acto-HMM ATPase Research Articles

Effects of the N-terminal Domains of Myosin Binding Protein-C in an in Vitro Motility Assay

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.

Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.

Methanol traps the troponin-tropomyosin-actin complex in an "off-state".

We have investigated the effect of methanol at concentrations below 15% (v/v) on acto-heavy meromyosin (HMM)- and tropomyosin (Tm)-troponin (Tn)-acto-heavy meromyosin-ATPase activities. Methanol slightly enhanced ATPase activity of acto-HMM alone. It had no apparent effect on normalized Tm-Tn-acto-HMM-ATPase activity in the absence of Ca2+. In the presence of Ca2+, however, methanol markedly inhibited normalized Tm-Tn-acto-HMM ATPase activity. These results show that methanol affects the troponin-tropomyosin regulation of acto-HMM ATPase: methanol suppresses the Ca(2+)-sensitivity of the regulatory system and traps it in an "off-state."

Requirement of the two-headed structure for the phosphorylation dependent regulation of smooth muscle myosin

It is known for smooth muscle myosin that while acto-HMM ATPase activity is regulated by phosphorylation, acto-S-1 ATPase activity is not regulated. To clarify the heavy chain structure required for the regulation, smooth muscle myosin containing 7 different lengths of the S-2 portion were expressed in Sf9 insect cells using Baculovirus expression system. Myosin containing longer than 991 residues of heavy chain formed a stable two-headed structure while myosin with shorter than 944 residues of heavy chain formed a single-headed structure, indicating that the residues Gln 945-Asp 991 are critical for the fomation of the two-headed structure. The actin activated ATPase activity of myosin mutants having a two-headed structure was activated by phosphorylation while that of myosin mutants that failed to form the two-headed structure was completely independent of phosphorylation. These results suggest that the two-headed structure is critical for the phosphorylation-dependent regulation.

Identification of the sequence of the regulatory light chain required for the phosphorylation-dependent regulation of actomyosin.

The amino acid structure of regulatory light chain which is essential to express the phosphorylation-mediated regulation of smooth muscle actomyosin ATPase was studied. Regulatory light chain of smooth muscle heavy meromyosin (HMM) was truncated by either lysylendopeptidase or trypsin. Lysylendopeptidase cleaved the regulatory light chain initially at the C-terminal side of lysine 6 (Lys C(1)-HMM) and subsequently at the C-terminal side of lysine 12 (Lys C(2)-HMM). On the other hand, trypsin cleaved at the C-terminal side of arginine 16 (tryp-HMM). While the actin activated ATPase activity of Lys C(1)-HMM and Lys C(2)-HMM was markedly activated by phosphorylation, that of tryp-HMM was not activated by phosphorylation. The exchange of cleaved regulatory light chain of tryp-HMM with undigested regulatory light chain restored the phosphorylation-mediated regulation on the actin activated ATPase activity. The regulatory light chain of the undigested HMM was also exchanged with the trypsin-digested regulatory light chain and this abolished the phosphorylation dependence of acto-HMM ATPase activity. These results show that the amino acid sequence arginine 13-arginine 16 is essential to express the regulation of actin activated ATPase of smooth muscle myosin which is mediated by the phosphorylation at serine 19 of the regulatory light chain.

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin.

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.

Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2

A monoclonal antibody against subfragment 2 (S-2) of smooth muscle myosin, designated MM-9, was generated and characterized. MM-9 potently inhibited subfragment 1 (S-1) release by papain proteolysis of myosin, suggesting that the epitope of MM-9 is at or very close to the S-1/S-2 junction. The depression of Ca 2+ - and Mg 2+-ATPase activities of myosin at low ionic strength was significantly reduced by MM-9. MM-9 increased the acto dephosphorylated HMM ATPase activity about 3-fold. On the other hand, the antibody had no effect on the KCl-dependence of viscosity of monomeric myosin. These results suggest that the folding of the myosin rod is not the direct determinant of enzymatic activity, and that the subtle conformational change at the S-1/S-2 junction (head-neck region) plays a critical role in determining enzymatic activities.

Thermal Activation of Actomyosin Mg2+‐ATPases from Flying Fish and Black Marlin Muscles

ABSTRACTThe effects of heat treatment (25°, 30°, 35°, 37°, and 40°C) on acto‐myosin Mg2+‐ATPase activity from flying fish and black marlin were studied with respect to setting of their meat pastes. The setting ability of flying fish had very high values but black marlin was low. Flying fish actomyosin Mg2+ATPase was activated more than three times by heating at 35°C, while the activation of black marlin was about two‐thirds at 37°C. For acto‐HMM ATPase, however, no appreciable difference in activation was observed between flying fish and black marlin. For both fishes, the acto‐S‐1 ATPase activities were not enhanced by the heat treatment. A possible role of myosin fragments in this activation is discussed.

Catalytic consequences of oligomeric organization: kinetic evidence for "tethered" acto-heavy meromyosin at low ATP concentrations.

The influence of the supramolecular organization of myosin on its ATPase activity was investigated at a range of ATP concentrations, using as a model system subfragment 1 (S1) and heavy meromyosin (HMM), which are respectively monomeric and dimeric proteolytic fragments of myosin. At low ATP levels in the presence of a molar excess of actin, dimeric HMM showed an increased rate of ATP hydrolysis relative to that for monomeric S1. This increased ATPase for HMM was inhibited by high concentrations of ATP, which reduced the acto-HMM ATPase rate to the lower level of acto-S1. This observation is consistent with the rapid ATP hydrolysis of acto-HMM at low ATP being due to rapid product release from a "tethered" acto-HMM species, which has product bound to one head group while the other head group remains bound to actin. At high concentrations of ATP, ATP binds to both head groups, resulting in net dissociation of HMM from actin. This model is supported by 18O exchange data. Acto-HMM hydrolyzed ATP with extensive exchange of water oxygens into Pi at high ATP levels, but not at low ATP levels. Acto-S1 exhibited extensive exchange at both high and low ATP levels. This result is consistent with rapid product release from a tethered acto-HMM intermediate at low ATP.

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.

Properties of tropomyosin from the dual-regulated obliquely striated body wall muscle of the earthworm (Lumbricus terrestris L.).

The obliquely striated body wall muscle of the earthworm Lumbricus terrestris L. possesses a dual actin-linked and myosin-linked regulatory system. Tropomyosin from this muscle has now been purified and its functional properties compared to tropomyosin from vertebrate skeletal muscle. Earthworm tropomyosin has a molecular weight of about 70 000 and is composed of two polypeptide chains of molecular weight of 34 000 and 37 000. Structural and functional similarities to skeletal muscle tropomyosin were demonstrated with respect to the formation and periodicity of paracrystals and nets and the potentiation of skeletal muscle acto-SF1 ATPase activity at low ATP concentration. Likewise, earthworm tropomyosin inhibited skeletal muscle acto-HMM ATPase activity at normal ATP concentrations but to a much greater extent than skeletal muscle tropomyosin; this inhibition was removed by skeletal muscle troponin, in the presence of Ca2+. In a system containing earthworm myosin and skeletal muscle actin, earthworm tropomyosin had no detectable influence on the actin-activated ATPase activity. It is concluded that earthworm tropomyosin plays an active role in the actin-linked troponin-dependent regulatory system and has no measurable effect on the regulation via myosin.

Comments on Bishop and Gray's criticism of the streaming driven by acto-heavy meromyosin.

The recent communication by Bishop and Gray (J. Biochem. 90, (1981)) on the streaming in the circular slit of stream cells is probably based on observations under conditions where no active streaming can be produced because of a substantial lack of F-actin. According to our experience, these conditions are similar to those obtained when actins are partly denaturated. Hence, for the sake of their criticism, detailed comparisons should be added of the stability of streaming, the distribution of streaming velocities as well as their sizes, acto-HMM ATPase activities under various streaming velocities and the amount of proteins fixed to the Millipore filter.

Myosin ATPase and acto-heavy meromyosin ATPase in normal and in pale,soft and exudative (PSE) porcine muscle.

An investigation was conducted on myosin and actin-activated heavy meromyosin(HMM) ATPase activities in normal porcine muscle stored for varying periods of time after death. Studies were also made on temperature dependent myosin ATPase, initial burst of ATPase and actinactivated HMM ATPase in normal and in pale, soft and exudative (PSE) porcine muscle. The Maximum velocity of acto-HMM ATPase of normal muscle decreased considerably with postmortem time, while the apparent dissociation constant decreased slightly. The maximum velocity of acto-HMM ATPase of postmortem normal muscle was approximately two-times larger than that of the corresponding PSE muscle. However, almost no difference was found in the apparent dissociation constant. The size of the initial burst of phosphate-liberation of myosin prepared from normal muscle was approximately 1.2 mol/mol of myosin and from PSE muscle 0. It is assumed that the lack of contractility of PSE muscle was brought about by two basic myosin malfunctions: one, the irreversible binding of myosin to actin filament and the other, the functional damage of myosin ATPase, responsible for the formation of phosphorylated complex, even when dissociable.

Mechanism of the Mg2+- and Mn2+-ATPase reactions of acto-H-meromyosin and acto-subfragment-1 in the absence of KCl at room temperature: direct decomposition of the complex of myosin-P-ADP with f-actin.

The extent of binding of myosin heads with F-actin was estimated by a light-scattering and an ultracentrifugal separation method, using well-homogenized F-actin. The extent of binding of HMM or S-1 with F-actin during the ATPase reaction was estimated in 2 mM K-PEP, 1 mM MgCl2 and 1 mM MgCl2 K-P1 at pH 7.4 and compared with the rate of the acto-HMM ATPase or acto-S-1 ATPase [EC 3.6.1.3] reaction in the steady state. The apparent first- order rate constant for recombination of the HMM-P-ADP complex, HMMPADP, or the S-l P-ADP complex, S-1PADP, with F-actin was also determined. At 20°C, the extent of binding (1—α) increased with increase in the F-actin concentration and approached 1.0 at a sufficiently high concentration of F-actin. The steady-state rate constant of the F-actin-dependent ATPase reaction, Δvo, was proportional to the value of (l-α). On the other hand, the apparent first-order rate constant for recombination of HMMPADP or S-1PADP with F-actin, vrecomb, was found to be 1/5–1/13 of Δvo. Therefore, we concluded that the main route of ATP hydrolysis at low ionic strength at 20°C is the one via direct decomposition of acto-MPADP and that the ATP hydrolysis cycle does not involve the dissociation step of actomyosin. This conclusion was supported by our finding that a high ATPase activity was observed immediately after adding ATP to acto-HMM, while a high ATPase rate in the steady state was observed after a lag phase required for binding of HMMPADP with F-actin, when the acto-HMM ATPase reaction was started by adding F-actin to a solution containing HMM and ATP. At 12°C, the rate constant of the acto-HMM ATPase reaction, Δvo was not proportional to the extent of binding of HMM with F-actin, 1-α, and Δvo was given by the equation Δ=(1-α)Δvo+αvrecomb;, where ΔVo is the value at sufficiently high concentrations of F-actin. The rate constant of the acto-HMM Mn2+ reaction at low ionic strength at 25°C was also accounted for by the above equation. The results obtained in this study, together with those described in one of the preceding papers (Ikebe, M., Inoue, A., & Tonomura, Y. (1980) J. Biochem. 88, 1653-1662), clearly demonstrate that in the actomyosin ATPase reaction, ATP is hydrolyzed via two routes: one via direct decomposition of acto-MPADP without dissociation of actomyosin and the othervia the route proposed by Lymn and Taylor (Lymn, R.W. & Taylor, E.W. (1971) Biochemistry 10, 4617-4624). The rate of the latter route is limited by the steps for recombination of MPADP with F-actin. The F-actin-concentration dependence of the apparent first-order rate constant for recombination of HMMPADP with F-actin could be accounted for by the Michaelis-Menten equation. Therefore, HMMPADP produced by the reaction of HMM with ATP may exist largely in a refractory state and may recombine with F-actin only after transformation into a nonrefractory state.

Reaction mechanism of Mn2+-ATPase of acto-H-meromyosin in 0.1 M KCl at 5 degrees C: evidence for the Lymn-Taylor mechanism.

The rate constants of a series of elementary steps in the H-meromyosin (HMM) Mn2+-ATPase [EC 3.6.1.3] and acto-HMM Mn2+-ATPase reactions were determined in 0.1 M KCl at 5 degrees C. We found that the rate-limiting step in the HMM Mn2+-ATPase reaction was the liberation of ADP from HMM.ADP, of which the rate constant was estimated to be 0.17 s-1. All the results obtained with the acto-HMM Mn2+-ATPase reaction could be quantitatively explained by a modified Lymn-Taylor mechanism (see Fig. 15). The second-order rate constant for the dissociation of acto-HMM induced by ATP was 3.0 X 10(5) M-1.s-1, and that for the Pi burst in the acto-HMM ATPase reaction was 1.7 X 10(5) M-1.s-1. The second-order rate constant for the binding of HMM.ADP with F-actin was 0.25 s-1.mg-1.ml, and the rate-limiting step in the acto-HMM Mn2+-ATPase reaction was the conversion of HMMPADP into HMM.ADP plus Pi, when the F-actin concentration was high.

Fluorometric method for estimating the kinetic parameters of beta-naphthyl triphosphate and ATP hydrolysis by acto-heavy meromyosin.

We have established a method to estimate the values of various kinetic parameters of acto-heavy meromyosin (acto-HMM) ATPase, using a fluorescent ATP analog, beta-naphthyl triphosphate (beta-NapP3); from the fluorescence intensity change accompanying beta-NapP3 hydrolysis, the various kinetic parameters of beta-NapP3 hydrolysis, including its product inhibition, were obtained. beta-NapPd3 hydrolysis is inhibited competitively by ATP, resulting in different time courses of fluorescence intensity change in the presence and absence of ATP. From this difference, the values of kinetic parameters of ATP hydrolysis, including its product inhibition, can be estimated. By extending this method to the acto-HMM system, seventeen parameters in a reaction scheme for the concurrent hydrolysis of ATP and beta-NapP3, including association constants between F-actin and substrate-free or substrate-bound HMM, were obtained. The kinetic-parameters estimated for ATP hjydrolsis were in good agreement with those in the literature.

Studies of the chemo-mechanical conversion in artificially produced streamings. I. Reconstruction of a chemo-mechanical system from acto-HMM of rabbit skeletal muscle.

Steady and uniform streamings (SUS) of HMM solutions were set up in the presence of Mg-ATP in a circular slit, on both side-walls of which a Millipore filter was fixed; F-actin filaments from rabbit skeletal muscle were bound onto the Millipore filter by cyanogen bromide in the flow. The direction of the SUS was specificially determined by that of the flow during the fixing of F-actin and was independent of the direction of the initial velocity applied externally to the HMM solutions. The SUS continued for about 90 min with a velocity of about 20 mum/s at 20 degrees C. There was a strong correlation between the acto-HMM ATPase activity and the velocity of SUS when the salt concentration was varied. Moreover, this was also the case when the ATPase activity was controlled by Ca2+, when native tropomyosin was bound to F-actin in the circular slit. Careful examination led to the conclusions that F-actin filaments are fixed on the Millipore filter with a specific polarity and that a chemo-mechanical system had been successfully reconstituted in our "stream cells," in which chemical energy from ATP is converted to the mechanical energy of streaming.

Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin.

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.

Correlation between the inhibition of the acto-heavy meromyosin ATPase and the binding of tropomyosin to F-actin: effects of Mg2+, KCl, troponin I, and troponin C.

When stoichiometric amounts of tropomyosin (TM) are bound to F-actin in the presence of 2 mM ATP, the MG2+-activated acto-heavy meromyosin (HMM) ATPase is inhibited by about 60% in 5 mM MgCl2-30 mM KCl. If the concentration of MgCl2 is reduced to 1 mM, the inhibition disappears because TM no longer binds to F-actin. Increasing the concentration of KCl to 100 mM restores both the binding and the inhibition. Thus, the binding of TM alone to F-actin causes significant inhibition of the ATPase provided that the HMM is saturated with ATP. (When the HMM is not saturated, TM activates the ATPase). When TM alone can bind stoichiometrically to F-actin, addition of troponin I (TN-I) increases the inhibition from 60% to about 85%, but the TM binding to F-actin is not affected. Under conditions such that TM alone neither inhibits the acto-HMM ATPase nor binds to F-actin, the inhibition caused by TN-I plus TM still approaches 100%. Direct binding studies under these conditions show that TN-I induces binding between TM and F-actin. A dual role for TN-I is proposed: first, TN-I can induce TM to bind to F-actin, causing inhibition of the ATPase; and second, TN-I can itself enhance the inhibition of the ATPase in a cooperative manner. The addition of TN-C in the absence of CA2+ has only a limited effect on the first role, but seems to be able to block completely the cooperative inhibition caused by TN-I such that the residual inhibition is a function only of the TM which remains bound.