ATP analogs and muscle contraction: mechanics and kinetics of nucleoside triphosphate binding and hydrolysis.

The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin (Vf) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis. Whereas all NTPs studied support force production and stiffness that vary by a factor 2 or less, the unloaded shortening velocity (Vu) of muscle fibers varies by almost 10-fold. 2-Deoxy ATP (dATP) was unique in that Vu was 30% greater than with ATP. Parallel behavior was observed between Vf and the steady-state maximum actin-activated HMM ATPase rate. Further comparisons suggest that the variation in force correlates with the rate and equilibrium constant for NTP cleavage; the variations in Vu or Vf are related to the rate of cross-bridge dissociation caused by NTP binding or to the rate(s) of product release.     

Steady-state studies of the actin-activated adenosine triphosphatase activity of myosin.

Reconstituted actomyosin (ATP phosphohydrolase, EC 3.6.1.3) (0.400 mg F-actin/mg myosin) in 10.0 muM ATP loses 96% of its specific ATPase activity when its reaction concentration is decreased from 42.0 mug/ml down to 0.700 mug/ml. The loss of specific activity at the very low enzyme concentrations is prevented by the addition of more F-actin to 17.6 mug/ml. It is concluded that at low actomyosin concentrations the complex dissociates into free myosin with a very low specific ATPase activity and free F-actin with no ATPase. The dissociation of the essential low molecular weight subunits of myosin from the heavy chains at very low actomyosin concentrations may be a contributing factor. Actomyosin has its maximum specific activity at pH 7.8-8.2. The Km for ATP is 9.4 muM, which is at least 20-fold greater than myosin's Km for ATP. The actin-activated ATPase of myosin follows hyperbolic kinetics with varying F-actin concentrations. The Km values for F-actin are 0.110 muM (4.95 mug/ml) at pH 7.4 and 0.241 muM (10.8 mug/ml) at pH 7.8. The actin-activated maximum turnover numbers for myosin are 9.3 s-1 at pH 7.4 and 11.6 s-1 at pH 7.8. The actomyosin ATPase is inhibited by KCl. This KCl inhibition is not competitive with respect to F-actin, and it is not a simple form of non-competitive inhibition.     

Mechanism of inhibition of relaxation by N-ethylmaleimide treatment of myosin.

It has remained unexplained why N-ethylmalaeimide (NEM) treatment of myosin can inhibit relaxation in actomyosin systems from rabbit skeletal muscle which appear to be regulated solely through tropomyosin and troponin. Since rigor complexes between (nucleotide-free) myosin and actin affect the tropinin-tropomyosin system, the possibility was explored that, as a result of NEM treatment, some of the myosin maintains rigor complexes with actin in the presence of ATP which might be responsible for inhibition of relaxation. Evidence is presented indicating that such a mechanism might account for the effects of NEM treatment. First, after exhaustive NEM treatment of heavy meromysin (HMM), acto-HMM complexes were no longer dissociated by ATP. Second, admixture of such NEM-treated, enzymatically inactive HMM or myosin to native regulated actomyosin or acto-HMM inhibited relaxation.     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.     

Effect of myosin DTNB light chain on the actin-myosin interaction in the presence of ATP.

The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3.6.1.3]. Although the Ca2+-, Mg2+-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HMM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.     

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.     

Elementary steps in the acto-H-meromyosin ATPase reaction to arterial smooth muscle.

Transient and steady state kinetics were studied in the interactions of ATP with acto-H-meromyosin reconstituted from bovine arterial heavy-meromyosin (HMM) and rabbit skeletal muscle F-actin. The results showed that the rate of dissociation of the hybrid acto-HMM induced by ATP was slower than the rate of the fluorescence enhancement of HMM, and that the rate of the P1 burst of HMM was unaffected by addition of skeletal muscle F-actin. The ATPase [EC 3.6.1.3] activity of arterial HMM was activated only slightly even with addition of high concentrations of skeletal muscle F-actin. Furthermore, the rates of dissociation of the hybrid acto-HMM induced by ATP and reassociation of dissociated arterial HMM with skeletal muscle F-actin after decomposition of ATP were much lower than those of skeletal muscle acto-HMM.     

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.     

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.     

The mechanism of ATP hydrolysis by smooth muscle myosin and subfragments using steady state titration and 18O exchange.

The mechanisms of increases in the ATPase rates of smooth muscle acto-myosin, acto-heavy meromyosin (HMM) and acto-subfragment 1 (S1) were investigated using steady state titration and 18O exchange. Phosphorylation increased the phosphate release rates both from acto-myosin and acto-HMM. Steady state titration at high enzyme concentrations and 18O exchange at substoichiometric ATP concentrations showed that gizzard myosin was kinetically homogeneous, whereas HMM and S1 prepared by various published methods were heterogeneous. At high ATP concentrations, a small population of HMM and S1 hydrolyzed ATP with a low amount of oxygen exchange.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Interaction of two heads of myosin with F-actin: binding of H-meromyosin with F-actin in the absence of nucleotide

The bindings of S-1 and the two heads of HMM with pyrene-labeled F-actin were studied using the change in light-scattering intensity or that in the fluorescence intensity of the pyrenyl group. At low ionic strength (50 mM KCl), both S-1 and HMM became bound tightly with F-actin (Kd less than 0.1 microM) and both heads of HMM became bound to F-actin. The affinities of S-1 and HMM for F-actin decreased with increasing KCl concentration. In 1 M KCl, the Kd values of S-1 and HMM for F-actin were 11 and 0.58 microM, respectively. Thus, HMM was bound to F-actin 19 times more tightly than S-1. We compared the extent of binding of HMM to F-actin measured by a centrifugation method with that measured by the fluorescence change of pyrenyl-group, and found that even in 1 M KCl, HMM became bound to F-actin with a two-headed attachment. We measured the kinetics of binding and dissociation of acto-S-1 and acto-HMM from the time course of the change in light-scattering intensity after mixing S-1 or HMM with F-actin at 1 M KCl and that after mixing 1 M KCl with acto-S-1 or acto-HMM formed at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal cation controls myosin and actomyosin kinetics

We have perturbed myosin nucleotide binding site with magnesium‐, manganese‐, or calcium‐nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time‐resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation‐specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.     

Competitive and uncompetitive effects of 2,4-dinitrophenol on ATPase activities of rabbit skeletal actomyosin and myosin.

A kinetic study of the ATPase reactions catalyzed by myosin and actomyosin was carried out by varying the concentrations of ATP and 2,4-dinitrophenol (DNP). Mg-ATPase of myosin in the initial burst and that of actomyosin were both inhibited competitively by DNP. The dissociation contants for the DNP-myosin interaction (Ki) were estimated to be very similar, that is, 4.2 mM in the initial burst of ATP splitting, and 3.3 mM for the actomyosin ATPase. It is therefore suggested that DNP acts at the same site when it inhibits the burst splitting of ATP and the actomyosin ATPase. In contrast, Mg,-Ca-, and EDTA-ATPase activities of myosin in the steady state were all affected uncompetitively by DNP. Moreover, the Ki value for Mg-ATPase of myosin in the steady state was found to be 31 mM, which is much higher than those mentioned above for the initial burst and actomyosin ATPase. It is therefore suggested that the site at which DNP acts to inhibit the burst splitting of ATP is different from the site at which DNP acts to affect Mg-, Ca-, and EDTA-ATPases in the steady state.     

Cryoenzymic studies on actomyosin ATPase. Evidence that the degree of saturation of actin with myosin subfragment 1 affects the kinetics of the binding of ATP

The initial steps of actomyosin subfragment 1 (acto-S1) ATPase (dissociation and binding of ATP) were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The dissociation kinetics were followed by light scattering in a stopped-flow apparatus, and the binding of ATP was followed by the ATP chase method in a rapid-flow quench apparatus. The data from the chase experiments were fitted to E + ATP in equilibrium (K1) E.ATP----(k2) E*ATP, where E is acto-S1 or S1. The kinetics of the binding of ATP to acto-S1 were sensitive to the degree of saturation of the actin with S1. There was a sharp transition with actin nearly saturated with S1: when the S1 to actin ratio was low, the kinetics were fast (K1 greater than 300 microM, k2 greater than 40 s-1); when it was high, they were slow (K1 = 14 microM, k2 = 2 s-1). With S1 alone K1 = 12 microM and k2 = 0.07 S-1. With acto heavy meromyosin (acto-HMM) the binding kinetics were the same as with saturated acto-S1, regardless of the HMM to actin ratio. The dissociation kinetics were independent of the S1 to actin ratio. Saturation kinetics were obtained with Kd = 460 microM and kd = 75 S-1. The data for the saturated acto-S1 could be fitted to a reaction scheme, but for lack of structural information the abrupt dependence of the ATP binding kinetics upon the S1 to actin ratio is difficult to explain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.     

Energetics and mechanism of actomyosin adenosine triphosphatase.

Rate constants were determined for the reaction of actin with subfragment 1 (S1), S1-product complex, heavy meromyosin (HMM), and HMM-products complex for a range of temperatures, pH's, and ionic strengths. For actin concentrations up to 10 muM, the rate of reassociation of the product intermediate was equal to the rate of actomyosin subfragment 1 (acto-S1) or acto-HMM adenosine triphosphatase (ATPase). Therefore, under these conditions, the only important pathway for adenosine triphosphate hydrolysis is through the dissociation and recombination of S1 or HMM. The apparent rate constants for the association of S1 and S1-product with actin showed a similar large ionic strength dependence. The S1-product reaction had a large temperature dependence paralleling the rate of acto-S1 ATPase, while the reaction with S1 had a much smaller variation with temperature. The low value of the rate constant for the S1-product reaction and its relationship to the s1 areaction suggests that the apparent rate constant does not measure a simple second-order reaction. A plausible mechanism is a rapid equilibrium for the binding step, followed by a transition (product release) which increases the association constant. A refractory state could also reduce the apparent rate constant of recombination. An approximate assignment of equilibrium constants for the acto-S1 ATPase reaction was made based on the interpretation of the present evidence and equilibrium constnats for the S1 ATPase.     

Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.