Effect of myosin DTNB light chain on the actin-myosin interaction in the presence of ATP.

The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3.6.1.3]. Although the Ca2+-, Mg2+-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HMM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one.

A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20,000-dalton and 17,000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20,000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration. 1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard "native" tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20,000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20,000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive. 2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phosphorylated HMM (in the presence of Ca2+ ions) were 5 S(-1) and 5.5 mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 S(-1), assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.     

Two calcium regulation systems in squid (Ommastrephes sloani pacificus) muscle. Preparation of calcium-sensitive myosin and troponin-tropomyosin.

The Ca-regulatory system in squid mantle muscle was studied. The findings were as follows. (a) Squid mantle myosin B (squid myosin B) was Ca-sensitive, and its Ca-sensitivity was unaffected by addition of a large amount of rabbit skeletal myosin (skeletal myosin) or rabbit skeletal F-actin (skeletal F-actin). (b) Squid myosin was prepared from the mantle muscle. It showed a heavy chain component and two light chain components in the SDS-gel electrophoretic pattern: the molecular weights of the latter two were 17,000 and 15,000. Actomyosin reconstituted from squid myosin and skeletal (or squid) actin showed Ca-sensitivity in superprecipitation and Mg-ATPase assays. EDTA- treatment had no effect on the Ca-sensitivity of squid myosin. (c) Squid mantle actin (squid actin) was prepared by the method of Spudich and Watt. Hybrid actomyosin reconstituted by using the pure squid actin preparation with skeletal myosin showed no Ca-sensitivity in Mg-ATPase assay, whereas that reconstituted using crude squid actin showed marked Ca-sensitivity. The crude squid actin contained four protein components which were capable of associating with F-actin in 0.1 M KCl, 1 mM MgCl2 and 20 mM Tris-maleate (pH7.5). (d) Native tropomyosin was prepared from squid mantle muscle, and it conferred Ca-sensitivity on skeletal actomyosin as well as on a hybrid actomyosin reconstituted from squid actin and skeletal myosin. (e) Squid native tropomyosin was separated into troponin and tropomyosin fractions by placing it in 0.4 M LiCl at pH 4.7. The troponin fraction was further purified by DEAE-cellulose chromatography. Squid troponin thus obtained was different in mobility from rabbit skeletal or carp dorsal troponin; three bands of squid troponin corresponded to molecular weights of 52,000, 28,000, and 24,000 daltons. It could confer Ca-sensitivity in the presence of tropomyosin on skeletal actomyosin as well as on a hybrid reconstituted from squid actin and skeletal myosin. (f) Squid myosin B, and two hybrid actomyosins were compared as regards Ca and Sr requirements for their Mg-ATPase activities. The myosin-linked regulatory system rather than the thin-filament-linked regulatory system was predominant in squid myosin B. Squid myosin B required higher Ca2+ and Sr2+ concentrations for Mg-ATPase activity; half-maximal activation of Mg-ATPase was obtained at 0.8 micron Ca2+ and 28 micron Sr2+ with skeletal myosin B, and at 2.5 micron Ca2+ and 140 micron Sr2+ with squid myosin B.     

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.     

Substructure of the myosin molecule: IV. Interactions of myosin and its subfragments with adenosine triphosphate and F-actin

The enzymic activity of several single-headed subfragments of myosin (HMM S-1 and single-headed HMM) has been compared to the double-headed derivative of myosin (HMM) both in the presence and absence of aetin. Under the assay conditions of our experiments, we find that HMM hydrolyses ATP at approximately twice the rate of any single-headed species. These results suggest a relatively independent functional role for each of the two heads of the myosin molecule.An attempt has been made to determine the stoichiometry of association between subfragments and actin, either in the absence of nucleotide or during the hydrolysis of ATP. It was originally thought that a comparison of the maximum turnover rate of HMM at infinite concentrations of actin with the maximum rate at infinite concentrations of enzyme (but with a fixed amount of actin) would yield the combining ratio of actin to HMM. However, the considerable variation of ATP turnover rates with the conditions of the experiment has made it impossible to reach any firm conclusions regarding stoichiometry. A more direct approach to the question of stoichiometry is possible in the absence of ATP. By reacting varying amounts of F-actin with a given concentration of subfragment and centrifuging the resulting complex, it is possible to determine the unbound concentration of subfragment in the supernatant. These data provide sufficient information to construct a Scatchard plot and show that twice as many moles of actin are bound by HMM as by HMM S-1. Furthermore, the association constant of actin for HMM is several orders of magnitude higher than that for the single-headed species.In connection with the question of why myosin has two “heads”, we have examined the ability of single-headed molecules to undergo the phenomenon of “superprecipitation”. We find that single-headed myosin (the preparation of which was discussed in the preceding paper) is able to superprecipitate in much the same manner as native myosin.We conclude from these studies that each head of the myosin molecule is able to function in a relatively independent fashion. These studies do not, of course, exclude the possibility of more subtle interactions between the heads of myosin which our techniques are not able to detect.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. II. Effect of modification of the Sa thiol group on superprecipitation and clearing.

The effect of Sa modification with NEM, which activates Mg2+-ATPase through an enhancement of the association of actin and myosin, was investigated on the superprecipitation, clearing and Mg2+-ITPase of myosin B with reference to the effect of S1-blocking. 1. Superprecipitation induced by ATP was markedly enhanced by Sa-blocking even at high concentrations of Mg2+ and substrate; this may be due to an increase in the affinity of myosin and actin on blocking Sa. 2. Nevertheless, neither ITP-induced superprecipitation nor Mg2+-ITPase was affected by Sa modification. 3. Blocking of S1 brought about the inhibition of ATP- and ITP-induced superprecipitation and Mg2+-ITPase activity, suggesting that S1-blocking decreases the affinity of myosin and actin. 4. Sa-blocked myosin B showed greater resistance to clearing by ATP, especially in the presence of Ca2+ ions, whereas in the clearing response of actomyosin gel to PPi no difference between Sa-blocked and unmodified myosins B was observed. On the other hand, the clearing response of myosin B became more sensitive to both ATP and PPi on blocking S1. Based on the above results and preliminary data suggesting that Sa is located in LMM, the interaction of myosin filaments and actin filaments under physiological conditions is discussed.     

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.     

Heavy meromyosin from skipjack tuna, Euthynus pelamis. Preparation and enzymic properties.

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin.     

Myosin-like protein and actin-like protein from Escherichia coli K12 C600.

Myosin-like protein was obtained from E. coli by extraction with a sucrose solution and by precipitation with rabbit skeletal actin. The preparation of E. coli myosin-like protein looked very similar, in the sodium dodecyl sulfate-gel electrophoretic pattern, to that of rabbit skeletal myosin. The myosin-like protein was able to reversibly bind to rabbit actin. It had the activities of EDTA-, Ca-, and Mg-ATPases. The product in the EDTA-ATPase reaction catalyzed by the myosin-like protein was identified as ADP by ion exchange chromatography. The Mg-ATPase activity of E. coli myosin-like protein was activated by either rabbit actin or E. coli actin-like protein though the activation was much stronger by the latter. However, the myosin-like protein did not exhibit superprecipitation either with rabbit actin or with E. coli actin-like protein. Actin-like protein was also obtained from E. coli by essentially the same procedures as those described for preparation of rabbit skeletal actin. E. coli actin-like protein was capable of activating Mg-ATPase of rabbit myosin, and also of superprecipitation with rabbit myosin. Extraction from both the whole cells and the membrane fraction of E. coli strongly suggested that the myosin-like protein and the actin-like protein are both localized in the membrane fraction rather than in the cytoplasmic fraction.     

Calcium binding and calcium-sensitivity of heavy meromyosin and subfragment-1 from squid (Todarodes pacificus) mantle and scallop (Patinopecten yessoensis) adductor muscles

1. HMM and S-1 both bind one mol of calcium per mole of head, and a half of the calcium binding was diminished upon magnesium addition (10 mM) at the low affinity site. 2. The Mg-ATPase activity of HMM (without actin) was fully activated by the binding of one mol of calcium bound per mol of HMM. 3. The calcium binding profile to S-1 is the same as that to HMM, however, the Mg-ATPase activity of S-1 is independent of calcium binding. It is suggested that there are two kinds of myosin head (or S-1) in molluscan myosin, functionally different in calcium binding properties.     

Evaluation of mechanical parameters of the mechanochemically 'active state' of actin filaments on the basis of purely chemical data

Oosawa and his collaborators (cf. F. Oosawa, Biophys. Chem. 11 (1980) 443), employing various optical techniques, have shown that the flexibility of actin filaments increases upon interacting with the enzymatically active myosin fragments, particularly heavy meromyosin (HMM). It has been reported (S. Hitchock, L. Carlsson and U. Lindberg, Cell 7 (1976) 53) that HMM can accelerate the DNase 1-induced depolymerization of F-actin, provided MgATP is also present. Since, as we have demonstrated (cf. J. Borejdo myosin, is endowed with mechanochemical capability, we made an attempt to correlate the enhanced rate of depolymerization with the decrease in rigidity of the G-G bonds in F-actin. On the basis of the chemical kinetic data of Hitchcock et al. we could derive the approximate value of the HMM-MgATP-induced change in rigidity which is a mechanical molecular parameter. Since interaction between HMM or HMM subfragment-1 and F-actin in the presence of MgATP leads to the movement of the myosin heads along the actin filaments, it is argued that the enzymic behavior of this system should not be analyzed on the basis of simple, equilibrium, complex formation.     

Interaction of rat brain microtubule proteins and 6S tubulin with rabbit skeletal muscle actomysin.

The interaction between actomyosin from rabbit skeletal muscle and microtubule proteins or 6S tubulin from rat brain was investigated with respect to the change in ATPase activity and physicochemical properties. Myosin bound to both microtubule proteins and 6S tubulin at low ionic strength. In the aggregates the molar ratio of microtubule proteins or 6S tubulin to myosin was 0.5–1.5 or 1.5–2.5. The superprecipitation of actomyosin was inhibited by 6S tubulin. The degree of superprecipitation inhibition was dependent on the mixing order of myosin, actin, 6S tubulin, and ATP. When myosin was preincubated first with 6S tubulin, the inhibition was most marked. The actin activation of myosin Mg-ATPase was inhibited by both microtubule proteins and 6S tubulin with stronger effects by the latter. The preincubation of myosin with 6S tubulin prior to the addition of actin induced not only greater inhibition of ATPase but also the binding of a larger quantity of 6S tubulin to myosin than the preincubation of myosin with actin. The similar results were obtained with microtubule proteins.     

The nucleoside triphosphate (NTP)-induced superprecipitation and NTPase reaction of chicken gizzard actomyosin as a function of the NTP concentration

The ATPase or ITPase reaction and ATP- or ITP-induced superprecipitation were studied as a function of the ATP or ITP concentration with suspensions of chicken gizzard "native" myosin B or "reconstituted" myosin B (a combination of actin, myosin, and native tropomyosin). The specific aim of the study was to answer the following questions: i) Is the superprecipitation or the ATPase reaction sensitive to calcium ions even at very low concentrations of ATP? ii) Is tropomyosin required for calcium sensitivity? iii) Does "native" myosin B from gizzard muscle behave differently from "reconstituted" myosin B? iv) Does the troponin-tropomyosin complex of rabbit skeletal muscle act as a regulatory protein for the contractile activity of acto-phosphorylated myosin? Considering the overall time course of reaction rather than single values of activity, we found that the answers to the first three questions were negative, while that to the last question was positive. These results favor the kinase-phosphatase mechanism of calcium regulation rather than the leiotonin mechanism.     

Hybrid with rabbit skeletal DTNB-light chains of heavy meromyosin, myosin, and glycerinated fibers from Akazara scallop adductor

It was shown in our previous report (Ojima et al. (1983) J. Biochem. 94, 307-310) that hybridization of Akazara scallop "desensitized" myosin with rabbit skeletal DTNB-light chains led to inhibition of the Mg-ATPase activity of acto-desensitized myosin but to enhancement of its superprecipitation activity. The following are now found: Development of tension in desensitized glycerinated fibers of Akazara adductor is significantly improved when DTNB-light chains are added to the fiber bath. The actin-affinity of desensitized heavy meromyosin in the presence of ATP but in the absence of Ca2+ is decreased by hybridization with chicken gizzard 20K dalton-light chains but significantly increased by that with DTNB-light chains. It is therefore suggested that the increase in actin-binding may account for the enhancing effect of DTNB-light chains on the superprecipitation and on the tension development.     

Heavy meromyosin and subfragment-1 from squid mantle myosin, and Ca-sensitivity of their Mg-ATPases

Heavy meromyosin (HMM) and subfragment-1 (S1) were obtained from squid mantle myosin by tryptic digestion and chymotryptic digestion, respectively. Squid HMM(T) and S1(CT) preparations contained stoichiometric amounts of the two types of light chain subunit; regulatory light chain, LC-2, and essential light chain, LC-1. No difference was detected in the chymotryptic digestibilities of squid mantle myosin in Ca-medium and in EDTA-medium. This is in contrast to the digestibility of scallop adductor myosin. The Mg-ATPase activity of HMM(T) alone and that of acto-HMM(T) were both sensitive to calcium ions. In contrast, the activity of S1(CT) alone and that of acto-S1(CT) were both insensitive to calcium ions. The affinity of HMM(T) for actin was not affected by calcium ions, but the amount of HMM(T) bound to actin was increased by calcium ions from 20% to 60% of the total amount of HMM(T). On the other hand, the actin affinity of S1(CT) and the amount of S1(CT) bound to actin were both unaffected by calcium ions. The role of calcium ions in the regulation of contraction in molluscan muscles is discussed.     

Calcium sensitivy of foot muscle myosin from clam (Meretrix lusoria).

1. It was found that Mg-ATPase of clam foot myosin is strongly activated by calcium or strontium ions and is as sensitive to those divalent cations as the Mg-ATPase and superprecipitation of rabbit skeletal acto-clam foot myosin are. 2. It was also found that desensitization and resensitization of clam foot myosin result in the loss of superprecipitation activity with acto-desensitized myosin and in its recovery with acto-resensitized myosin, respectively. However, the ATP-ASE activity in the absence of calcium ions rises with acto-desensitized myosin and falls again with acto-resensitized myosin. 3. It is thus proposed that the primary role of the EDTA-light chain component in calcium regulation is to inhibit myosin-ATPase rather than to inhibit the actin-myosin interaction.     

The causes of the nonuniform thermostability of Mg-ATPase and of the contractility of muscle models

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.