Hybrid formation between scallop myofibrils and foreign regulatory light-chains

Scallop myofibrils (Placopecten magellanicus) from which regulatory light-chains had been completely removed by EDTA treatment at 30 °C were hybridized with regulatory light-chains of different myosins. Pure hybrids, containing only foreign regulatory light-chains with a stoichiometry of two moles per myosin, were readily formed with all the light-chains tested. Some of the regulatory light-chains restored regulatory functions to desensitized myofibrils by selectively inhibiting the actin activated Mg-ATPase in the absence of calcium. Light-chains from Mercenaria, Spisula Loligo and Urechis behaved as scallop regulatory light-chains, were inhibitory in the absence of calcium, and restored high-affinity calcium binding sites. Regulatory light-chains of Limulus, cricket, chicken gizzard and platelet were also inhibitory; however, calcium binding was restored with a lowered affinity and the hybrids required higher calcium concentrations for ATPase activation. Hybrids formed with the regulatory light-chains of vertebrate striated (rabbit, chicken, skate), bovine cardiac and lobster tail and claw muscles remained insensitive to calcium, their ATPase activity was not selectively depressed in the absence of calcium and specific high-affinity calcium binding sites were not restored. Phosphorylation of the light-chains (rabbit, cardiac and gizzard) has no effect on ATPase activity. The behaviour of the hybrids supports the interpretation that in vertebrate striated muscles myosin does not function as a regulatory switch.Foreign regulatory light-chains (Spisula, Loligo, Mercenaria, rabbit) bind to desensitized myofibrils with a similar or slightly higher affinity as scallop regulatory light-chains. The two light-chain binding sites of myosin are equivalent and differences in affinity appear to be the result of an interaction between the two halves of the myosin molecules.     

Essential light chain exchange in scallop myosin

The exchange of essential light chains (SH-LCs) of scallop myosin was followed with the aid of scallop SH-LC alkylated with 14C-labeled iodoacetate. More than 70% of the SH-LCs were exchanged in myosin preparations that were desensitized by removal of both regulatory light chains (R-LCs) with ethylenediaminetetraacetic acid (EDTA) treatment. Although desensitized myosin solubilized with 0.6 M NaCl or with 10 mM adenosine 5'-triphosphate (ATP) in the absence of salt equilibrated rapidly with SH-LCs even in the cold, exchange in myosin filaments required elevated temperatures. Equilibration of the SH-LCs in desensitized preparations did not depend on ATP or magnesium ions but was significantly accelerated by actin. The desensitized myosin preparations containing alkylated SH-LCs (approximately 1 mol of thiol alkylated/mol of SH-LC) readily recombined with R-LCs. The preparations regained fully the calcium dependence of the actin-activated magnesium adenosinetriphosphatase (Mg-ATPase), contained R-LCs and SH-LCs in equimolar amounts, and had an ATPase activity similar to that of untreated myosin preparations. R-LCs interfered with the equilibration of the SH-LCs. In intact myosin preparations, the exchange of SH-LCs was slow and was frequently associated with the dissociation of the R-LCs. The blocking action of the R-LC on SH-LC exchange agrees with evidence showing that the two light chain types interact and suggests that parts of the SH-LC may lie between the R-LC and the heavy chain of myosin.     

Position of Mercenaria regulatory light-chain Cys50 site on the surface of myosin visualized by electron microscopy

Mercenaria regulatory light-chains, specifically labelled at cysteine 50 with N-iodoacetyl-N'-biotinylhexylenediamine, were rebound to regulatory light-chain denuded scallop myosin, and the hybrid myosin formed was decorated with avidin. These hybrid myosins were visualized by rotary-shadowing electron microscopy. Three distinct images of avidin-decorated hybrid myosin molecules were obtained. These comprise singly decorated molecules, where the avidin is bound symmetrically or asymmetrically with respect to the two heads of myosin, in addition to "figures-of-five", where two myosin molecules associate with a centrally placed avidin molecule. Analysis of these images indicates that the Mercenaria regulatory light-chain Cys50 site is located 15 to 35 A from the head-rod junction when the light-chain is bound in situ to myosin. Implications with respect to head topology and probe studies are discussed.     

Regulatory light chains in myosins.

Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity.Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA.There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.     

Comparative studies on amino and thiol groups in myosins from different sources.

Myosins from rabbit white and red skeletal, rabbit heart, fish skeletal and chicken gizzard muscles, as well as from human platelets were subjected to trinitrophenylation by trinitrobenzene sulfonate and alkylation by N-ethylmeleimide which affected their amino and thiol groups, respectively. The blocking of amino groups was carried out in the presence or in the absence of Mg-ADP and was followed both spectrophotometrically and enzymatically. Essential amino groups, whose modification throughly changes the enzymic characteristics of myosin, were found in heart and in all skeletal muscle myosins but were absent in myosins from chicken gizzard muscle and from human platelets. The reaction of these amino groups was highly retarded in the presence of Mg-ADP. Alkylation of thiols led to loss of the K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in all myosins. However, the rate of loss of activity varied from one myosin to another and, for a given myosin, was affected by the presence of nucleotides and by the value of the ionic strength. The change in Ca(2+)-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) on alkylation was influenced by the presence of Mg - ADP during the reaction. In the absence of this nucleotide, the Ca(2+)-ATPase activity increased and reached a plateau as a consequence of modification. The extent of activation largely depended on the origin of the myosin. When alkylation was carried out in the presence of Mg-ADP, the Ca(2+)-ATPase activity as a function of time exhibited a maximum but the descending part of the curve was absent in myosins from heart and gizzard muscles.     

Comparative studies on 5,5'-dithiobis-(2-nitrobenzioc acid)-treated myosins.

Myosins prepared from chicken and rabbit fast and slow muscles were treated with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2). About half of the thiol groups of the fast muslce myosins reacted with Nbs 2, but in slow muscle myosins, only about 10-20% of the thiol groups reacted. This treatment removed 50-60% of the L2 components, Nbs2 light chain, from fast muscle myosins, but did not result in specific dissociation of the light chains in slow myscle myosins. The treatment sometimes released L4 component from chicken muscle myosins instead of L2 component. The changes of myosin ATPase [EC 3.6.1.3] activities caused by this treatment did not correlate with the release of Nbs2 light chain, but were dependent upon the species, chicken or rabbit.     

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.     

An immunological approach to myosin light-chain function in thick filament linked regulation. 2. Effects of anti-scallop myosin light-chain antibodies. Possible regulatory role for the essential light chain

Specific antibodies directed against the regulatory light chains (R-LC) or essential light chains (SH-LC) of scallop myosin abolished calcium regulation in myofibrils, myosin, and heavy meromyosin by elevating the actin-activated Mg2+-ATPase activity in the absence of calcium. Calcium dependence was completely eliminated at molar ratios of 2.5-3 antibodies bound per myosin. Monovalent anti-R-LC Fab and anti-SH-LC Fab fragments also desensitized myofibrils fully. High Ca2+-ATPase activity remained unaffected by the antibodies. Anti-SH-LC IgG reduced to about one-half the actin-activated Mg2+-ATPase in the presence of calcium and the potassium-activated ethylenediaminetetraacetic acid (EDTA)-ATPase activities. Anti-SH-LC Fab, however, desensitized without inhibiting the actin-activated Mg2+-ATPase. The desensitizing effect of both antibodies was abolished by prior absorption with the homologous myosin light chain. Calcium binding and R-LC and anti-SH-LC IgG's and by anti-SH-LC Fab. The anti-R-LC Fab fragment induced a significant (70%) dissociation of R-LC from myofibrils and myosins with concomitant losses in calcium binding. In contrast, anti-R-LC IgG prevented the dissociation of R-LC from myosin by EDTA. Binding of anti-R-LC IgG to myofibrils was proportional to thier R-LC content. Increased amounts of anti-SH-LC IgG were bound by myofibrils devoid of R-LC. Bound anti-SH-LC antibody significantly inhibited the reuptake of R-LC by EDTA-treated myofibrils as well as the full binding of anti-R-LC antibody. Certain rabbits produced a population of anti-SH-LC antibodies which were specific for this light chain and bound extensively to myosin but failed to desensitize it (nondesensitizing anti-SH-LC antibody). The desensitizing and nondesensitizing anti-SH-LC populations bound to different regions of the SH-LC on the myosin, and the binding of the two types of antibody to the SH-LC was nearly additive. The nondesensitizing SH-antibody inhibited the reuptake of R-LC less, and its binding to myofibrils was not influenced by the absence of R-LC. These studies indicate a direct or indirect involvement of the SH-LC's in myosin-linked regulation, raise the possibility of an interaction between the R-LC and SH-LC, and confirm the regulatory function of the scallop R-LC. A model for a relative location of the two types of light chains and the involvement of the subfragment-2 region of myosin linked regulation is discussed.     

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.     

Evidence for a single essential thiol in the yeast hexokinase molecule.

In yeast hexokinase B, two thiols per monomer appeared to be essential when enzymic inactivation was produced by the concurrent alkylation of both of them, by several reagents including the affinity reagent N-bromoacetyl-2-D-galactosamine. However, it is shown that only one of these thiols is actually essential. Three of the four thiols present can be blocked by alkylation in the presence of a substrate in appropriate conditions, without loss of enzymic activity. Subsequently, in the absence of substrate, the affinity reagent reacts at the one remaining thiol, with complete inactivation. The same behavior can be obtained by reaction with iodoacetamide or by the formation of the -SCN group. The affinity reagent inactivates hexokinase B faster than does the isomeric glycosidic compound (glycosides being nonsubstrates), although the latter has twice the reactivity of the former toward glutathione. The reactions with alkylating agents, with or without substrate present, are used to classify the four thiols in the monomer. The temperature dependence of the alkylation of the essential thiol provides evidence for a transition in the molecule at about 31 degrees C. The inactive monomer containing the -SCN group can regenerate, by thiolysis, active enzyme with the thiol free. It can also perform an intramolecular cleavage of the chain. The latter reaction was used to locate the essential cysteine residue in the chain, at 80% of the length from the N terminus.     

The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.     

Radioactive labeling and location of specific thiol groups in myosin from fast, slow and cardiac muscles.

1. Based on incorporation of radioactively labeled N-ethylmaleimide, the readily reactive thiol groups of isolated myosin (EC 3.6.1.3) from fast, slow and cardiac muscles could be classified into 3 types. All 3 myosins contain 2 thiol-1, 2 thiol-2 and a variable number of thiol-3 groups per molecule. Both thiol-1 and thiol-2 groups which are essential for functioning of the K+-stimulated ATPase, are located in the heavy chains in all 3 myosin types. 2. The variation in the incorporation pattern of N-ethylmaleimide over the 3 thiol group classes under steady-state conditions of Mg(2+) - ATP hydrolysis allowed different conformations of some reaction intermediates to be characterized. In all 3 types of myosin the hydrolytic cycle of Mg(2+) - ATP was found to be controlled by the same step at 25 degrees C. In all three cases, this rate-limiting step is changed in the same way by lowereing temperature. 3. Using the chemically determined molecular weights for myosin light chains, their stoichiometry was found on the basis of sodium dodecyl sulfate electrophoresis to be 1.2 : 2.1 : 0.8 for light chain-1: light chain-2:light chain-3 per molecule of fast myosin, 2.0 : 1.9 for light chain-1:light chain-2 per molecule of slow myosin and 1.9 : 1.9 for light chain-1:light chain-2 per molecule of cardiac myosin. This qualitative difference in light subunit composition between the fast and the two types of slow myosin is not reflected in the small variations of the characteristics exhibited by the isolated myosins, but rather seems to be connected with their respective myofibrillar ATPase activities.     

Essential light chain modulates phosphorylation-dependent regulation of smooth muscle myosin

To examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH(4)(+)/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin subfragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.     

Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease

Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.     

Proximity of regulatory light chains in scallop myosin

The distance between the regulatory light chains of the two heads of the scallop myosin molecule was estimated with the aid of two photolabile cross-linkers, benzophenone maleimide and p-azidophenacylbromide. These cross-linkers selectively alkylate thiol groups and have a maximum length of about 9 A. One of the two regulatory light chains of scallop myosin was removed by treatment of myofibrils at 10 degrees C with EDTA and replaced with a foreign regulatory light chain carrying a cross-linker. Cross-linking between the scallop and foreign regulatory light chains was effected by photolysis. This was demonstrated by incubating nitrocellulose transfers of sodium dodecyl sulfate/polyacrylamide gels of the photolyzed hybrid myofibrils with specific antibodies against the different light chains, followed by fluorescein isothiocyanate-125I-labeled secondary antibody. Scallop regulatory light chains cross-linked extensively (20 to 50%) with Mercenaria regulatory light chains (cysteine in position approximately 50) in solutions that induce rigor in skinned fibers (no ATP) and in relaxing solutions (ATP but no Ca2+). Neither the regulatory light chains of chicken skeletal myosin (cysteines 129 and 157) nor those of gizzard myosin (cysteine 108) were cross-linked to scallop regulatory light chains in either medium. These results indicate that the N-terminal portions of the myosin regulatory light chains can approach each other within 9 A or less, while the distance between the C-terminal halves exceeds 9 A, and support the view that the N termini of the regulatory light chains point toward the myosin rod. Since the relative distance between the regulatory light chains of the two myosin heads is not altered between rigor and rest, we suggest that motion of the essential light chains is mainly responsible for the observed difference in the relative positions of the regulatory and essential light chains between conditions of rigor and rest.     

Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

Regulatory properties of single-headed fragments of scallop myosin.

Calcium control was studied in single-headed myosin and subfragment-1 (S1) preparations obtained by papain digestion of scallop myosin. Single-headed myosin, containing light chains in stoichiometric amounts, was calcium regulated; in contrast, the actin-activated Mg-ATPase of all S1 species lacked calcium sensitivity. Both regulatory and essential light chains were retained by S1 and single-headed myosin preparations provided divalent cations were present during papain digestion, although a peptide amounting to 10% of the mass was removed from regulatory light chains. The modified regulatory light chain retained its ability to confer calcium binding and restore calcium sensitivity to the ATPase of desensitized myofibrils. Regulatory light chains protected the essential light chains from fragmentation by papain. S1 bound regulatory light chains with a uniformly high affinity and appeared to consist of a single species. The results demonstrate that head to head interactions are not obligatory for calcium control, although they may occur in the intact myosin molecule, and suggest a role for the subfragment-2 region in calcium regulation of myosin.     

The role of thiol groups in the structure and mechanism of action of arginine kinase

1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.     

Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS.

The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca²⁺-ATPase activity and inhibition of the K⁺-EDTA-ATPase activity of myosin. The stoichiometry of the [³H]1,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH₁” groups responsible for the catalytic properties of myosin. Moreover, it has been unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with [³H]1,5 IAEDANS or iodo[1-¹⁴C]acetamide indicates strongly that the identical thiol was labeled by both reagents.