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.     

Reaction of myosin with salicylaldehyde II. Effect of salicylalation on the ATPase activity of myosin

The effect of salicylalation on the biological properties of myosin was studied.

1. 1. The ATPase activity of myosin is affected by salicylalation if the treatment is carried out at higher pH than 6.5. The Mg²⁺-activated ATPase shows a maximal curve with 250–380% maximal activation when 25–70 moles of salicylaldehyde are bound per mole of myosin. The EDTA-activated ATPase decreases with increasing salicylalation. Ca²⁺-activated ATPase shows a small increase with increasing salicylalation.

2. 2. Less salicylaldehyde is bound if the treatment is carried out in the presence of ATP, while that of PP_i does not affect the degree of salicylalation. The enzymic properties of myosins salicylalated in the presence of ATP or PP_i are not different from those of the samples treated in their absence.

3. 3. Salicylalation decreases ATP sensitivity of ATPase and superprecipitation of actomyosins reconstituted from salicylalated myosins only if more than 50 moles of salicylaldehyde are bound per mole 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.     

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.     

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.     

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.     

DNA-stimulated ATPase activity on the lon (CapR) protein.

The gene product of the pleiotropic lon (also called capR) locus in Escherichia coli, the CapR protein, is an ATP hydrolysis-dependent protease and a nonspecific nucleic acid-binding protein. We demonstrated that it is also a DNA-stimulated adenosine triphosphatase (ATPase). This new activity is distinct from the protease-associated ATPase activity and occurs in the absence of proteolytic substrate. The reaction requires the presence of a divalent cation and has a pH optimum of 8.0. The products of the reaction are ADP and inorganic phosphate. No adenylation or phosphorylation of the DNA or proteins was detected. The maximum rate of ATP hydrolysis occurs in the presence of supercoiled (form I) DNA. Relaxed circles (form II), double-stranded DNA, and single-stranded DNA are less effective in promoting ATPase activity, whereas RNA is inactive. The DNA-stimulated ATPase activity is inhibited by a mutationally altered form of the CapR protein called the CapR9 protein. The interaction of the CapR and CapR9 subunits suggests that this enzymatic activity of the CapR protein is oligomeric in the presence of DNA. Our in vitro experiments indicate a possible role for nucleic acids in the regulation of all lon (capR) activity.     

肌球蛋白轻链激酶CaM结合位点突变体对肌球蛋白ATP酶活性的影响

目的:探讨肌球蛋白轻链激酶(MLCK)钙调蛋白(CaM)结合位点突变体对肌球蛋白ATP酶活性的影响.方法:构建牛胃重组全长野生型MLCK CaM结合位点突变型蛋白(△CaM/MLCK);孔雀绿方法检测△CaM/MLCK对肌球蛋白的Mg2+-ATP酶活性的影响.结果:在无Ca2+/CaM存在时,随着△△CaM/MLCK浓度的增加,非磷酸化肌球蛋白的Mg2+-ATP酶活性明显增加;而磷酸化肌球蛋白的Mg2+-ATP酶活性明显降低.结论:△CaM/MLCK对肌球蛋白Mg2+-ATP酶活性的影响表明MLCK具有非激酶活性.     

The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity

The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDeltaLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin-activated ATPase activity of Myo1d was approximately 75% inhibited by Ca2+-binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+-dependence, we propose that Ca2+-binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif(s). Interestingly, a similar but Ca2+-independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2, fused directly to the Myo1d-head, was rotated through 200 degrees by the deletion of two amino acids in the lever arm alpha-helix N-terminal to the IQ-motif.