The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins
2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês
10.1074/jbc.m608191200
ISSN1083-351X
AutoresBogdan Iorga, Nancy Adamek, Michael A. Geeves,
Tópico(s)Cardiovascular Effects of Exercise
ResumoFast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the β-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to ∼1000 s–1, a very similar rate constant to MHC-2, but 10–15% of the complex must go through a slow isomerization (∼20 s–1) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin·ADP and provide evidence of three closely related actin-myosin·ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (∼20 s–1), and a third complex that releases ADP too slowly (∼6 s–1) to be on the main ATPase pathway. The role of these actin-myosin·ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release. Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the β-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to ∼1000 s–1, a very similar rate constant to MHC-2, but 10–15% of the complex must go through a slow isomerization (∼20 s–1) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin·ADP and provide evidence of three closely related actin-myosin·ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (∼20 s–1), and a third complex that releases ADP too slowly (∼6 s–1) to be on the main ATPase pathway. The role of these actin-myosin·ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release. Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes (1Cope M.J. Whisstock J. Rayment I. Kendrick-Jones J. Structure. 1996; 4: 969-987Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 2Sellers J.R. Goodson H.V. Wang F. J. Muscle Res. Cell Motil. 1996; 17: 7-22Crossref PubMed Scopus (66) Google Scholar, 3Sellers J.R. Sheterline P. Myosins. 2nd Ed. Oxford University Press, London1999Google Scholar, 4Sellers J.R. Biochim. Biophys. Acta. 2000; 1496: 3-22Crossref PubMed Scopus (618) Google Scholar, 5De La Cruz E.M. Ostap E.M. Curr. Opin. Cell Biol. 2004; 16: 61-67Crossref PubMed Scopus (220) Google Scholar, 6Goodson H.V. Dawson S.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3498-3499Crossref PubMed Scopus (14) Google Scholar). During the myosin ATPase cycle, the myosin motor domain (or cross-bridge) undergoes a series of conformational changes coupled to the binding of nucleotide and actin, which results in a translocation of the myosin cargo domain relative to the actin track (7Geeves M.A. Holmes K.C. Adv. Protein Chem. 2005; 71: 161-193Crossref PubMed Scopus (303) Google Scholar). The sequence of molecular events in the actin-myosin cross-bridge cycle appears essentially the same for all myosins so far studied. The different mechanochemical properties of each myosin are attributed to a modulation of the rates and equilibrium constants of individual molecular events to match each myosin to its physiological role. The most widely studied myosins are the vertebrate, striated-muscle myosins, which define the class II myosins. This class of myosins have a dimerization domain that forms a long coiled-coil and which can assemble further to form the backbone of the bipolar thick myosin filament. Of the class II myosin, the myosin heavy chain 2 (MHC-2) 4The abbreviations used are: MHC-2, myosin II heavy chain isoform 2; solS1, rabbit soleus myosin subfragment 1; psoS1, rabbit psoas myosin subfragment 1; MHC-1, myosin II heavy chain isoform 1; MOPS, 4-morpholinepropanesulfonic acid. isoform found primarily in white, anaerobic, fast-contracting, skeletal muscle has been most thoroughly described at both the molecular and physiological levels. In adult, mammalian, muscle tissue the MHC-2 is found as various isoforms (e.g. 2a, 2b, 2x) and it has been established that the essential mechanical properties of the muscle fiber contraction (e.g. maximum velocity of shortening, force per cross-bridge) are properties of the MHC present in the tissue, the light chain isoforms play a minor modulatory role (8Bottinelli R. Pflugers Arch. 2001; 443: 6-17Crossref PubMed Scopus (167) Google Scholar, 9Schiaffino S. Reggiani C. Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1271) Google Scholar). The larger subclass of myosin II motors includes slow, cardiac, and smooth muscle isoforms, as well as developmental isoforms and non-muscle myosin II. Non-muscle isoforms are found throughout eukaryotic organisms from vertebrates to single cell organisms such as yeast, Acanthamoeba and Dictyostelium. One of the outstanding issues of the myosin family of motors is how the closely related myosin isoforms express such a wide range of physiological properties. We recently undertook a study of mammalian striated muscle myosin isoforms (10Weiss S. Rossi R. Pellegrino M.A. Bottinelli R. Geeves M.A. J. Biol. Chem. 2001; 276: 45902-45908Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Nyitrai M. Rossi R. Adamek N. Pellegrino M.A. Bottinelli R. Geeves M.A. J. Mol. Biol. 2006; 355: 432-442Crossref PubMed Scopus (97) Google Scholar) and concluded that the maximum velocity of muscle fiber contraction was limited by the rate of cross-bridge detachment from the actin filament at the end of cross-bridge ATPase cycle. This was in agreement with earlier studies of a wider spectrum of myosin II isoforms (12Siemankowski R.F. Wiseman M.O. White H.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 658-662Crossref PubMed Scopus (319) Google Scholar). For the fastest muscle myosins of the MHC-2 group, cross-bridge detachment was limited by the rate of ATP binding to the cross-bridge and inducing actin detachment. This was clearest at 12 °C where both the rate of ATP binding to actin-S1 in vitro and the muscle fiber shortening velocity could be well defined. This was not true for the MHC-1 group of myosin isoforms which predominate in slow-skeletal, aerobic, muscle fibers. This group of myosin isoforms is also known as the β isoform when expressed from the same gene in cardiac tissue. For these MHC-1 isoforms, we concluded that ADP release, which occurs before ATP binding, may well limit the velocity of muscle contraction because the ATP-induced dissociation of actin from myosin is much too fast. Thus, the earlier conclusion of Siemankowski et al. (12Siemankowski R.F. Wiseman M.O. White H.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 658-662Crossref PubMed Scopus (319) Google Scholar) that ADP release defines the velocity of muscle contraction probably remains true for all but the fastest muscle myosins. Our initial studies of myosin and heavy-mero-myosin (HMM) isolated from slow muscle fibers indicated that ADP release was a complex process and, therefore, we thought it is timely to investigate more thoroughly the solution biochemical kinetic properties of a slow muscle myosin isoform. The results of this work for the MHC-1 isoform prepared from rabbit soleus muscle are reported here. While this work was underway, the optical trap studies of Capitanio et al. (13Capitanio M. Canepari M. Cacciafesta P. Lombardi V. Cicchi R. Maffei M. Pavone F.S. Bottinelli R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 87-92Crossref PubMed Scopus (141) Google Scholar) were published. This work demonstrated for the first time that, with a high time resolution optical trap, the mechanical events of myosin II isoforms from fast and slow muscle fibers can be resolved into two distinct steps, as had previously been reported for smooth muscle myosin II and some non-muscle myosins (14Veigel C. Coluccio L.M. Jontes J.D. Sparrow J.C. Milligan R.A. Molloy J.E. Nature. 1999; 398: 530-533Crossref PubMed Scopus (264) Google Scholar, 15Veigel C. Molloy J.E. Schmitz S. Kendrick-Jones J. Nat. Cell Biol. 2003; 5: 980-986Crossref PubMed Scopus (268) Google Scholar, 16Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Crossref PubMed Scopus (333) Google Scholar). These two steps have been interpreted as a force-generating power stroke that is coupled to Pi release from the cross-bridge followed by a second, smaller mechanical event associated with ADP release. This second step has been described as either an additional force generating event or as a strain sensing mechanism (17Barsoti R.J. Dantzig J.A. Goldman Y.E. Nat. Struct. Biol. 1996; 3: 737-739Crossref PubMed Scopus (12) Google Scholar, 18Whittaker M. Wilson-Kubalek E.M. Smith J.E. Faust L. Milligan R.A. Sweeney H.L. Nature. 1995; 378: 748-751Crossref PubMed Scopus (340) Google Scholar, 19Nyitrai M. Geeves M.A. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2004; 359: 1867-1877Crossref PubMed Scopus (146) Google Scholar). Because this second event is associated with ADP release, the mechanism of ADP release is of particular interest for these muscle myosins. The in vitro expression of defined skeletal muscle myosin isoforms has not yet been achieved and therefore we have used tissue purified myosin. In rabbit, there are four major adult, skeletal muscle fiber types with the corresponding MHC isoforms: type 1 or slow and three fast types, 2a, 2b and 2x or 2d, respectively (20Schiaffino S. Gorza L. Sartore S. Saggin L. Ausoni S. Vianello M. Gundersen K. Lomo T. J. Muscle Res. Cell Motil. 1989; 10: 197-205Crossref PubMed Scopus (775) Google Scholar). Most mammalian muscle expresses a mixture of myosin isoforms. However, rabbit psoas muscle contains only the fast isoforms MHC-2x (∼92%) and MHC-2b (∼8%), whereas rabbit soleus muscle contains almost exclusively MHC-1 (∼97%) with some MHC-2a (∼3%) (21Tikunov B.A. Sweeney H.L. Rome L.C. J. Appl. Physiol. 2001; 90: 1927-1935Crossref PubMed Scopus (56) Google Scholar). Myosin S1 prepared from rabbit soleus muscle (solS1) is used here and compared with myosin S1 prepared from rabbit psoas muscle (psoS1). We show that the nucleotide binding properties of solS1 are quite distinct from those of psoS1 and, in terms of ADP release from actin-solS1, the soleus protein has much in common with the smooth muscle and slow tension-sensing non-muscle myosins. This suggests that the coupling of biochemical and mechanical properties of myosins shows a broad spectrum of behaviors with the fast muscle myosin being at one extreme end of the spectrum and with slow and smooth muscle myosins isoforms showing properties similar to many other myosins. Preparation of Psoas and Soleus Myosin Subfragment 1 (solS1 and psoS1)—Myosin was extracted from bulk rabbit psoas and soleus muscle using the method of Margossian and Lowey (22Margossian S.S. Lowey S. Methods Enzymol. 1982; 85: 55-71Crossref PubMed Scopus (825) Google Scholar) and digested with α-chymotrypsin to produce S1 (23Weeds A.G. Taylor R.S. Nature (London). 1975; 257: 54-56Crossref PubMed Scopus (931) Google Scholar). The term S1 will be used as a generic name and the specific S1 prepared from rabbit soleus and psoas referred to as solS1 and psoS1. The S1 was purified by anion exchange chromatography on a Sepharose FF column and then freeze-dried from 5 mm potassium phosphate buffer with 1% sucrose and stored at –80 °C. The protein was characterized by SDS-PAGE and a scanned image of a gel is shown in Fig. 1. This shows the purified soleus myosin and solS1 along with psoS1 for comparison. Soleus myosin shows the presence of two light chains (MLC) the regulatory LC2s and the essential LC1s. As expected, both S1 preparations have only the essential light chain; the regulatory light chain is lost during digestion by chymotrypsin. The psoS1 contains two isoforms of the essential LC, LC1f, and LC3f. Column purification separated the two isoforms and the LC1f isoform is used in the studies presented here. For experimental use, the S1 was dissolved and dialysed in the desired experimental buffer to remove phosphate and sucrose. The concentration of S1 was determined from the absorbance at 280 nm (ϵ1% = 0.79 cm–1, MW = 115 kDa), using a Varian Cary 50 Bio UV spectrophotometer. The S1 solution was stable at 4 °C for up to 2 weeks. Preparation of Actin—Actin was prepared from rabbit skeletal muscle as described by Spudich and Watt (24Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). For the stopped-flow experiments, the actin was selectively labeled with pyrene at cysteine 374 (pyr·actin) as described by Criddle et al. (25Criddle A.H. Geeves M.A. Jeffries T. Biochem. J. 1985; 232: 343-349Crossref PubMed Scopus (180) Google Scholar) (the average labeling content was ∼90%). In experiments using F-actin at concentrations below 1 μm, the F-actin was stabilized by incubating a stock solution at 10 μm with equimolar concentrations of phalloidin for at least 4 h. Experimental Conditions—All kinetic experiments were performed either in a low salt (30 mm KCl) or high salt (100 mm KCl) buffer at pH 7.0 containing 5 mm MgCl2, 1 mm NaN3, and either 20 mm MOPS (at a fixed temperature) or 20 mm cacodylate for experiments varying the temperatures. Stopped-flow Experiments—Rapid kinetic stopped flow experiments were carried out on a stopped-flow system (model SF-61 DX2, Hi-Tech Ltd., Salisbury, UK) fitted with a 75 watt Xe/Hg lamp and monochromator. Pyrene was excited at 365 nm and emission was monitored at 389 nm (KV389 cut-off filter). Intrinsic protein fluorescence was excited at 295 nm, and emission was monitored at 320 nm (WG320 cut-off filter). Unless otherwise stated, the concentrations used to describe the experimental conditions in this article refer to the final concentrations in the stopped-flow reaction mixture (after mixing in the ratio 1:1). Typically, working volumes of 700 μl were used to obtain one data set. All of the transients shown are the average of 3–10 shots of the stopped-flow apparatus, and the best fit to a single, A(1 – exp{kobst}), or double exponential function, Afast(1 – exp{–kfastt}) + Aslow(1 – exp{–kslowt}), is shown superimposed. Data were stored and analyzed using KinetAsyst software provided by Hi-Tech Scientific. The standard error of the exponential fits is typically less than 1% of the values given unless otherwise stated. This grossly over estimates the precision of the measurement. Each measurement was repeated 2–3 times on different preparations of S1 and actin. All values of measured exponentials are considered to be defined to no better than 20% unless specific controls are described. Kinetic Data Analysis—The kinetic scheme for the interaction of myosin S1 (M) with ATP (T), ADP (D) and phosphate (Pi) is shown in Scheme 1, the ATPase pathway initially proposed by Bagshaw and Trentham (26Bagshaw C.R. Trentham D.R. Biochem. J. 1974; 141: 331-349Crossref PubMed Scopus (335) Google Scholar). In Scheme 1, asterisks represent different protein conformations as detected by intrinsic protein fluorescence and the equilibrium constants are defined as: Ki = k+i/k–i. We investigated the ATP-induced actin-S1 dissociation, the inhibition of the ATP reaction by ADP and have interpreted the data in terms of models described in Scheme 2. This is based on the scheme originally proposed by Geeves, Perrault, and Coluccio (27Geeves M.A. Perreault-Micale C. Coluccio L.M. J. Biol. Chem. 2000; 275: 21624-21630Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) for non-muscle myosin class I. In Scheme 2, the equilibrium constants are defined in the direction from A·M′ and A·M′·D toward M·T. Thus, KAD is a dissociation constant and K1 is an association constant. A very rapid equilibrium is reached between A·M and ATP after mixing actin-S1 with ATP, defined by the association constant K1 and followed by isomerization of the ternary complex (A·M·T), which limits the maximum rate of actin dissociation from the complex (k+2). Thus, the observed rate constant for the ATP-induced actin-S1 dissociation is defined by Equation 1. kobs=k+2K1[ATP]1+K1[ATP](Eq. 1) For some myosins, an additional slow time course component is seen with a rate constant defined by k+α and the relative amplitudes of the fast and slow phases defined by Equation 2. AfastAslow=[A·Mopen][A·Mclosed]≡Kα(Eq. 2) For psoS1, if the closed form exists, the term Kα ≫ 1 and only a single phase is observed. The equilibrium constant KαD for psoS1 is also ≫ 1 and the binding of ADP is a rapid equilibration event controlled by KAD. Under these conditions, ADP and ATP compete effectively for A·M binding site and the kobs, for a fixed ATP concentration, is given by Equation 3, kobs=ko1+[ADP]KAD(Eq. 3) where ko is the observed rate constant of ATP-induced actin-S1 dissociation in the absence of ADP and defined by Equation 1. KAD represents the affinity of actin-S1 complex for ADP, and it is expressed as dissociation constant: KAD = k+AD/k–AD. For solS1, the ADP binding is not in rapid equilibrium and the two terms Kα and KαD are small, where Kα = k+α/k–α and KαD = k+αD/k–αD. Therefore, a different approach was required to define the interaction of ADP and ATP with soleus actin-myosin. The appropriate method and equations are introduced in the text. ATP-induced Actin-S1 Dissociation—ATP-induced dissociation of actin-myosin subfragment 1 complex can be conveniently followed by monitoring the increase in fluorescence of pyrene-labeled actin (pyr·actin). Fig. 2A shows the fluorescence transients observed at 15 °C when 0.5 μm actin-S1 is mixed with 20 μm ATP in the stopped-flow fluorimeter. The observed transient for psoS1 was well described by a single exponential equation, as has been previously reported (28Millar N.C. Geeves M.A. FEBS Lett. 1983; 160: 141-148Crossref PubMed Scopus (66) Google Scholar), with an observed rate constant (kobs) of 103 s–1 and amplitude of 77%. For solS1, the transient in Fig. 2A shows two components: a fast phase and a slow phase. The total amplitude was similar for soleus and psoas S1 (∼77%) with the ratio of fast to slow phase amplitude of 4:1 (see Equation 2), while the kobs values were 108 s–1 for the fast phase (kfast) and 22 s–1 for the slow phase (kslow). Note that in Fig. 2, the two transients are scaled to give the same amplitude for the fast phase as this shows the two components more clearly. At 15 °C, for both proteins the kobs values showed a hyperbolic dependence on increasing ATP concentration as can be seen in Fig. 2B. For psoS1, the hyperbola defined the K50% value (1/K1) of 185 μm and a kmax (k+2) value of 720 s–1 (see Scheme 2). A hyperbolic fit for the fast phase of the soleus data gave an equilibrium constant 1/K1 of 192 μm and a k+2 value of 980 s–1 (see Scheme 2). The slow phase was only clearly separable from the fast phase at ATP concentrations above 20 μm where it was independent of the ATP concentration with an observed kmax value of ∼35 s–1 and an amplitude of ∼15% of the total fluorescence signal. The ATP-induced dissociation experiment was performed with both soleus and psoas actin-S1 at several temperatures between 4 and 15 °C. At each temperature, the solS1 transients had two distinct phases when the ATP concentration was above 250 μm and the observed rate constant of the slow phase (kslow) was independent of ATP concentration. With psoS1, no clear evidence of a slow phase could be found at any temperature. The kobs values for psoS1 and those of the fast phase (kfast) for solS1 were plotted as a function of Mg·ATP concentration in Fig. 3, A and B, respectively, for each temperature used. At temperatures above 15 °C, the kobs values at high ATP concentrations became too fast to be measured with any precision and, therefore, the hyperbola was not well defined. Hyperbolic fits to each data set, below 15 °C, gave estimates of both k+2 and 1/K1. The maximum dissociation rate constant k+2 increased from ∼400–500 s–1 at 4 °C to ∼700–900 s–1 at 15 °C for both isoforms. In fact, the slow solS1 showed slightly higher values of k+2 than psoS1 at all temperatures studied. An Arrhenius plot of the data is shown in Fig. 3D and the derived activation parameters ΔH ‡ and ΔS‡ are listed in Table 1. The activation enthalpy (ΔH‡ ∼40 kJ mol–1) and activation entropy (ΔS‡ ∼ 200JK–1 mol–1) were positive and similar for both isoforms. The maximum observed rate constant of the slow phase (kslow) seen for solS1 showed a temperature dependence about half of that of k+2. kslow increased from ∼25 s–1 at 4 °C to ∼45 s–1 at 15 °C (see Fig. 5).TABLE 1Thermodynamic parameters of the ATP-induced actin-S1 dissociationpsoassoleussoleusK1K1ΔH° (kJ·mol-1)-1.9 ± 3.6-4.4 ± 1.5ΔS° (J·K·mol-1)37.0 ± 12.828.4 ± 5.2k+2k+2k+αk+D2k+D3k+6aMeasured at 100 mm KCl.ΔH‡ (kJ·mol-1)35.7 ± 7.244.1 ± 9.318.9 ± 3.172.8 ± 3.457.7 ± 4.7159 ± 2ΔS‡ (J·K·mol-1)178.7 ± 25.3209.9 ± 33.098.5 ± 10.8283.6 ± 11.7219.5 ± 16.3538 ± 8a Measured at 100 mm KCl. Open table in a new tab The equilibrium constant K1 for the formation of the collision complex A·M·T (see Scheme 2) was about the same (1/K1 ∼200 μm) for both psoS1 and solS1, and was almost independent of temperature as shown in the Van't Hoff plot of Fig. 3C. The derived thermodynamic parameters ΔH°, ΔS° (see Table 1) were small for both isoforms. The presence of the second phase of ATP-induced actin-S1 dissociation was specific to the solS1 and could originate from various sources: contamination by other slower myosins, contamination by damaged myosin, or the presence of ADP limiting the ATP access to the nucleotide site. Extensive treatment with apyrase did not alter the slow phase suggesting that ADP contamination was not cause of the observation. Elimination of the other two causes is not simple but the slow phase has been detected having the same relative magnitude for each rabbit soleus preparation that has been used. A similar phase was also seen in S1 prepared from the soleus of the rat or pig and S1 from bovine masseter muscle, all predominantly containing MHC-1 slow isoform. 5M. Bloemink, C. Reggiani, and M. A. Geeves, manuscript in preparation. The myosin in each of these muscles is ≥90% isoform 1, the rest being isoform 2a (29Aigner S. Gohlsch B. Hamalainen N. Staron R.S. Uber A. Wehrle U. Pette D. Eur. J. Biochem. 1993; 211: 367-372Crossref PubMed Scopus (120) Google Scholar, 30Janmot C. d'Albis A. FEBS Lett. 1994; 353: 13-15Crossref PubMed Scopus (39) Google Scholar). A contamination with the fast 2a isoform could not produce the observed slow phase. The presence of two conformations of the actin-myosin complex have been reported for non-muscle myosins: rat myo-1b and myo-1c and Dictyostelium myo-E (19Nyitrai M. Geeves M.A. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2004; 359: 1867-1877Crossref PubMed Scopus (146) Google Scholar, 27Geeves M.A. Perreault-Micale C. Coluccio L.M. J. Biol. Chem. 2000; 275: 21624-21630Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 31Batters C. Arthur C.P. Lin A. Porter J. Geeves M.A. Milligan R.A. Molloy J.E. Coluccio L.M. EMBO J. 2004; 23: 1433-1440Crossref PubMed Scopus (70) Google Scholar, 32Fujita-Becker S. Durrwang U. Erent M. Clark R.J. Geeves M.A. Manstein D.J. J. Biol. Chem. 2005; 280: 6064-6071Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In each case, these myosins dissociate from actin in two steps: a fast step from the conformer to which ATP can bind readily and a slow step from a conformer that must undergo a relatively slow isomerization before ATP can bind (Scheme 2; Equation 2). We will assume that this kinetic model (Scheme 2) applies to soleus myosin (MHC-1) and return to review the evidence on this point under "Discussion." Interaction of ADP with Psoas Actin-S1 (Actin-psoS1) and Soleus Actin-S1 (Actin-solS1)—One of the characteristics of slow muscle myosin isoforms compared with fast muscle isoforms is the tighter binding of ADP to actin-myosin (11Nyitrai M. Rossi R. Adamek N. Pellegrino M.A. Bottinelli R. Geeves M.A. J. Mol. Biol. 2006; 355: 432-442Crossref PubMed Scopus (97) Google Scholar, 12Siemankowski R.F. Wiseman M.O. White H.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 658-662Crossref PubMed Scopus (319) Google Scholar, 33Marston S.B. Taylor E.W. J. Mol. Biol. 1980; 139: 573-600Crossref PubMed Scopus (173) Google Scholar). If ADP is in rapid equilibrium with actin-S1 compared with the net rate constant of ATP-induced actin-S1 dissociation (k+AD + [ADP]k–AD ≫ K1k+2[ATP]), then ADP acts as a competitive inhibitor of the ATP-induced dissociation reaction as defined by Equation 2 (see Scheme 2). Plotting the kobs as a function of ADP concentration allows the actin-S1 affinity for ADP (KAD) to be estimated. The result of one such experiment is shown in Fig. 4. In this case, 0.5 μm pyr·actin-S1 was mixed with 20 μm ATP at 20 °C, and the dissociation reaction followed. At this low ATP concentration, the kobs values are relatively slow: 65.8 and 49.3 s–1 for psoas and soleus, respectively. In addition, the reaction appears as a single phase for both psoS1 and solS1 and simplifies the analysis. Premixing the ATP with various concentrations of ADP results in a marked slowing of the observed reaction as shown in Fig. 4. The data are well described by Equation 3 and a fit to this equation is shown superimposed and gives a value for KAD of 124 and 8.5 μm for psoas and soleus actin-S1, respectively. Thus, as expected, actin-solS1 binds ADP > 10-fold more tightly than muscle actin-psoS1 does. The above experiment assumes that ADP is in rapid equilibrium, with actin-S1 on the time scale of the measurement. This is well established for psoS1 under all conditions but is not true for solS1; in fact, the rate of ADP release is known to be relatively slow for myosin S1 prepared from slow or cardiac muscle myosin (33Marston S.B. Taylor E.W. J. Mol. Biol. 1980; 139: 573-600Crossref PubMed Scopus (173) Google Scholar, 34Siemankowski R.F. White H.D. J. Biol. Chem. 1984; 259: 5045-5053Abstract Full Text PDF PubMed Google Scholar). To measure the rate constant of ADP release, the actin-solS1 complex was saturated with ADP (50 μm) and then ADP displaced by addition of a large excess of ATP (5 mm). The observed transients were biphasic at all temperatures between 5 and 30 °C and were independent of ATP concentration above 1 mm. The observed transients at 5 and 30 °C are shown in Fig. 5A. The kobs values for the fast phase were 207 s–1 at 30 °C and 15.6 s–1 at 5 °C and for the slow phase were 30 and 4.5 s–1, respectively. The relative amplitudes of the fast and slow phases were ∼3:1 (i.e. KαD = 3) at 30 °C and 1:1.3 (i.e. KαD = 0.77) at 5 °C. Thus, both the rates and amplitudes show significant temperature dependence. The temperature dependence of the two kobs values (kfast and kslow) is shown in Fig. 5B and the activation parameters are given in Table 1. The fast phase is too slow to be the rate at which ATP dissociates any actin-solS1 free of ADP (Fig. 2D) and both phases are quite distinct from the slow phase observed in the ATP-induced dissociation reaction free of ADP. To make this clear, the temperature dependence of the kmax values for the slow phase of the ATP-induced actin-solS1 dissociation in the absence of ADP is also shown in Fig. 5B. The simplest interpretation of these results is that there are two actin-myosin·ADP conformations present (A·M·D and A·M′·D) and that lower temperature favors the more tightly bound ADP conformation: A·M′·D (see Scheme 2). At 5 °C and 30 mm KCl, the two states have a similar occupancy (KαD = 0.77) and the two phases (fast and slow) of the ADP displacement represent k+AD (= 15.6 s–1, ADP off rate constant) and k+αD (= 4.5 s–1, A·M·D isomerization rate constant), respectively. The overall dissociation constant of ADP for actin-S1 (KADP) is given by Equation 4. KADP=KADKαD1+KαD(Eq. 4) The equilibrium between the two forms of bound ADP (A·M·D and A·M′·D, Scheme 2) was also ionic strength dependent: at 100 mm KCl two phases were seen in the displacement of ADP by ATP from actin-solS1 but the phases were more difficult to fit to a unique set of parameters. The values are therefore less reliable than at 30 mm KCl. The value of KαD (at 100 mm KCl) was 1.25 at 12 °C and 3.3 at 20 °C, with k+AD and k+αD having values of 63 s–1 and 15 s–1 at 12 °C, and 100 s–1 and 15 s–1 at 20 °C, respectively. Under the conditions of the ADP affinity measurements used in Fig. 3 (100 mm KCl), the two values for ADP displacement (k+AD and k+αD) were 61 s–1 and 17.9 s–1, which ar
Referência(s)