Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.     

Preparation of myosin rods from myofibrils

A simple and fast procedure is described for producing myosin rods with a good yield. The method is based on the digestion of myofibrils by soluble papain. After purification by alcoholic precipitation and gel filtration, rod segments exhibited physical-chemical parameters identical to previously reported data.     

The two pathways for oxygen exchange by actomyosin and myofibrils and their dependence on temperature

At an intermediate stage in the hydrolysis of MgATP by actomyosin there is an exchange of oxygen between water and the terminal phosphoryl group of MgATP, tightly bound to the myosin active site. This intermediate oxygen exchange results from the reversible hydrolysis of the bound MgATP. The rate of the exchange cycle (hydrolysis and the reverse) is assumed to be determined by the rate of reverse hydrolysis; and the average time available for exchange is determined by the post-exchange reaction that immediately follows the cycle. Past analytical studies of the exchange, using actomyosin mixtures and myofibrils at room temperature, have revealed two pathways for hydrolysis, operating at a comparable flux but differing greatly in the extent of exchange they support. It is shown here that these pathways also appear over a range of temperatures from 5 to 30 degrees C and that temperature had little effect on their relative fluxes. At each temperature, the flux ratio (%) for the low exchange pathway: high exchange pathway was near 50:50 for actomyosin mixtures and 60:40 for myofibrils. Apparently, the rate-limiting steps that determine the fluxes of the two pathways have a similar temperature dependence. However, the analysis indicates that one or both of the steps that determine the extent of exchange (reverse-hydrolysis and/or the post-exchange reaction) shows a different temperature dependence for the two pathways. We interpret this to reflect a difference in the temperature dependence of the post-exchange reaction, which we propose is exceedingly fast and independent of actin concentration along the low exchange route, but slow and dependent on the actin concentration along the high exchange route. Thus at all temperatures over a broad range of actin concentration there are two pathways of comparable flux that differ primarily in the time available for exchange.     

A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization

A new protein component of skeletal myofibrils has been isolated and characterized. It is prepared from impure myosin preparations and corresponds to band C, the principal contaminant observed in sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of such preparations (Starr and Offer, 1971).The C-protein, as we term it, is deduced to be a component of the skeletal myofibril because (i) glycerinated or fresh myoflbrils contain a component with a mobility identical to C-protein on sodium dodecyl sulphate gels, (ii) this component is extracted from myofibrils by the same solvent which extracts C-protein and (iii) C-protein may be prepared from preparations of isolated myofibrils. It is presumed to be a component of the thick filaments because it binds strongly to myosin at low ionic strength; immunological evidence which confirms this view is presented elsewhere.The quantity of C-protein in the myofibril has been estimated to be 2.0% by densitometry of sodium dodecyl sulphate gels of glycerinated myofibrils using actin as an internal reference. About forty molecules of C-protein are present in a thick filament.The properties of C-protein distinguish it from the other well-characterized myoflbrillar proteins. The C-protein molecule contains a single polypeptide chain of molecular weight 140,000. The intrinsic viscosity of 13.6 ml/g suggests that the molecule is neither completely globular nor as elongated as molecules like paramyosin or tropomyosin. The α-helical content is very low and the proline content higher than the other myofibrillar proteins. The molecule associates at low ionic strength.C-protein has no ATPase activity, nor does it affect the ATPase of pure myosin. But it reduces the activity of the actin-activated myosin ATPase by about half, this inhibition being independent of the level of Ca²⁺. C-protein does not bind Ca²⁺ in the presence of Mg²⁺. Its possible location and function are discussed.     

Actin antibodies. Preparation and characterization of antibodies specific for smooth-muscle actin isoforms.

We have determined the specificity of sera elicited by glutaraldehyde-stabilized bovine aortic actin. This modification induces a high titre of antibodies directed against the N-terminal (residues 1-39) and the C-terminal region of smooth-muscle actins. The crude antisera were purified on peptide (corresponding to the 1-9 or 1-8 N-terminal sequences of smooth-muscle isoactins)-polyacrylic-resin columns. By fractionating the antisera we obtained oligoclonal antibody populations specific for each isoactin.     

A modelized distribution of actomyosin interactions in the vertebrate cardiac muscle

Preliminary assumption of this model is that interactions between actin and myosin presupposes an exact three-dimensional geometrical correspondence between sites, due to the very short time constants present under physiological conditions. Only small and controlled torsions of the actin filaments are accepted. The model uses geometrical information concerning orientations and dimensions of myosin crossbridges and actin monomeres to modelize the distribution of their inter-actions. An orientation map of actin sites in the cross-section perpendicular to the filament axis is proposed, adapted to the specific filament array of vertebrate muscle. Orientation of myosin crossbridges follows Luther's rules. According to the model, any interaction between actin and myosin implies the superimposition of their respective cross-sectional planes. The axial length of actin monomere is 55 A; the distance between two crossbridges along the myosin filament axis is 143 A. The following properties are derived: 1) The shortening step of the sliding actin filament must be a multiple of 11 A (highest common factor). Taking into account the staggered disposition of the two actin strands and the presence of two heads for each cross-bridge, the most probable value for this shortening step is equal to 99 A. A specific scheme is proposed to describe the shortening process. The behavior of the modelized crossbridge does not need any elastic structure--2) Planes situated at 715 A (lowest common multiple) of actin and myosin coinciding planes are also in coincidence. In a hemi-sarcomere the maximal number of these planes, referred to as simultaneously activable planes, is 10 (20 if both myosin heads are considered). The proportion of interactions authorized by the site orientations is 1/12. In the model, the concept of randomly recruited crossbridges is replaced by a discretized recruitment, based on geometrical properties at an ultrastructural level. The proposed distribution is homogeneous: it can be extended radially in the sarcomere and authorizes the actin filament sliding in the whole physiological range under the control of a dual activation function, reproducing Ca++ temporal and spatial distribution.     

Stochastic properties of actomyosin motor

The epoch-making techniques for manipulating a single myosin molecule have recently been developed, and the unitary mechanical reactions of a single actomyosin, muscle motor molecule, are directly measured. The data show that the unitary mechanical step during sliding along an actin filament of approximately 5.5 nm, but groups of two to five rapid steps in succession produce displacements of approximately 11-30 nm. The instances of multiple stepping are produced by single myosin heads during one biochemical cycle of ATP hydrolysis. Thus, the coupling between ATP hydrolysis cycle and mechanical step is variable, i.e. loose-coupling. Such a unique operation of actomyosin molecules is different from that of man-made machines, and most likely explains the flexible and effective mechanisms of molecular machines in the biosystems.     

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.     

Microscopic analysis of the elastic properties of nebulin in skeletal myofibrils.

The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers.     

Electrical properties and transmembrane ion currents of single smooth-muscle cells

Current and voltage clamp investigations of freshly isolated smooth muscle cells from guinea-pig ileum and taenia coli were performed using single suction micropipette technique. Specific membrane capacity of smooth muscle cells was calculated and accounted for 1.6 microF/cm2, with specific resistance varying from 50 to 150 k omega X cm2. Transmembrane currents consisted of two inward components, inactivating and noninactivating ones, carried by Ca2+ ions, overlapping with early activated potassium outward current. Time constant of inward current activation was not only voltage-sensitive but also ion-dependent. When Ca2+ ions in Krebs solution were replaced by Ba2+, both the rate of activation and inactivation of inward current were significantly reduced. Estimation of intracellular Ca2+ concentration increase has indicated that inward calcium current transports enough Ca2+ for direct contraction activation.     

Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles

The mechanism of ATP hydrolysis by myosin and actomyosin was investigated for the four major classes of vertebrate muscles: fast white (posterior latissimus dorsi), slow red (anterior latissimus dorsi), cardiac and smooth (gizzard). The kinetic behavior of all classes of muscle was consistent with the scheme developed previously for rabbit fast white muscle, but quantitative differences were observed for the rate constants of some of the steps in the hydrolysis cycle. The rate of the hydrolysis step of myosin subfragment-1 was similar for the striated muscles and two to three times smaller for smooth muscle. Two isomerizations of the enzyme occurred in the pathway leading to the formation of the myosin-products intermediate. The rate of dissociation of acto S–1 by ATP was slower for slow muscles and a maximum rate was observed at low temperature. The rate of association of the S-1-products intermediate with actin was equal to the turnover rate of acto S–1 ATPase at low concentrations of actin. The rate of dissociation of ADP from an acto S–1-ADP complex was also much slower for slow muscle. It was shown by Barany (1967) that the maximum turnover rate of actomyosin ATPase (V_M) is proportional to the velocity of contraction of the muscle. The only step in the mechanism that is correlated with V_M is the apparent second-order rate constant for the formation of a complex of the S-1-product state with actin. The evidence is discussed in terms of a mechanism in which the release of reaction products from actomyosin is the step that is of primary importance in determining the value of V_M and the velocity of contraction.     

Contractile properties of compressed monolayers of actomyosin

1. Surface-spread actomyosin, compressed into fibers, shows biological properties of contractility and enzymic activity. 2. In unloaded contractions, wet and dry weight determinations show no appreciable water loss in contraction. The fibers also evince a strong ATP-ase activity. 3. A structural continuity in the fibers by intermolecular linkages of the component actomyosin molecules is established during the formation of the fibers. Evidence includes their visible longitudinal structural organization, the lack of elongation effect of ATP when under tension, and their ability to lift appreciable loads, so that, like muscle, they can transform chemical energy into mechanical work. 4. Up to a limiting critical weight, the fibers perform more work with increasing imposed weight load. 5. Theoretical aspects are discussed, including the possibility that surface-spread protein is involved in the formation of cell structures. Possible explanations for the relative slowness of the fiber contractions are offered.     

Autophosphorylation of smooth-muscle caldesmon.

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.