Behaviour of the crossbridges in stretched or compressed muscle fibres.

The maximum chord of the myosin heads is comparable to the closest surface-to-surface spacing between the myofilaments in a muscle at the slack length. Therefore, when the sarcomere length increases or when the fibre is compressed, the surface-to-surface myofilament spacing becomes lower than the head long axis. We conclude that, in stretched or compressed fibres, some crossbridges cannot attach, owing to steric hindrance. When the amount of compression is limited, this hindrance may be overcome by a tilting of the heads in the plane perpendicular to the filament axes; in this case, there is no consequence as concerns the crossbridge properties. In highly compressed fibres, the crossbridges become progressively hindered and all the crossbridges are hindered for an axis-to-axis spacing representing about 60% of the spacing observed under zero external osmotic pressure. In this case, both the isometric tension and the ATPase activity of the fibre are zero. In fibres stretched up to 3.77 microns (sarcomere length corresponding to the disappearance of the overlap between the thick and the thin filaments), the ratio of hindered crossbridges over the functional crossbridges may be estimated at about 55%. In stretched fibres, a noticeable proportion of crossbridges are sterically hindered and the crossbridges performance (e.g. constants of attachment and detachment) depends on filament spacing, i.e. on sarcomere length. Therefore, we think it is probably impossible to consider the crossbridges as independent force converters, since this idea requires that the crossbridge properties are independent of sarcomere length. In this connection, all the experiments performed on osmotically compressed fibres are of major importance for the understanding of the true mechanisms of muscle contraction.

Cryoenzymic studies on myosin: transient kinetic evidence for two types of head with different ATP binding properties.

The two step tight binding of ATP to myosin, heavy meromyosin and myosin subfragment 1 was investigated, under cryoenzymic conditions by the unlabeled ATP chase method: M + ATP in equilibrium K1 M.ATP k2 in equilibrium k-2 M*.ATP where M is myosin. k-2 is close to zero. In multi-turnover experiments, one obtains the constants for the binding process together with the concentration of ATPase sites. Here the kinetics of the formation of M*.ATP are first order. Inversion of the reagent concentrations (i.e., single-turnover experiments) should give identical kinetics but such experiments often give biphasic curves. This biphasicity depends upon the myosin preparation used and it is directly related to the active site titration. The simplest explanation for these results is one involving 2 sites for ATP: one site hydrolyzes ATP by the Bagshaw-Trentham scheme (tight binding preceding hydrolysis) but the second site binds ATP loosely without significant hydrolysis. This heterogeneity in ATP binding may explain certain difficulties, such as questions concerning the non-identity of the myosin heads and the number of steps involved in nucleotide binding. Attempts were made to determine the cause of the head heterogeneity but these were unsuccessful. We cannot exclude the possibility that the heterogeneity is relevant to muscle contraction.

Muscle contraction and movement of cellular organelles: are there two different types of mechanisms for their generation?

It has been shown that myosin molecules attached to Covaspheres can "walk along" polar actin filament in vitro. The driving force for this movement seems to explain only about 1% of the isometric tension developed by a muscle fibre. Therefore, the driving force for the bead movement seems to be incompatible with that found in muscle, and the bead movement cannot be considered as a model for muscle contraction. The origin of the bead movement may be related to a "molecular jet" process, resulting from the rapid ejection of the MgATP splitting products. This "molecular jet" might also explain the movements of many cellular organelles.

Absence of functional activity of tissue plasminogen activator in patients with severe forms of von Willebrand's disease.

Some patients with von Willebrand's disease do not respond to stimuli such as venous occlusion and infusion of a vasopressin analogue DDAVP. In these patients, fibrinolytic activity is not enhanced and von Willebrand's factor is not released into the blood. Skin biopsies and cryostat sections were used to study the fibrinolytic activity of skin vessels and localization of tissue plasminogen activator (t-PA) in three patients with severe form of von Willebrand's disease. On fibrin films, no fibrinolysis developed around the skin vessels of the patients; however, using specific polyclonal and monoclonal antibodies to t-PA, and peroxidase coupled specific IgG, presence of t-PA antigen was demonstrated in endothelial cells (EC) of all of them. In plasma no t-PA activity was detected either before or after venous occlusion although t-PA inhibitor activity was in a normal range. Small amounts of t-PA antigen was measured in blood by ELISA. From these results, it is concluded that in patients with severe forms of von Willebrand's disease, t-PA present in EC is not functional and can not transform plasminogen into plasmin.

MgATPase activity of myosin subfragment 1. The dimer is more active than the monomer.

The MgATPase activity of the rabbit skeletal myosin subfragment 1 (S1), in the steady state, was measured by means of the intrinsic fluorescence of tryptophan. This technique gave results similar to those obtained by other methods (linked or radioactive assays). The activity was measured under conditions that effect the monomer/dimer ratio. It is shown that there is a close correlation between MgATPase activity and the proportion of dimer. At 20 degrees C, for pH 6.9 to 8.1 and for [KCl] less than or equal to 1 M, the observed activity (kobs) can be linearly related to the proportion of dimer (Ed/Eo) by: kobs(s-1) = 0.016-7 X 10(-3)[KCl] + 0.031(Ed/Eo), where [KCl] is expressed in M. We deduce that, at 20 degrees C and for [KCl] = 0 M, the activity of the monomer is kmobs = 0.016 s-1 (Ed/Eo = 0) and that of the dimer kdobs = 0.047 s-1 (Ed/Eo = 1), i.e. a ratio kdobs/kmobs approximately equal to 3. Beyond pH approximately equal to 8.3, the activities of both the monomer and the dimer increased steeply with increasing pH value. In the standard conditions (pH 8.0, [KCl] = 0 to 100 mM), S1 is mainly in the form of a dimer, and such conditions are not appropriate for study of the S1 monomer. For studying the pure monomer, the conditions required at 20 degrees C and in bis-Tris-propane are: S1 concentration approximately equal to 0.2 mg/ml, pH 6.9 to 7.8, [KCl] approximately equal to 300 mM. For studying the pure dimer, the conditions required are: S1 concentration greater than or equal to 0.2 mg/ml, pH 7.8 to 8.1 and [KCl] approximately equal to 0. In both cases the MgATP concentration is about 50 microM. Finally, if great care is taken concerning the age of the S1 solutions and the evaluation of the proportion of dimer, the values of kobs are extremely precise: the uncertainty regarding the values of kobs, as determined by means of intrinsic fluorescence, does not exceed +/- 0.001 s-1. Beyond this error bar conditions are uncontrolled.

Rabbit skeletal myosin heads in solution, as observed by ultracentrifugation and freeze-fracture electron microscopy: dimerization and maximum chord.

The use of analytical ultracentrifugation and freeze-fracture electron microscopy in solution allowed us to observe the monomeric and dimeric forms of Mg.71. This subfragment of the myosin molecule contains the LC2 light chain and is comparable to a "native" myosin head. Sedimentation-diffusion equilibrium ultracentrifugation shows that it is necessary to use slightly different conditions in order to obtain a pure Mg.S1 dimer, as compared to the case of chymotryptic S1 (LC2-free S1). For example, in a buffer leading to a complete dimerization of chymotryptic S1, Mg.S1 is only in the form of a monomer-dimer mixture, with comparable proportions of monomer and dimer. The freeze-fracture technique, applied to solutions containing Mg.S1 or chymotryptic S1, revealed that the monomeric species both have the same maximum chord (about 120 A) and that both dimeric species also have the same maximum chord (about 250 A). The maximum chord of the monomer is comparable to the surface-to-surface spacing between the myosin and actin filaments, in a fiber at the slack length. In sharp contrast this chord is higher than this spacing in a stretched fiber. The consequences of this fact are discussed, with particular reference to the sarcomere length-tension relationship.