Calponin-Like Protein from Mussel Smooth Muscle Is a Competitive Inhibitor of Actomyosin ATPase.

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.

[Modulation of S-1 conformation and inhibition of the skeletal muscle S-1-ATPase by calponin of the mussel].

A novel 40 kDa protein has been detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. In this study, using skeletal muscle actin and S-1, we investigated the effects of the mussel 40-kDa actin-binding protein on the acto · S-1 ATPase activity. On increasing the 40-kDa actin-binding protein (CaP-40) concentration, the actin-activated ATPase activity decreased, and was inhibited 80% at a CaP-40 to actin ratio of 0.5. Polarized fluorimetry technique and glycerinated muscle fibers were used to study effects of CaP-40 on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to CyS-707 of myosin subfragment-1 in the absence of nucleotide, and in the presence of MgADP or MgATP. We have concluded that CaP-40 binding to actin affects the strong binding of myosin to actin but has no effect on the weak binding. Thus, the influence of the CaP-40 on the formation of strong actomyosin binding forms A · M and A · M · ADP manifests itself by a decrease in the relative content of myosin cross-bridges strongly bound with actin, which probably results in a decrease in the relative content of "switch on" actin monomers in thin filaments. This suggests that, as calponin CaP-40 selects its target the phase of strong actomyosin binding binding which preceded by a phase generating power stroke.

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle.

Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.

40-kDa actin-binding protein of thin filaments of the mussel Crenomytilus grayanus inhibits the strong bond formation between actin and myosin head during the ATPase cycle.

Mobility and spatial orientation of a novel 40-kDa actin-binding protein from the smooth muscle of the mussel Crenomytilus grayanus was studied by polarized fluorometry. The influence of this protein on orientation and mobility of the myosin heads was investigated during modeling the different stages of the ATPase cycle. The 40-kDa actin-binding protein affected the strong actin-myosin binding. We suggest that the 40-kDa actin-binding protein is involved in regulation of the actin-myosin interaction in the smooth muscle of the mussel.

[Thin filaments of molluscan catch muscles contain a calponin-like protein].

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.

[N-ethylmaleimide inhibits polymerization of myorod, a contractile protein of thick filaments of molluscan smooth muscles]. / N-étilmaleimid ingibiruet polimerizatsiiu mioroda--sokratitel'nogo belka tolstykh nitei gladkikh myshts molliuskov.

The effect of N-ethylmaleimide on the polymerization of myorod, a protein of molluscan smooth muscles, which is colocalized with myosin on the surface of paramyosin core of thick filaments and is a product of the alternative splicing of the gene of heavy myosin chains, was studied. It was shown that myorod modified by N-ethylmaleimide completely loses the polymerization ability but acquires the ability to aggregate in the presence of Mg2+. At the same time, treatment of molluscan myosin with N-ethylmaleimide did not affect its polymerization. It was supposed that the effect of N-ethylmaleimide on myorod polymerization is related to the modification of the myorod SH domain containing Cys722.

Optical properties of myofibril and actomyosin suspensions. 1. Angular dependence of light scattering by myofibril suspensions and its changes under myofibril contraction.

The angular dependence of light scattering by myofibril suspensions has been measured within the angle range of 0.05 degrees and 160 degrees at different suspension concentrations and wavelengths. Angular diagrams appear to be highly asymmetry, and extended forward in the direction of the primary light propagation. A suspension with the concentration of 0.1 mg/ml at 550 nm scatters, for example, no less than 90% light at angles of less than 6 degrees. The scattering diagram becomes less asymmetric upon increasing the suspension concentration and decreasing the wavelength of the light. Upon addition of MgATP in the presence of Ca2+, a myofibril suspension undergoes two kinds of optical change, namely, fast ones corresponding to myofibril contraction, and slow ones correlating with the subsequent aggregation of the contracted myofibrils. Fast changes are believed to be due to the increase of the refractive index of the contracting myofibrils, and slow changes to the increase in the size of the suspension particles resulting from aggregation. Light scattering by myofibril and actomyosin suspensions seems to be due not only to the particle as a whole, but also to its internal structure.

[Aggregation of isolated myofibrils stimulated by their contraction. II. Aggregation mechanism]. / Agregatsiia izolirovannykh miofibrill, stimuliruemaia ikh sokrashcheniem. II. Mekhanizm agregatsii.

Aggregation of contracted myofibrils was studied as a function of experimental conditions at myofibril contraction. The aggregation rate increased at higher concentrations of suspensions and at increased ionic strength of the medium to achieve the maximum at 0.1 M KCL in the last case. The aggregate sizes grow with an increase of ionic strength and concentration of MgATP and reduce with addition of F-actin. Aggregation of myofibrils develops only in the case of their complete or significant contraction. It was suggested that aggregation is stimulated by dehydration of myofibril at contraction.

[Aggregation of isolated myofibrils stimulated by their contraction. I. Origin of the second phase of optical changes during myofibril contraction]. / Agregatsiia izolirovannykh miofibrill, stimuliruemaia ikh sokrashcheniem. I. Proiskhozhdenie vtoroi fazy opticheskikh izmenenii pri sokrashchenii miofibrill.

Aggregation of contracted myofibrils was shown to be responsible for spontaneous: slow reduction of optical density of myofibril suspension on the final stage of their contraction. This effect faded with an increase in light wavelength, with increased angle of view of the photocell and diminished sizes of myofibrils.

[The causes of the nonuniform thermostability of Mg-ATPase and of the contractility of muscle models]. / Prichiny neodinakovoi teploustoichivosti Mg-ATFazy i sokratimosti myshechnykh modelei.

With longer periods of preliminary heat-treatment of actomyosin suspension the decrease in the rate of superprecipitation (SPP) is followed by that in the extent of SPP, and, finally, in the Mg-ATPase activity. A similar uncoupling of mechanical and enzymatic activities is observed when the ratio between the native and the inactivated myosin in reconstructed actomyosin varied. This uncoupling is supposed to result from the formation during heat-treatment of myosin bridges incapable of dissociating in the presence of Mg-ATP. The bridges affect largely the mechanical properties of actomyosin, and in a lesser degree, its enzymatic properties.

[Interrelated effects of ATP and ionic strength on Ca2+-sensitivity of isolated myofibrils]. / Vzaimozavisimoe vliianie ATF i ionnoi sily na Ca2+-chuvstvitel'nost' izolirovannykh miofibrill.

Ability of isolated myofibrils for relaxation was studied in a medium with KCl concentrations of 6 to 250 mM with ATP content varying from 0.01 to 1.0 mM. It was shown that myofibrils can relax at all the studied values of the ionic strength, but ATP concentration required for the relaxation is to increase with decreasing the ionic strength A stable state of Ca2+-sensitive system of myofibrils is maintained at approximately the same values of ATP and KCl concentrations. A possible mechanism of this phenomenon is discussed.

[Changes in the mechano-chemical properties of actomyosin during desensitization]. / Izmenenie mekhanokhimicheskikh svoistv aktomiozina pri desensibilizatsii.

The loss of Ca2+-sensitivity by natural actomyosin (desensitisation) after treatment with low ionic strength solutions results in marked deceleration of protein superprecipitation. This phenomenon is not due to the removal of minor proteins, since a similar effect was observed during "desensitisation" of synthetic actomyosin containing only myosin and actin. However, addition to desensitised actomyosin of tropomyosin, especially in combination with alpha-actinin markedly restores the initial parameters of superprecipitation and ATPase activity. It was assumed that desensitisation has a direct modifying influence on actomyosin, whose effect is weakened in the presence of tropomyosin and alpha-actinin.

[A high molecular proteolysis-resistant actin fragment]. / Vysokomolekuliarnyi ustoichivyi k proteolizu fragment aktina.

The proteolysis-stable actin fragment (m. w. 36300 as determined by SDS polyacrylamide gel electrophoresis) was obtained by actin treatment with a non-identified bacterial protease. This fragment is larger than the trypsin-stable actin fragment and is similar to the unstable trypsin intermediate fragment. The obtained actin fragment is not polymerized, does not activate the ATPase activity of myosin and does not form a superprecipitating complex with it. Thus, the functional properties of actin are lost during the split-off of a relatively small, apparently, the N-terminal part of the polypeptide chain.

[Influence of C-protein on mechano-chemical properties and structure of reconstituted actomyosin]. / Vliianie C-belka na mekhanokhimicheskie svoistva i strukturu rekonstruirovannogo aktomiozina.

C-protein on the mechano-chemical properties (ATPase activity and superprecipitation) of actomyosin systems has been investigated. The presence of C-protein in AM-complexes has been shown to decrease the rate of superprecipitation (SPP) and simultaneously increase the ATPase activity. Both effects of C-protein are dependent on its quantity in the system. Tropomyosin decreased considerably but does not eliminate completely the inhibitory influence of C-protein on the SPP. Electron microscopy does not reveal considerable structural differences in the initial AM-complexes depending on the presence or absence of C-protein. It is supposed that the discovered effects of C-protein on the behaviour of AM-systems are determined by the fine local structural and (or) charge changes produced by C-protein in the region of myosin cross-bridges, which in its turn results in a modification of the actin-myosin interaction. Possible participation of C-protein in the regulation of the interaction of thin and thick filaments in the muscle is discussed.

[Protein makeup of rabbit myofibrils determined by a disc eletrophoretic method in the presence of sodium dodecyl sulfate]. / Belkovyi sostav miofibrill krolika, opredelennyi metodom disk-élektroforeza v prisutstvii dodetsilsul'fata natriia

The protein subunit composition of isolated myofibrils of rabbit skeletal muscle is studied by polyacrylamide gel disc-electrophoresis in the presense of sodium dodecyl sulfate (SDS). The method of disc-SDS-electrophoresis is described in detail. The electrophoretic patterns of SDS-solubilized myofibrils obtained by disc-SDS-electrophoresis and by SDS-electrophoresis in continuous buffer system according to Weber and Osborn are compared. The former results in a markedly improved resolution and allows to discover some additional protein components, the origin of these additional components being discussed. A standard curve is given for determination of polypeptide chain molecular weights by disc-SDS-electrophoresis.