Difference between subfragment-1 and heavy meromyosin in their interaction with F-actin

To elucidate the difference between subfragment-1 and heavy meromyosin in their interaction with F-actin, we used limited tryptic digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binding of actin to subfragment-1 lowers the susceptibility of the 50K-20K junction of its heavy chain to tryptic digestion. At a molar ratio of one actin to one subfragment-1, all the sites were gradually cleaved by trypsin whereas the sites were completely protected in the presence of a 2-fold molar excess of actin over subfragment-1. In the case of heavy meromyosin, nearly half of the sites were protected completely by the presence of an equimolar amount of actin to its heads suggesting that the two heads of heavy meromyosin bound actin in a different manner. The rate of the cross-linking reaction between subfragment-1 heavy chain and actin with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide also depended on the molar ratio of actin to subfragment-1. The rate was maximum at a molar ratio of about 5 actin to 1 subfragment-1. When heavy meromyosin was cross-linked to actin, the maximum rate was observed at a molar ratio of about 3 actin to 1 heavy meromyosin head, the level being about 60% that for subfragment-1 and actin. It was suggested that the presence of the subfragment-2 portion of heavy meromyosin caused these differences by restricting the motion of the two heads.     

Investigation, by cross-linking, of conformational changes in F-actin during its interactions with myosin

The hypothesis that the subunits of F-actin rotate during interactin with myosin and ATP has been tested by using the specific cross-linking reagent p-phenylene-N,N'-bis(maleimide) (PM). The insertion of cross-links between F-actin subunits does not change the ability of the F-actin to activate the ATPase of either myosin subfragment-1 (S-1) or heavy meormyosin, and its ability to superprecipitate with myosin is unimpaired. We conclude that large-scale rotations of actin subunits are not required for activity. The cross-linking of F-actin by PM is, however, inhibited in a noncooperative fashion by S-1 binding, suggesting that a small local change in actin structure may accompany the binding of S-1 or that S-1 sterically blocks the cross-linking by binding near the contact region between actin subunits.     

Evidence for the association between two myosin heads in rigor acto-smooth muscle heavy meromyosin

The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and 60K were generated. These products were not obtained by EDC treatment of HMM in the absence of F-actin. The HMM fragments that participated in cross-linking were identified by fluorescent labeling and amino acid composition studies. The 115K peptide was determined to be a covalently cross-linked complex that formed between actin and the COOH-terminal 68K fragment of the HMM heavy chain. Our results are in agreement with a previous study which proposed that the site of cross-linking between HMM and F-actin resides within the COOH-terminal 22K fragment of the myosin subfragment 1 heavy chain [Marianne-Pépin, T., Mornet, D., Bertrand, R., Labbé, J.-P., & Kassab, R. (1985) Biochemistry 24, 3024-3029]. The 60K peptide, however, was not a product of cross-linking between HMM and F-actin. On the basis of its amino acid composition, we concluded that this 60K peptide was a cross-linked dimer of the NH2-terminal 24K fragments of the HMM heavy chain. The cross-linking of acto-gizzard HMM significantly increased the Mg-ATPase activity of gizzard HMM without any observable phosphorylation of the regulatory (20K) light chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.     

Characterization of nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015. A novel copper protein

Covalent cross-linking reaction between SH1 and SH2 groups in myosin subfragment-1 (S-1) by N,N'-p-phenylenedimaleimide (pPDM) was followed by the degree of inactivation of NH4+-EDTA ATPase activity. The rate of the cross-linking reaction decreased to less than a 20th in the presence of F-actin. The inhibitory effect of F-actin was not observed in the presence of MgATP. Binding of F-actin to S-1 was measured using ultracentrifugation. S-1 whose SH1 and SH2 were covalently cross-linked by pPDM or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not bind F-actin. After the DTNB-cross-linked S-1 is reduced by dithiothreitol, the ability to bind F-actin is recovered. These results suggest that S-1 has a binding site for F-actin in the region between SH1 and SH2. This site appears to determine the high affinity of acto-S-1 complex at the rigor while decreasing the affinity more than 10(2) times in the presence of MgATP.     

Further characterization of the structural and functional properties of the cross-linked complex between F-actin and myosin S-1

Several structural and functional properties of the covalent complex, formed upon cross-linking of the myosin heads (S-1) to F-actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, were characterized. The elevated Mg2+-ATPase activity was measured during a 1-month storage of the complex under various conditions. In aqueous medium it showed a rapid time-dependent decrease but it was significantly more stable in the presence of 50% ethylene glycol at -20 degrees C. The ATPase loss most likely reflects a progressive conformational change within the S-1 ATPase site resulting from its greater exposure to the medium, induced by the permanently bound F-actin. The covalent acto-S1 complex was submitted to depolymerization-repolymerization experiments using different depolymerizing agents (0.6 M KI; 4.7 M NH4Cl; low-ionic-strength solution). The depolymerization led to an immediate loss of the enhanced Mg2+-ATPase activity; this activity was almost entirely recovered upon repolymerization of the complex. The protein material formed upon depolymerization of the covalent acto-S1 was analyzed by gel chromatography, gel electrophoresis, analytical ultracentrifugation and electron microscopy. It comprised mainly small-sized actin oligomers associated with the covalently bound S-1 and only a limited amount of free G-actin. The results illustrate the relationships between the filamentous state of actin and its ability to stimulate the Mg2+-ATPase activity of S-1. They also indicate that the binding of S-1 to F-actin is transmitted to several neighbouring actin subunits and strengthens the interactions between actin monomers. Acto-S1 cross-linked complexes were prepared in the presence of tropomyosin and the tropomyosin-troponin system. Under the conditions employed, the regulatory proteins were not cross-linked to actin or S-1 and did not affect the extent or the pattern of S-1 cross-linking to F-actin. Measurements of the elevated Mg2+-ATPase activity of the cross-linked preparations revealed that tropomyosin and the tropomyosin-troponin complex, in the absence of Ca2+, inhibit ATP hydrolysis; the extent of ATPase inhibition (up to 50%) was dependent on the amount of covalently bound S-1, being larger at low level of S-1 cross-linking; the addition of Ca2+ restored the ATPase activity to the control value. The data provide direct evidence that the regulatory proteins can modulate directly the kinetics of ATP hydrolysis by the covalent acto-S1 complex as has earlier been suggested for the reversible complex [Chalovich, J. M. and Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437].(ABSTRACT TRUNCATED AT 400 WORDS)     

A 72,000-mol-wt protein from tomato inhibits rabbit acto-S-1 ATPase activity

Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP.     

The mechanism of inhibition of the actin-activated myosin MgATPase by calponin.

Calponin inhibits the actin-activated ATPase of smooth muscle myosin and thus has been proposed as a thin filament-based regulatory component in smooth muscle. To obtain information on the mechanism of inhibition by calponin we have used chemical modification of actin and cross-linking of actin and subfragment 1. Modification of Lys 61 of actin had no effect on the inhibition by calponin of acto-heavy meromyosin ATPase, i.e. different from tropomyosin-troponin. In addition, modification of the acidic N-terminal region of actin did not impair the ability of calponin to bind to F-actin. Finally, calponin was effective in inhibiting ATPase activity of cross-linked acto-subfragment 1. Therefore the mechanism of inhibition by calponin is distinct from troponin-tropomyosin and caldesmon in that it does not involve either the N-terminal acidic region of actin nor the area around Lys 61 and does not fit a simple steric blocking model.     

Binding between maleimidobenzoyl-G-actin and myosin subfragment 1.

It is well known that the structural interactions between S-1 moieties of myosin molecules ("cross bridges") and actin molecules in polymerized ("F") form are thought to underlie muscle contraction. It is surmised that such interactions are unitary (actin:S-1 = 1:1), but actual demonstration thereof is handicapped by intrinsic properties of the proteins. Recently, it has been reported that chemically modified [with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)] actin maintains its monomeric ("G") form and makes a stable unitary complex with S-1 but does not activate the S-1 ATPase [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. However, we recently showed that when MBS-G-actin and S-1 are covalently cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), ATPase activity is restored [Hozumi, T. (1991) Biochem. Int. 23, 835-843]. Here we investigated the interface between MBS-G-actin and S-1 using the techniques of tryptic digestion and EDC-cross-linking. MBS-G-actin specifically protected the N-terminal region of S-1 heavy chain against tryptic cleavage at the 25 kDa/50 kDa junction, which is different from the effect that a protomer within F-actin has on the protection of the 25 kDa/50 kDa junction. In addition, the cross-linking pattern between MBS-G-actin and S-1 was different from that between F-actin and S-1. When MBS-G actin was cross-linked to trypsin-treated S-1, no cross-linked product was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.     

The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.     

Immunochemical evidence for the binding of caldesmon to the NH2-terminal segment of actin

The binding of caldesmon and its actin-binding fragments to actin was studied by using peptide antibodies directed against two actin sites implicated in actomyosin interactions. Antibodies against residues 1-7 on skeletal alpha-actin strongly inhibited the binding of caldesmon to actin and perturbed to a smaller extent the interaction between actin and the actin binding fragments. Carbodiimide coupling of ethylenediamine to the NH2-terminal acidic residues on actin inhibited the binding of caldesmon and its fragments to actin to a similar extent as the (residues 1-7) antibodies. Antibodies against residues 18-28 showed only limited competition with caldesmon for the binding to actin. These results lead to the following conclusions. (i) The NH2-terminal residues on actin play an important role in the binding of caldesmon to actin, (ii) residues 18-28 on actin do not form a major caldesmon interaction site, and (iii) the actin-binding fragments do not contain the full actin-binding interface. These conclusions and other literature data suggest that caldesmon regulates the actomyosin ATPase by competing with myosin.ATP for the NH2-terminal segment on actin.     

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.     

The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements

Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-1 as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site. From static fluorescence polarization measurements, the rotational relaxation time of FITC-labelled actin filaments was calculated to be 20 ns as the value reduced in water at 20 degrees C, while any rotational relaxation time of 1,5-IAEDANS bound to Cys-374 on F-actin in the presence of a twofold molar excess of phalloidin could not be detected by static polarization measurements under the same conditions. This indicates that the Lys-61 side chain is extremely mobile even in the filamentous structure. Fluorescence resonance energy transfer between the donor 1,5-IAEDANS bound to SH1 of myosin subfragment-1 and the acceptor fluorescein-5-isothiocyanate bound to Lys-61 of actin in the rigor complex was measured. The transfer efficiency was 0.39 +/- 0.05 which corresponds to the distance of 5.2 +/- 0.1 nm, assuming that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime and that the transfer occurs between a single donor and an acceptor.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Disulfide cross-linking of caldesmon to actin.

Treatment of a solution of actin and smooth muscle caldesmon with 5,5'-dithiobis(2-nitrobenzoic acid) results in the formation of a disulfide cross-link between the C-terminal penultimate residue Cys-374 of actin and Cys-580 in caldesmon's C-terminal actin-binding region. Therefore, these 2 residues are close in the actin-caldesmon complex. Since myosin also binds to actin in the vicinity of Cys-374 and since caldesmon inhibits actomyosin ATPase activity by the reduction of myosin binding to actin, then the inhibition might be by caldesmon sterically hindering or blocking myosin's interaction with actin. [Ca2+]Calmodulin, which reverses the inhibition of the ATPase activity, decreases the yield of the cross-linked species, suggesting a weakening of the caldesmon-actin interaction in the cross-linked region. It is possible to maximally cross-link one caldesmon molecule/every three actin monomers, in the absence or presence of tropomyosin, clearly ruling out an elongated, end-to-end alignment of caldesmon on the actin filament in vitro, and raising the possibility that the N-terminal part of caldesmon projects out from the filament. Reaction of 5,5'-dithiobis(2-nitrobenzoic acid)-modified actin with caldesmon leads to the same disulfide cross-linked product between actin and caldesmon Cys-580, enabling the specific labeling of the other caldesmon cysteine, residue 153, in the N-terminal part of caldesmon with a spectroscopic probe.     

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.     

Actin-fragmin interactions as revealed by chemical cross-linking

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.     

Hyperthyreosis inhibits the ability of actin to form strong-binding with myosin

The effect of hyperthyreosis development induced by the increase in thyroid hormones in rats (during 2-4 weeks) on the orientation and mobility of fluorescent probe N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) specifically bound to Cys 374 of actin in ghost muscle fibers isolated from fast (EDL) and slow (SOL) rat muscles was studied. It was found that the binding of myosin subfragment-1 (S1) to F-actin induced the typical for the formation of strong binding actomyosin decrease in mobility of actin subdomain 1 and its rotation towards thin filament periphery. Development of hyperthyreosis markedly inhibited these phenomena. The maximal effect was observed after 21 days of disease development. It is suggested that one of the reasons of the contractile deficit of muscle in hyperthyreosis is inhibition of the strong binding between actin and myosin during ATPase cycle.     

Fluorescence energy transfers between points in acto-subfragment-1 rigor complex

Fluorescence energy transfer was measured by time-resolved and steady-state fluorimetry in order to investigate the spatial relationships between the nucleotide binding site of actin, the Cys-373 residue of actin, and the SH1 of myosin subfragment-1 in the rigor complex of acto-subfragment-1. N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to the Cys-373 of actin or the fluorescent ADP analogue 1-N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) bound to F-actin was used as a donor and 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazo le (IANBD) or 5-iodoacetamidofluorescein (IAF) bound to SH1 of myosin subfragment-1 was used as an acceptor. Assuming the random orientation factor, K2, to be 2/3, the distance between Cys-373 residue of actin and SH1 of myosin subfragment-1 was calculated to be about 50 A, in agreement with the values previously reported, 60 A (Takashi, R. (1969) Biochemistry 18, 5164-69) and 50 A (Trayer, H.R. and Trayer, I.P. (1983) Eur. J. Biochem. 135, 47-59). The distance between the nucleotide binding site of actin and SH1 of myosin subfragment-1 was calculated to be about 70 A or greater.