Diphosphorylation of platelet myosin by myosin light chain kinase.

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.     

Unshadowed myosin molecules: STEM mass-maps of myosin heads.

Myosin molecules were directly visualized without heavy metal shadowing by scanning transmission electron microscopy (STEM) under low dose conditions. The general appearance and dimensions of heavy metal-free molecules were similar to those of shadowed myosin, either after freeze-drying without or air-drying with glycerol. Two characteristic configurations of myosin head regions were found, a first type showing two pear-shaped heads with narrow necks and a second type showing two heads connected by an extra mass in the central regulatory domain where the light chains are located. The mass of the latter type (mol. wt. = 265 +/- 39 kd) is in excellent accordance with biochemical data whereas the mass of the first type is somewhat lower (mol. wt. 219 +/- 44 kd).     

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.     

Substructure of the myosin molecule. 3. Preparation of single-headed derivatives of myosin

Native myosin has two globular regions attached to an a-helical rod. Papain is able to cleave the globular “heads” from the rod, leading to the formation of a variety of single-headed molecules. Among these subfragments are isolated globules (HMM S-1) and single globules attached to helical rods of lengths varying from 500 to 1400 Å. These subfragments can be separated from the other products of the proteolytic digestion by salt elution from a DEAE-cellulose column. Some of the properties of single-headed heavy meromyosin and myosin have been determined by hydrodynamic methods, and shadow-cast preparations of these subfragments have been directly visualized by electron microscopy. In addition to providing further evidence for the presence of two similar halves in myosin, these new subfragments can be used in studies related to the question of why myosin has two active “heads”.     

Macromolecular assemblies of myosin.

The self-assembly of myosin into filamentous structures is a highly cooperative and rapid process. Nevertheless, the presence of nonequivalent bonding interactions within the filament permits differential stabilization of several macromolecular assemblies of myosin under well-controlled ionic conditions in citrate/Tris buffer at pH 8.0. We have detected and characterized bipolar myosin minifilaments, myosin octamers, and tetramers by using light scattering, analytical ultracentrifugation, and viscosity techniques. These structures have molecular weights of 8.0 X 10(6), 3.9 X 10(6) g/mol, sedimentation coefficients of 32S, 22S, and 18S, and radii of gyration of 990 A, 890 A and 790, A, respectively. The similar radii of gyration indicate similar bipolar geometry for all these particles. The 32S minifilaments in 10 mM citrate/Tris buffer (pH 8.0) are the most stable species. The smaller 18S and 22S assemblies in 2 mM and 5 mM citrate/Tris, pH 8.0, are readily affected by low concentrations of KCl and fuse into the minifilament particles. The instability of the 18S and 22S forms of myosin assembly is also revealed by their titration with ATP. These structures are dissociated at lower ATP concentrations than the minifilaments and do not show the cooperative dissociation transitions characteristic of filaments and minifilaments. Sedimentation velocity analysis of the 18S and 22S species in the presence of ATP reveals the involvement of 10S myosin dimer in the dissociation of assembled myosin. The different forms of assembled myosin are discussed in the context of formation of myosin minifilaments.     

The myosin power stroke.

Optical trapping technology now allows investigators in the motility field to measure the forces generated by single motor molecules. A handful of research groups have exploited this approach to further develop our understanding of the actin-based motor, myosin, an ATPase that is capable of converting chemical energy into mechanical work during a cyclical interaction with filamentous actin. In this regard, myosin-II from muscle is the most well-characterized myosin superfamily member. By combining the data obtained from optical trap assays with that from ensemble biochemical and mechanical assays, this review discusses the fundamental properties of the myosin-II power stroke and, perhaps more significantly, how these properties are governed by this molecule's atomic structure and the biochemical transitions that define its catalytic cycle.     

Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.     

Internal motions in myosin.

High-resolution proton nuclear magnetic resonance (1H NMR) measurements were made on myosin, heavy meromyosin (HMM), myosin subfragment 1 (S1), light meromyosin (LMM), and actin. A strong signal from amino acid side chains undergoing motions too fast to be accounted for by simple rotations of groups on a rigid backbone was obtained from myosin. Comparison of myosin, HMM, S1, and LMM showed that the mobile region is located almost entirely in S1 and accounts for approximately 22% of its structure. Adenosine triphosphate (ATP) and ATP analogues had no measurable effect on the S1 spectrum. Actin, on the other hand, quenched the internal motions of S1. When S1 was titrated with actin, an association was obtained which was in agreement with other measured values. The actin effect was reversed by adding magnesium pyrophosphate (MgPPi) or adenyl-5'-yl imidophosphate (MgAMPPNP). Quantitative treatment of the broad signals from myosin and its subfragments substantiated the existence of two flexible regions in myosin. The highly mobile portion of myosin may be located in the "swivel" between S1 and the rest of myosin or in the actin binding site or in both. These possibilites are discussed, and a new possible mechanism for muscle cross bridge elasticity is proposed.     

Selective modification of myosin SH1 with 1,2,4-trinitrobenzene. VIII. Thiols of myosin

Myosin has 2 mol of the most reactive thiol, named SH1. 1,2,4-Trinitrobenzene (TNB), a novel dinitrophenyl(DNP)ating reagent [Takahashi et al. (1983) Chem. Lett. 1445-1448], was found to react only with SH1 without any other amino acid residues in myosin under the conditions used. Its reaction with myosin SH1 was about 30 times faster than that with N-acetylcysteine (NAC). The reaction rate of TNB with SH1 was about twice compared with that of NEM, the most reactive selective reagent for SH1 so far found, although its rate with NAC was only one sixtieth that of NEM. As to the lambda max of the absorption spectrum of SH1-DNP-myosin, a large red shift of as much as 20 nm was observed compared with low molecular S-DNP derivatives. This red shift disappeared in 8 M urea. This outstanding feature of SH1 modification with TNB was discussed in terms of affinity labeling by interaction with an aromatic amino acid near SH1.     

General model of myosin filament structure. 3. 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.     

Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.     

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.