N-Ethylmaleimide modified heavy meromyosin in only 3-fold activated by actin rather than 200-fold as is normal heavy meromyosin (Silverman, R., Eisenberg, E., and Kielley, W. W. (1972), Nature (London) 240, 207). Ultracentrifuge studies demonstrated that in the absence of ATP the N-ethylmaleimide modified heavy meromyosin binds to actin at a ratio of 2 actins to 1 N-ethylmaleimide modified heavy meromyosin. However, it was found that most of the N-ethylmaleimide modified heavy meromyosin was not bound to actin during ATP hydrolysis. Ultracentrifuge studies demonstrated that in the presence of 25 or 50 mM KCl under conditions where the ATPase is maximally activated by actin, less than 5% of the N-ethylmaleimide modified heavy meromyosin was bound to actin. In the absence of KCl there was limited binding but even this binding did not appear to correlate with the N-ethylmaleimide modified heavy meromyosin ATPase rate. Turbidity and viscosity studies also indicated that in the presence of ATP under conditions of maximal actin activation the N-ethylmaleimide modified heavy meromyosin and actin are almost completely dissociated, whereas there is a marked increase in turbidity and viscosity after all of the ATP is hydrolyzed. These results suggest that in the presence of ATP and actin N-ethylmaleimide modified heavy meromyosin exists most of the time in a refractory state unable to bind to actin and only a small part of the time in a nonrefractory state which can interact with actin. It follows that the major rate-limiting step during actin activation is the transition from the refractory to the nonrefractory state. Since the actin activation of N-ethylmaleimide modified heavy meromyosin is lower than that of normal heavy meromyosin this transition may be slower for N-ethylmaleimide modified heavy meromyosin than for normal heavy meromyosin.

Viscosity, turbidity, and laser-light fluctuation autocorrelations of acto-heavy merymyosin (HMM) and acto-subfragment 1 (S-1) solutions were measured under conditions where the actin-activated ATPase is close to its maximal value. The results were compared to similar data obtained in the absence of ATP where the actin and myosin fragments were completely domplexed, and in the presence of ATP but at 0.1 M KLC where the actin and HMM or S-1 were almost completely dissociated. It was found that at maximal actin activation, the viscosity, turbidity, and autocorrelation data were all much closer to the values for the completely dissociated systems than to the values for the completely complexed systems. Assuming that viscosity, turbidity, and autocorrelation measurements approximate a linear measure of binding between actin and HMM or S-1, the results suggest that at maximal actin activation less than 10% of the HMM or S-1 are bound to the actin. Therefore as was suggested previously by ultracentrifuge and kinetics studies, it appears that under conditions of maximal actin activation, most of the HMM and S-1 occur in a refractory state unable to bind to actin.

By means of new or modified procedures troponin-tropomyosin complex was fractionated into three components plus tropomyosin, and then reconstituted in the absence of urea.Tropomyosin was separated from troponin by hydroxyapatite column chromatography, a method giving sharper separation than the previously used technique of isoelectric precipitation.The chromatography also separated the tropomyosin into two fractions.One gave a single band on sodium dodecyl sulfate gel electrophoresis, and the other a double band; and the complete absence of proline suggested that each was a different form of pure tropomyosin.Troponins I, T, and C were separated by DEAE-Sephadex chromatography in 6 M urea as previously, but the separation was found to be much improved if the troponin was treated with a CaZ+ chelator before being applied to the column.The activity of the troponin fractions was assayed at varying ratios to actin both with and without tropomyosin present.It was found that near physiologic ratios to actin, either the combination of troponin I plus tropomyosin or troponin I plus troponin T inhibited the acto-heavy meromyosin ATPase.However, in both cases when troponin C, which is necessary for Ca2+ sensitivity, was added, the inhibition was reversed not only in the presence but also in the absence of Ca2+.Only when both troponin T and tropomyosin were present in addition to troponin I and C did inhibition occur in the absence but not in the presence of Ca2+.We therefore conclude first, that if troponin T is present tropomyosin may not be required for inhibition by troponin I at physiologic ratios to actin, second, that all three components plus tropomyosin are necessary to restore full Ca*+ sensitivity, and third, that this reconstitution can be accomplished by combining the individual components in the absence of urea.

The sliding filament theory, first proposed in 1954 (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) is now generally accepted as the overall basis for muscle contraction, and the elegant structural studies of H. E. Huxley and his coworkers suggest that the key event in the sliding process is the cyclic interaction of myosin bridges with F-actin and ATP (Huxley, 1969). Kinetic studies on the interaction of actin, myosin, and ATP in vitro should provide useful information about the cyclic interaction occurring in vivo; but unfortunately, because myosin occurs as insoluble filaments at low ionic strength, these studies have been difficult to interpret in a quantitative manner (Eisenberg and Moos, 1967). On the other hand, heavy meromyosin (HMM), a tryptic digestion product of myosin which retains the two-headed (two-site) structure of the myosin, and subfragment-1 (S-l), a further proteolytic digestion product which consists of single myosin heads (Lowey et al...

NUMEROUS studies have been performed on the effect of blocking the SH groups of myosin with sulphydryl reagents1–8. In particular, the effect of NEM has become quite clear; on blocking one specific SH group (referred to as SH1) per myosin subunit, the EDTA-ATPase of the myosin is completely inhibited whereas the Ca-ATPase is markedly activated9. However, the interaction of SH1-blocked myosin and actin has not been as thoroughly investigated. Sekine and Yanoguchi have reported that blocking SH1 with NEM does not affect the ATPase activity and superprecipitation of actomyosin10. On the other hand, the effect of native tropomyosin on the actomyosin ATPase has been reported to be abolished by NEM under certain conditions although differences were reported depending on whether myosin or actomyosin was exposed to the NEM11.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBinding of actin to heavy meromyosin in the absence of adenosine triphosphateEvan Eisenberg, Louis Dobkin, and W. Wayne KielleyCite this: Biochemistry 1972, 11, 25, 4657–4660Publication Date (Print):December 1, 1972Publication History Published online1 May 2002Published inissue 1 December 1972https://pubs.acs.org/doi/10.1021/bi00775a003https://doi.org/10.1021/bi00775a003research-articleACS PublicationsRequest reuse permissionsArticle Views24Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The binding of actin to heavy meromyosin (HMM) in the presence of ATP was studied by analytical ultracentrifuge and ATPase studies. At 0 degrees C, at very low ionic strength, the double-reciprocal plot of HMM ATPase against actin concentration is linear. If one assumes that all of the HMM is bound to actin when the ATPase activity equals V(max), then, at an actin concentration where the actin-HMM ATPase is 85% of V(max), all but 15% of the HMM should be complexed with actin. However, when the binding of HMM to actin in the presence of ATP was measured with the analytical ultracentrifuge, more than 60% of the HMM was not bound to actin. From experiments with EDTA- and Ca-ATPases it seemed unlikely that the unbound HMM was denatured. It is thus possible that during the steady-state hydrolysis of ATP, HMM spends more than 50% of its cycle of interaction with actin and ATP in a "refractory state," unable to bind to actin, i.e., while an HMM molecule goes through one cycle of interaction with actin and ATP, an actin monomer could bind and release several HMM molecules so that the turnover rate per mole of added actin would be considerably greater than that per mole of added HMM. Comparison of the rate of ATPase activity at very high actin concentration with that at very high HMM concentration shows that this is indeed so. Therefore, both kinetic and ultracentrifuge studies suggest that the HMM exists in a refractory state during a large part of its cycle of interaction with actin and ATP.

In the absence of Ca++, native tropomyosin markedly inhibits the acto-subfragment-1 ATPase as well as the acto-heavy meromyosin ATPase despite the fact that subfragment-1, in contrast to heavy meromyosin, has only a single site for ATP. Double reciprocal plots of acto-heavy meromyosin ATPase vs. actin concentration both in the presence and absence of native tropomyosin, show that the native tropomyosin has little effect on the acto-HMM-ATPase but acts mainly by affecting the binding of actin to the HMM-ATP complex.

The catalytic properties of native myosin and of myosin in which one SH-group per subunit chain has been blocked with N-ethylmaleimide have been compared.

This study investigates possible sources for the variance of more than two orders of magnitude in the published values for the shear moduli of purified actin filaments. Two types of forced oscillatory rheometers used in some of our previous work agree within a factor of three for identical samples. Polymers assembled in EGTA and Mg2+ from fresh, gel-filtered ATP-actin at 1 mg/ml typically have an elastic storage modulus (G′) of ∼1 Pa at a deformation frequency of 0.1–1 Hz. G′ is slightly higher when actin is polymerized in KCl with Ca2+ and Mg2+. Gel filtration removes minor contaminants from actin but has little effect on G′ for most preparations of actin from acetone powder. Storage of actin monomers without frequent changes of buffer containing fresh ATP and dithiothreitol can result in changes that increase the G′ of filaments by more than a factor of 10. Frozen storage can preserve the properties of monomeric actin, but care is necessary to prevent protein denaturation or aggregation due to freezing or thawing.

It is now widely recognized that under certain conditions of temperature and pH (1, 2)) the Ca++-activated hydrolysis of adenosine triphosphate (ATP) by myosin exhibits a biphasic response to titration of the sulfhydryl groups of the protein by organic mercurials and by N-ethylmaleimide (3-5). On the other hand, titration by these reagents results only in inhibition if inosine triphosphate or guanosine triphosphate are the substrates or if ethylenediaminetetraacetate (EDTA) is used as the activator with ATP (3-6). It has become clear, also, that this protein is built up from physically and chemically identical subunits or monomers with a molecular weight of approximately 200,000 (7-9). Although participation of some low molecular weight component in the structure is not excluded, it appears that the basic form of the molecule is a three-stranded rope built up from these highly helical subunits (7). With the development of the radioactive labeling and “fingerprint” technique employed in demonstrating that the subunits are probably of identical chemical structure (9)) it became possible to approach the problem of the structural segments involved in these sulfhydryl-binding effects. Early realization that the reaction of N-ethylmaleimide with myosin -SH was quite selective in inhibition of the EDTA-activated adenosine triphosphatase (3) encouraged us to utilize partial labeling with W-N-ethylmaleimide in an effort to locate that portion of the structure containing the cysteine residue or residues involved in loss of the EDTA activation, with the hope that eventually the structure of the active site or sites might be identified. The present report demonstrates that the sensitivity of the EDTAactivated adenosine triphosphatase is associated with the masking of one of two SH groups present in one of the peptides resulting from exhaustive trypsin digestion of myosin.

Gel electrophoresis of myosin in concentrated urea solutions demonstrates that the dissociated chains of myosin migrate as a monodisperse electrochemical species. At lower urea concentrations a complex dissociation-association system is obtained. Sedimentation, viscosity, optical rotation and gel electrophoresis studies under the latter conditions indicate the equilibrium nature of the system.

1.The molecular weight of myosin has been determined in 0.5 M KCl and 5 M guanidine · HCl.2.1. The molecular weight, using the Archibald approach to equilibrium method is strongly dependent on protein concentration in both solvents giving infinite dilution values of 6.19·105 in 0.5 M KCl and 2.06·105 in 5 M guanidine·HCl.3.2. Sedimentation, diffusion and viscosity studies on myosin in 5 M guanidine·HCl provide estimates of the molecular weight in reasonable agreement with the Archibald studies.4.3. Preferential binding of guanidine·HCl by myosin has been determined and shown to be of the order of 5% or less.5.4. Utilizing these molecular weight determinations and available X-ray diffraction, light scattering and optical rotatory data, a model for the myosin molecule is proposed, based on three equal weight polypeptide chains in the form of a three-stranded α rope.