Introduction 1 , 2 3 4 5 6 6 7 8 9 (1) Unlike MHC-II-2, the MHC-II-1 gave biphasic dissociation of actomyosin by ATP. (2) The tight binding of ADP to actomyosin was confirmed for MHC-II-I with at least three conformations of actomyosin–ADP present in equilibrium. (3) The rate constant for ADP release from the predominant actomyosin–ADP complex was the correct size to limit the shortening velocity of MHC-II-1 containing soleus muscle fibres. (4) MHC-II-1 has a significantly reduced rate constant for the ATP hydrolysis step. Discussion 8 , 10 BC k +3 k - −1 11,12 RS k +3 k -3 −1 9 RS 10 13 14 V 0 −1 V 0 −1 13 V 0 −1 13 , 15 V 0 10 , 11 K AD K D K AD K D BM −1 BC −1 et al 16 17 18 19 Results BM Materials and Methods Figure 1 BM Figure 2 BM F k obs −1 F −1 F 6 −1 −1 k obs K 0.5 k max −1 Figure 2 k obs −1 k obs −1 Figure 2 F k obs −1 F k obs −1 F k obs −1 F Figure 2 −1 −1 −1 BM K 0.5 k max K 1 k +2 Scheme 1 k +3 k -3 Scheme 1 20 , 21 BM Figure 3 k obs −1 BM Scheme 1 K 3 22 , 23 k obs k +3 k -3 −1 k obs −1 9 Table 1 k obs −1 Discussion k +2 k +3 k -3 K 1 K 1 k +2 k +3 k -3 BM F k obs −1 Figure 2 k +6 k -D k +6 −1 k +6 k -6 k max,ADP −1 K 7 K 0.5,ADP k -6 K 7 k +D k max,ADP K 0.5,ADP 6 -1 −1 Table 1 K D 6 K 7 PS K D Table 1 k +6 k -D BM Figure 4 BM k obs −1 −1 −1 BM −1 −1 BM Figure 4 −1 −1 −1 −1 BM Figure 4 A fast A medium A medium k obs A fast A medium A fast A medium K D Figure 2 K D K D 11 Nucleotide binding to MHC1 myosin S1 in the presence of actin BM Figure 5 Figure 5 BM k obs −1 F −1 F k obs −1 −1 Figure 5 k slow −1 −1 BM 13 BM k obs k +α −1 BM K α K α k +α k -α k +α −1 k -α −1 BM k obs E a −1 BM k obs Figure 6 (4) K AD 7 −1 −1 k -AD −1 K AD −1 BM k obs k obs versus Figure 6 K AD k obs BM k obs −1 F k obs −1 F Figure 7 Scheme 2 −1 −1 Scheme 2 k -ADP Scheme 2 k +αD −1 Figure 6 E a A fast A slow A f A s K AD K D BM K AD K D K DA K A BM BM k obs k obs −1 BM k obs −1 Figure 8 K A K DA K DA K A K AD K D PS Tables 1 and 2 PS k max −1 k max −1 A fast A slow K α k +α −1 k -α −1 Table 2 PS K AD PS k obs,f −1 k obs,s −1 A fast A slow K αD Table 2 Discussion BM BM BC 10 , 11 Tables 1 and 2 BM BC 12 k +3 k -3 Scheme 1 20 , 21 k +3 k - k +3 k -3 −1 k +2 BM k +3 k -3 −1 −1 9 −1 24 BM K D BC K D BC 11 BC 12 K D Table 1 +6 BM BC k +6 −1 −1 k- 6 K 7 6 −1 −1 6 −1 −1 K D K AD K D Table 1 k +3 k -3 k +6 k +6 −1 k +6 −1 k -6 K 7 K D Figure 2 Figure 4 Table 2 K DA K A K A K DA K AD K D K DA K A 25 , 26 Table 2 K 1 k +2 k +2 10 BC 27 26 K αD k -ADP k +αD −1 Table 2 Table 3 K αD k -ADP V 0 V 0 Table 3 V 0 −1 13 k min k min = V 0 d V 0 −1 d 8 k min −1 d −1 d Table 3 k -ADP −1 k min k min k -ADP 9 k +αD k min 28 et al 9 29–32 17–19 33 16 34–36 34 , 37 38 k detach = K 1 k + 2 [ ATP ] / ( 1 + K 1 [ ATP ] + [ ADP ] / K AD ) K AD k -ADP Table 3 −1 −1 K 1 k +2 Table 2 k detach −1 K AD In summary we have shown that the MHC-II-1 isoforms isolated from slow skeletal muscles from the cow and the pig are similar in general properties to MHC-II-1 isolated from bovine cardiac tissue and to the MHC-II-1 of the rabbit and these are quite distinct from the MHC-II-2 isoforms. Of significance is the slow ATP-hydrolysis rate, the low thermodynamic coupling between ADP and actin binding, the slow rates of ADP release (which are of the correct size to limit the maximum shortening velocity) and multiple actomyosin ADP complexes. Nucleotide access to and release from the binding site requires relatively slow isomerisations and these may be load-dependent. These properties are distinct from the fast muscle MHC-II-2 isoforms and similar to smooth and some non-muscle myosin isoforms, e.g. mammalian myosins 1b and 1c, and non-muscle myosin II. However, unlike the non-muscle isoforms, the MHC-II-1 isoforms have a low duty ratio and can produce relatively rapid movement in motility assays or in muscle fibres. These parameters suggest that slow muscle myosin isoforms may form a distinct functional group from the fast muscle isoforms and the slower group which we have called the strain-sensors; non-muscle myosins particularly a sub-set of class I myosins (myo1b and 1c) that appear to operate less as transport motors than as load-bearing and strain-sensing molecules. Slow muscle myosin, like fast muscle myosin, is required for movement but also has an additional role in efficient load bearing in postural muscles. Thus, the molecular properties of the MHC-II-1 motor are consistent with its physiological role. Materials and Methods Proteins 39 BM 40 BM BM i Figure 1 BM g PS BM PS BM PS 2 41 42 ATPase assay 2 A 340 −1 −1 2+ −1 −1 12 2+ BM −1 Transient kinetics 2 BM PS etal 43 k +i k -i K i k +i k -i Scheme 1 26 , 44 Scheme 2 K α k +α k -α K αD k +αD k -αD Tables 1 and 2 Scheme 2 (1) k obs , fast = K 1 k + 2 [ ATP ] / ( 1 + K 1 [ ATP ] ) k obs,slow k +α K α k +α k -α k -α (2) A fast / A slow = [ A . M ′ ] / [ A . M ] = K α sk pso K α K AD (3) (3) K AD = K ADP · K α D / ( 1 + K α D ) K ADP K αD K ADP k obs (4) (4) k obs = k 0 / ( 1 + [ ADP ] / K AD ) k 0 (1) via k +αD k +α k +αD k obs k +αD BM (5) Amp fast = Amp 0 / ( 1 + [ ADP ] / K AD ) + C fast 0 K AD C C (6) Amplitude = ( Δ F / F t ∞ ) × 100 F F t ∞ Quenched-flow experiments 2 BM g i BM