RESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , Proteínas dos Microfilamentos/farmacologia , Proteínas Musculares/farmacologia , Miosinas/antagonistas & inibidores , Mytilidae , Animais , Cinética , Músculo Liso , CalponinasRESUMO
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.
Assuntos
Actinas/química , Adenosina Trifosfatases/metabolismo , Bivalves/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bivalves/química , Bivalves/enzimologia , Proteínas de Ligação ao Cálcio/química , Proteínas dos Microfilamentos/química , Proteínas Musculares/química , Conformação Proteica , CalponinasRESUMO
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.
Assuntos
Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Fibras Musculares Esqueléticas/química , Miosinas/metabolismo , Mytilidae/química , Actinas/química , Animais , Fibras Musculares Esqueléticas/metabolismo , Mytilidae/metabolismo , Espectrometria de FluorescênciaRESUMO
Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas de Ligação a Calmodulina/química , Músculo Liso/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Músculo Esquelético/metabolismo , Músculo Liso/citologia , Subfragmentos de Miosina/metabolismo , Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Coelhos , Espectrometria de Fluorescência , Fatores de Tempo , Tropomiosina/química , Tropomiosina/metabolismo , Troponina/metabolismoRESUMO
To discriminate between the influences of a motoneuron and muscle activity on the conformation of actin filaments, the extrinsic polarized fluorescence [of rhodamine-phalloidin and N-(iodoacetylamine)-1-naphthylamine-5-sulfonic acid attached to F-actin] was measured in "ghost" fibers from intact rat soleus muscles and atrophying muscles after denervation, immobilization, or tenotomy. The results show that the conformation of F-actin changed in all the atrophying muscles, but differently. In the denervated muscle, the flexibility of the actin filaments decreased, whereas in the other experimental muscles it remained as in the intact muscle. In the denervated muscle, the mobility of the C-terminus of the actin polypeptide increased. Attachment of myosin subfragment-1 influenced the F-actin conformation differently in the denervated muscle than in the other muscles studied. These results suggest that changes in the conformation of the actin filament are induced by the lack of connection with the motoneuron rather than by muscle inactivity.
Assuntos
Actinas/ultraestrutura , Imobilização/fisiologia , Neurônios Motores/fisiologia , Denervação Muscular , Músculo Esquelético/fisiologia , Animais , Feminino , Corantes Fluorescentes , Microscopia Eletrônica , Músculo Esquelético/inervação , Músculo Esquelético/ultraestrutura , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/ultraestrutura , Naftalenossulfonatos , Ratos , Ratos Wistar , Tendões/fisiologiaRESUMO
We studied the conformational changes in actin filaments induced by the binding of calponin or a 38-kDa fragment of caldesmon, two actin-binding proteins known to inhibit actin-activated ATP hydrolysis by phosphorylated smooth muscle myosin. The F-actinin myosin-free muscle fibers (ghost fibers) was labeled with fluorescein-5-maleimide and the conformational change in actin was determined by polarized fluorimetry. Data show that both calponin and the 38-kDa caldesmon fragment inhibit the conformational changes in F-actin that are compatible with the "strong-binding" state between myosin heads and actin. Tropomyosin slightly reduced the effect produced by calponin, but enhances the effect produced by the 38-kDa caldesmon fragment.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Animais , Proteínas de Ligação ao Cálcio/farmacologia , Proteínas de Ligação a Calmodulina/farmacologia , Galinhas , Reagentes de Ligações Cruzadas/farmacologia , Etilmaleimida/farmacologia , Moela das Aves , Maleimidas/farmacologia , Proteínas dos Microfilamentos , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Músculo Liso/fisiologia , Miosinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Coelhos , CalponinasRESUMO
The influence of myosin regulatory light chains (LC2s) lacking the 2kD N-terminal portions of these chains, on internal organization of myosin heads and on actin-myosin interaction was studied with limited proteolysis and polarized fluorescence methods. For these studies heavy meromyosin (HMM) preparations were used: HMM containing intact LC2s (phosphorylated or dephosphorylated) and HMM containing LC2s lacking the 2kD N-terminal portions (including serine which can be phosphorylated). It was found that the susceptibility of the heavy chain cleavage site to trypsin and the alkali light chain (LC1) site to papain of myosin containing shortened regulatory LC2s, is not dependent on saturation of LC2s with Ca2+ or Mg2+ ions. This is in contrast to the myosin containing intact LC2s where Ca2+ or Mg2+ ion saturation does demonstrate a dependence. Similarly, in spectroscopic experiments, dephosphorylated HMM containing intact LC2s causes decrease or increase of actin filament flexibility depending on whether Mg2+ or Ca2+ are bound to LC2s. Correspondingly, HMM with shortened LC2s induces only increase of actin flexibility despite cations being bound. We conclude that the N-terminal fragment of LC2 is important for ensuring a proper Ca2+ dependent conformation of myosin head in the course of its actin-activated ATP hydrolysis.
Assuntos
Actinas/metabolismo , Miosinas/química , Miosinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Cálcio/farmacologia , Polarização de Fluorescência , Magnésio/metabolismo , Músculo Esquelético/química , Subfragmentos de Miosina/metabolismo , Papaína/metabolismo , Fosforilação , Coelhos , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-Atividade , Tripsina/metabolismoRESUMO
Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Galazkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.
Assuntos
Actinas/metabolismo , Proteínas de Ligação a Calmodulina/farmacologia , Músculos/metabolismo , Miosinas/metabolismo , Marcadores de Afinidade , Animais , Proteínas de Ligação a Calmodulina/fisiologia , Polarização de Fluorescência , Músculos/efeitos dos fármacos , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/fisiologia , Naftalenossulfonatos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Tropomiosina/metabolismoRESUMO
It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, associated with the fast-reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronounced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.
Assuntos
Magnésio/farmacologia , Músculos/análise , Miosinas/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Densitometria , Eletroforese em Gel de Poliacrilamida , Polarização de Fluorescência , Corantes Fluorescentes , Glicerol , Músculos/efeitos dos fármacos , Naftalenossulfonatos/análise , Fosforilação , Conformação Proteica , Coelhos , Triptofano/análiseRESUMO
Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Miosinas/metabolismo , Tropomiosina/metabolismo , Troponina/metabolismo , Animais , Galinhas , Eletroforese em Gel de Poliacrilamida , Polarização de Fluorescência , Músculo Liso/enzimologia , Músculos/enzimologia , CoelhosRESUMO
The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.
Assuntos
Actinas/metabolismo , Proteínas de Ligação a Calmodulina/farmacologia , Músculos/efeitos dos fármacos , Miosinas/metabolismo , Animais , Galinhas , Polarização de Fluorescência , Moela das Aves/análise , Músculos/enzimologia , CoelhosRESUMO
The interaction of regulated and unregulated actin of myosin-free ghost single fibre with myosin subfragment-1 free of 5,5'-dithiobis(2-nitrobenzoic acid) light chains was investigated by polarized microphotometry. The anisotropy of intrinsic tryptophan fluorescence of regulated actin is Ca2+-dependent and has a maximal value at low (pCa greater than or equal to 7) and a minimal value at high (pCa less than or equal to 6) concentrations of Ca2+. The interaction of myosin subfragment-1 with actin induces cooperative changes in actin structure, which manifest themselves in a decrease in the anisotropy of tryptophan fluorescence. The cooperativity of conformational changes in actin, induced by myosin subfragment-1, is high for regulated actin in the absence of Ca2+ and low for unregulated and regulated actin in the presence of Ca2+. The data obtained suggest that the decrease of the flexibility of actin filaments, induced by tropomyosin or by Ca-unsaturated troponin-tropomyosin complex, results in increased cooperativity of conformational changes of actin induced by myosin subfragment-1.
Assuntos
Actinas , Cálcio , Miosinas , Fragmentos de Peptídeos , Tropomiosina , Troponina , Animais , Birrefringência , Fenômenos Químicos , Química , Polarização de Fluorescência , Subfragmentos de Miosina , Ligação Proteica , Conformação Proteica , Coelhos , Relação Estrutura-Atividade , TriptofanoRESUMO
The fluorescent ADP analogue, 1:N6-ethenoadenosine 5'-diphosphate, was incorporated into F-actin in a myosin-free ghost single fibre. Polarized fluorescence measurements of tryptophan residues and 1:N6-ethenoadenosine 5'-diphosphate were performed under a microspectrophotometer to investigate the conformation of F-actin and the changes induced in it by myosin subfragment-1 with 5,5'-dithiobis(2-nitrobenzoic acid) light chains and without them. A relation was found between the conformational state of F-actin and the presence of 5,5'-dithiobis(2-nitrobenzoic acid) light chains. The conformational changes were shown to be controlled by Ca2+ in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) light chains.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Ácido Ditionitrobenzoico/metabolismo , Miosinas/análise , Nitrobenzoatos/metabolismo , Difosfato de Adenosina/análogos & derivados , Animais , Fenômenos Químicos , Química , Fragmentos de Peptídeos , Ligação Proteica , Conformação Proteica , Coelhos , Espectrometria de FluorescênciaRESUMO
By means of polarized ultraviolet fluorescence microscopy the structural changes of F-actin and myosin were discovered at the changing a functional state of a living muscle fibre and during spreading degeneration (Zenker's necrosis). The character of conformational changes of F-actin and myosin at activation, contraction, contracture and rigor is similar, but the number of changed macromolecules depends on a fibre state. At fibre local damage in its morphological intact parts there was found an alternation of zones, reflecting two states unusual for a fibre. During spreading degeneration these states transform into irreversible contracture and then into rigor. Similar changes were observed in muscle fibres obtained from denervated muscles.
Assuntos
Actinas/análise , Músculos/citologia , Miosinas/análise , Animais , Anuros , Polarização de Fluorescência , Microscopia de Fluorescência/métodos , Contração Muscular , Relaxamento Muscular , Rigidez Muscular , Músculos/análise , Músculos/patologia , Necrose , Raios UltravioletaRESUMO
By means of polarized ultraviolet fluorescence microscopy the conformational changes of F-actin occuring in glycerinated muscle fibers of rabbit and barnacle (Balanus rostratus Hock.) under the influence of adenosine triphosphate in the presence of ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid were discovered. These changes seem to be located near the surface of the globules thus hampering the penetration of univalent iones and neutral molecules into the F-actin macromolecule. It is suggested that similar changes of F-actin take place in thin myofilaments of living muscle fiber during the contraction-relaxation process.