Reaction of myosin with salicylaldehyde I. The effect of salicylaldehyde on the physicochemical properties of myosin

The specificity and nature of the reaction between salicylaldehyde and myosin and the effect of salicylalation on the molecular parameters of myosin were studied. The following observations were made.

1. 1. The reaction of salicylaldehyde with the lysyl residues of myosin is specific, since no salicylaldehyde is bound if the lysyl residues of myosin are trinitrophenylated.

2. 2. Salicylaldehyde is bound by myosin through the formation of an azomethine linkage (Schiff's base). This was established from the measured difference absorption spectrum of the myosin-salicylaldehyde complex.

3. 3. Three groups of lysyl residues can be distinguished with respect to the reaction with salicylaldehyde, namely, (a) residues with high association constant (K_ass = 1.8 ± 0.9·10⁵ M^-1), (b) residues with moderate association constant (K_ass = 2.2·10³ M^-1) and (c) residues that react with salicylaldehyde only after the denaturation of the protein. Their numbers could be estimated as 10 ± 5, 130 ± 5 and 260 ± 5 per mole myosin, respectively. The first group of residues was found to be absent from heavy and light meromyosin, the proteolytic fragments of myosin.

4. 4. The reaction is reversible. The complex formation rate constant, evaluated from the formula for second order reaction, is 2.2 sec^-1·M^-1, and the decomposition rate constant for first order reaction is 1.1·10^-3 sec^-1 at 22°.

5. 5. The reaction is pH dependent, the reaction yield increasing at higher pH.

6. 6. The solubility of myosin at low ionic strength decreases with increasing degree of salicylalation at slightly alkaline pH.

7. 7. The intrinsic viscosity of myosin does not change on salicylalation.

8. 8. A second peak due to polymerization appears on the sedimentation profile of the protein if more than 70 lysyl residues are salicylalated per mole of myosin.

Effect of calponin on actin-activated myosin ATPase activity

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.     

Effect of medium viscosity on the activity of myosin ATPase

The influence of increased medium viscosity on the activity of myosin Ca2+-ATPase has been studied in 28, 45 and 60% water-sucrose solutions at 25 degrees C. In the wide range of viscosities (10 divided by 430 mp) the rate constant of ATP hydrolysis displays the negative power-law dependence on solution viscosity with an index approximately -0,5. The obtained data confirm an idea about the existence of direct connection between the low-frequency liquid relaxations and structural dynamics of proteins and enzymes.     

Effect of the filamentous structure of myosin on the actomyosin ATPase activity

The ATPase activities of acto-heavy meromyosin and of acto-myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KC1 concentrations. The activities, which are strongly salt-dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06-0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto-myosin minifilaments than for acto-heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponintropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F-actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.     

Effect of trinitrophenylation on myosin ATPase

1. The enzymic and actin binding properties of myosins trinitrophenylated to different extents in the presence or absence of ATP have been studied.

2. The enzymic properties of myosin trinitrophenylated in the absence of ATP are different from those of myosin treated in the presence of ATP even on trinitrophenylating an equal number of lysyl residues. On trinitrophenylation in the absence of ATP the EDTA-(K⁺-)activated ATPase and Ca²⁺-activated ATPase decrease while the Mg²⁺-activated ATPase considerably increases. In the presence of ATP the enzymic properties of myosin are much less affected by trinitrophenylation.

3. The actin binding capacity of trinitrophenylated myosin does not change, although its enzymic properties may be greatly altered, and even if its property to be activated by actin is completely lost.     

Effect of swimming training on cardiac function and myosin ATPase activity in SHR

Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) were subjected to swimming training 6 times/wk, commencing at 4 wk of age, to determine whether this type of endurance exercise might alter contractile proteins and cardiac function in young adult SHR. The total duration of exercise was 190 h. Myofibrillar adenosinetriphosphatase (ATPase) activity was assayed at various free [Ca2+] ranging from 10(-7) to 10(-5) M. Ca2+-stimulated ATPase activity of actomyosin and purified myosin was determined at various Ca2+ concentrations both in the low and high ionic strength buffers. Actin-activated myosin ATPase activity of purified myosin was assayed at several concentrations of actin purified from rabbit skeletal muscle. Under all these conditions the contractile protein ATPase activity was comparable between trained and untrained WKY and SHR. Analysis of myosin isoenzymes on pyrophosphate gels showed a single band corresponding to V1 isoenzyme, and there were no differences between swimming-trained and nontrained WKY and SHR. Ventricular performance was assessed by measuring cardiac output and stroke volume after rapid intravenous volume overloading. Both cardiac index and stroke index were comparable in nontrained WKY and SHR but were significantly increased in the trained groups compared with their respective nontrained controls. These results suggest that myosin ATPase activity and distribution of myosin isoenzymes are not altered in the moderately hypertrophied left ventricle whether the hypertrophy is due to genetic hypertension (SHR) or to exercise training (trained WKY). Moreover, the data indicate that SHR, despite the persistence of a pressure overload, undergo similar increases in left ventricular mass and peak cardiac index after training, as do normotensive WKY.     

ADP inhibition of myosin V ATPase activity

The kinetic mechanism of myosin V is of great interest because recent evidence indicates that the two-headed myosin V molecule functions as a processive motor, i.e., myosin V is capable of moving along an actin filament for many catalytic cycles of the motor without dissociating. Three recent publications assessing the kinetics of single-headed myosin V provide different conclusions regarding the mechanism, particularly the rate-limiting step of the cycle. One study (, Proc. Natl. Acad. Sci. USA. 96:13726-13731) identifies ADP release as the rate-limiting step and provides a kinetic explanation for myosin V processivity. The others (, J. Biol. Chem. 274:27448-27456;, J. Biol. Chem. 275:4329-4335) do not identify the rate-limiting step but conclude that it is not ADP release. We show experimental and simulated data demonstrating that the inconsistencies in the reports may be due to difficulties in the measurement of the steady-state ATPase rate. Under standard assay conditions, ADP competes with ATP, resulting in product inhibition of the ATPase rate. This presents technical problems in analyzing and interpreting the kinetics of myosin V and likely of other members of the myosin family with high ADP affinities.     

ATPase activity of myosin in hair bundles of the bullfrog''s sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.     

The effect of myosin light chain phosphorylation on the actin-stimulated ATPase activity of myosin minifilaments

It has been shown that in the absence of KCl, the actin-stimulated Mg2+-ATPase activity of rabbit skeletal myosin minifilaments with phosphorylated regulatory lights chains (LC2) exceeds 3-4-fold that of myosin minifilaments with dephosphorylated LC2. Addition of KCl leads to a decrease in the difference between the two ATPase activities. LC2 phosphorylation considerably increases the rate of ATPase reaction and only slightly decreases the affinity of myosin minifilaments for F-actin. It is suggested that the unusual effect of LC2 phosphorylation on the kinetic parameters of the actin-stimulated ATPase reaction of myosin minifilaments can be accounted for by its influence on the interaction between myosin heads which results in the ordered self-assembly of minifilaments.     

A myosin II mutation uncouples ATPase activity from motility and shortens step size

It is thought that Switch II of myosin, kinesin and G proteins has an important function in relating nucleotide state to protein conformation. Here we examine a myosin mutant containing an S456L substitution in the Switch II region. In this protein, mechanical activity is uncoupled from the chemical energy of ATP hydrolysis so that its gliding velocity on actin filaments is only one-tenth of that of the wild type. The mutant spends longer in the strongly bound state and exhibits a shorter step size, which together account for the reduction in in vitro velocity. This is the first single point mutation in myosin that has been found to affect step size.     

Effect of staphylococcus active substances on ATPase activity of smooth muscle actomyosin and myosin

The effect of staphylococcus active substances--protein A (PA) and peptidoglican (PG) at concentrations 10(-6)-10(-2) mg/ml on the ATPase activity of pig stomach natural actomyosin and myosin was studied. It was shown that PA and PG at direct contact with smooth muscle contractile proteins caused the activation and inhibition of ATPase activity, respectively. On the basis of this investigation it was assumed that staphylococcal active substances were able to modify of the ATPase activity smooth muscle contractile proteins perhaps via direct action on the myosin molecule, which could be accompanied by conformational changes of the active center of myosin ATPase.     

Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains--I. Wild-type fly

1. Two types of myosins with phosphorylated and dephosphorylated myosin light chains were prepared from Drosophila flies. The former had ATPase (Ca2(+)- and Mg2(+)-activited) activities twice those of the latter. 2. The myosin phosphorylated with crude myosin light chain kinase from flies showed ATPase (Ca2(+)- and Mg2(+)-activated) activaties twice those of the dephosphorylated myosin. 3. It is suggested that phosphorylation of myosin light chains several hours after emergence stimulates myosin ATPase activity so as to facilitate the flight function of the fruitfly.     

Measurement of ATPase activity of immobilized myosin heads

Myosin, heavy meromyosin, and myosin subfragment-1 (S-1) were immobilized on the inner surfaces of glass capillary tubes, the inside walls of which had been coated with nitrocellulose. The ATPase activities of the immobilized proteins were measured using radiolabeled ATP and electrophoretic separation of the reaction products. The activity was proportional to the amount of immobilized protein. Activation by actin of the ATPase was also observed.