosin filament structure: III. Molecular packing arrangements in myosin filaments

Following the original proposals about myosin filament structure put forward as part of a general myosin filament model (Squire, 1971, 1972) it is here shown what the most likely molecular packing arrangements within the backbones of certain myosin filaments would be assuming that the model is correct. That this is so is already indicated by recently published experimental results which have confirmed several predictions of the model (Bullard and Reedy, 1972; Reedy et al., 1972; Tregear and Squire, 1973). The starting point in the analysis of the myosin packing arrangements is the model for the myosin ribbons in vertebrate smooth muscle proposed by Small & Squire (1972). It is shown that there is only one reasonable type of packing arrangement for the rod portions of the myosin molecules which will account for the known structure of the ribbons and which is consistent with the known properties of myosin molecules. The dominant interactions in this packing scheme are between parallel myosin molecules which are related by axial shifts of 430 Å and 720 Å. In this analysis the myosin rods are treated as uniform rods of electron density and only the general features of two-strand coiled-coil molecules are considered. Since the general myosin filament model is based on the assumption that the structures of different types of myosin filament must be closely related, the packing scheme derived for the myosin ribbons is used to deduce the structures of the main parts (excluding the bare zones) of the myosin filaments in a variety of muscles. It is shown in each case that there is only one packing scheme consistent with all the available data on these filaments and that in each filament type exactly the same interactions between myosin rods are involved. In other words the myosin-myosin interactions involved in filament formation are specific, they involve molecular shifts of either 430 Å or 720 Å, and are virtually identical in all the different myosin filaments which have been considered. Apart from the myosin ribbons, these are the filaments in vertebrate skeletal muscle, insect flight muscle and certain molluscan muscles. In the case of the thick filaments in vertebrate skeletal muscle the form of the myosin packing arrangement in the bare zone is considered and a packing scheme proposed which involves antiparallel overlaps between myosin rods of 1300 Å and 430 Å. It is shown that this scheme readily explains the triangular profiles of the myosin filaments in the bare zone (Pepe, 1967, 1971) and many other observations on the form of these myosin filaments. Finally it is shown that the cores of several different myosin filaments, assuming they contain protein, may consist of different arrangements of one or other of two types of core subfilament.

Comparative Studies of Myosin Rods Isolated by Enzymatic and Non-enzymatic Cleavage of Myosin

Tryptic (LMM Fr 1) and CNBr-treated (LMM-C) rod portion of myosin molecule were prepared from rabbit skeletal muscle myosin. An attempt was made to clarify the difference between LMM Fr 1 and LMM-C as measured by solubility and some physico-chemical techniques. The rigid structure of LMM Fr 1 melted into a random coil as temperature of the solution increased and the value of b0 and reduced viscosity did not show full recovery upon gradual cooling. The situation of LMM-C, on the other hand, showed the higher thermostability than LMM Fr 1. As stated in our previous paper,1) we have considered that the rod portion of myosin molecules is substantially thermostable.

Isolation and characterization of myosin and two myosin fragments from human blood platelets.

Platelet myosin (thrombosthenin M) and two additional proteins corresponding to the head and rod portion of the myosin molecule have been prepared from human blood platelets. Characterization of these proteins by SDS-polyacrylamide gel electrophoresis, actin binding studies, assay of enzymic ATPase activity, and electron microscopy has shown that the platelet contractile proteins closely resemble the corresponding muscle proteins. Platelet myosin and platelet myosin-head bind to both muscle and platelet actin and have an EDTA + K-stimulated ATPase activity, which is suppressed by Mg(2+) in high salt concentration, whereas platelet rod does not possess either of these properties; platelet myosin and platelet myosin rod aggregate to form thick filaments at low ionic strength. Both intact platelet myosin and myosin head form typical arrowhead-shaped complexes with either platelet or muscle F-actin.

Segments from vertebrate smooth muscle myosin rods

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 Å overlap as well as a 430 Å axial translation.

Segments from myosin rods

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.

Substructure of the myosin molecule: I. Subfragments of myosin by enzymic degradation

The formation of light meromyosin and heavy meromyosin by trypsin first demonstrated that myosin had regions of high α-helix content which could be separated from the ATPase and actin-binding centers of the molecule. Here we report the action of papain on myosin. Under suitable experimental conditions, low concentrations of papain can split the enzymic globular regions of myosin from the structural α-helical rod, leaving the latter intact along its length. Further digestion cleaves the rod along two-thirds of its length to form light meromyosin and the rod portion of heavy meromyosin. The size and shape of these proteolytic fragments as deduced from hydrodynamic measurements are compared to the structure seen in electron micrographs of shadow-cast preparations. From these studies we conclude that the myosin molecule consists of two globular units, each about 70 Å in diameter, attached to a double-stranded α-helical rod about 1350 Å long. The effect of Ca- and Mg-actin on the ATPase activity of the cleaved isolated globules is similar to the effect of these cations and actin on the globules in the native or attached state existing in heavy meromyosin. The rod portion of myosin is insoluble at low ionic strength and is therefore responsible for filament formation under physiological conditions. However, only the light meromyosin end of the rod is insoluble in water at neutral pH; the remainder of the rod is soluble at all ionic strengths. There is no evidence for an extensive amorphous region connecting these two subfragments. The intact rod has an α-helical content consistent with a fully helical molecule, and no proline residues could be detected by amino acid analysis.

The ultrastructure of myofilaments. II. Further investigation of the thick filaments of crab muscles.

Abstract Investigations on cross-sections of cross-striated muscles of two crabs ( Paralithodes camtschatica and Carcinus maenas ) showed that intensive staining and araldite embedding reveal the fine structure of thick filaments. There were from 13–15 to 18 “subunits” observed in cross-sections of thick filaments. There is reason to suppose that the maximum number of subunits (18) reflects the actual situation. The subunits, or at least most of them, appear to be the rod portions of myosin molecules. Whether or not this situation occurs in muscles of other species remains unresolved.