Comparison of ATPase activities and heavy chains of rabbit atrial and thyrotoxic ventricular myosin subfragment-1

Summary Subfragment-1 of rabbit atrial and thyrotoxic ventricular myosin (V₁ isomyosin) has been prepared and purified by DEAF-cellulose column chromatography. Pyrophosphate-polyacrylamide gel electrophoretic patterns and column chromatographic profile of the atrial subfragment differ from those of thyrotoxic ventricular myosin subfragment-1. On the other hand, Ca²⁺, Mg²⁺ and actin-activated ATPase activities of these subfragments are identical. Comparison of the peptide mapping by limited proteolysis in the presence of sodium dodecyl sulfate of the heavy and the light subunits of these subfragments reveals that the patterns for the heavy chain peptides of these subfragments are substantially similar but their light chain peptide patterns differ. The results suggest that the enzymatic and structural similarities that have been recognized between these isoenzymes using intact myosin hold true for the myosin subfragment-1.The differences between these subfragments are due to the differences in the light chains associated with them.Abbreviations EDTA Ethylene Diamine Tetra-acetic Acid - SDS Sodium Dodecyl Sulfate - S₁ myosin subfragment-1 - HC Heavy Chain - LC Light Chain     

Two different preparations of subfragment-1 from chicken skeletal myosin and from porcine cardiac myosin

Two different subfragment-1 preparations were obtained from either skeletal or cardiac myosin. They were identical in the heavy chain and light chain compositions but different in the pH dependence of the Ca-ATPase activity and in the relationship with "reactive lysine residues" (RLR).     

Enzymatic comparisons between light chain isozymes of human cardiac myosin subfragment-1

Human cardiac ventricular myosin subfragment-1 (S-1) was prepared by chymotryptic digestion of myosin purified from adult and fetal hearts. The enzymatic properties of adult S-1 were compared to those of two light chain isozymes of fetal S-1 which were separated by ion-exchange chromatography. One fetal isozyme contained a light chain (LC) indistinguishable from the adult ventricular LC1 and the other fetal isozyme contained the LC1 variant that is a component of intact fetal myosin. The fetal isozymes had identical actin-activated Mg2+ ATPase rates at all actin concentrations, as well as the same K+EDTA, Ca2+, and Mg2+ATPase rates. Furthermore, both fetal isozymes had the same actin-activated Mg2+ATPase rates as S-1 purified from adult hearts. The K+EDTA and Ca2+ATPase rates of adult S-1 were only slightly different from those of fetal S-1. These observations are consistent with other available data suggesting that human fetal and adult ventricular myosin differ only in light chain content, not in heavy chain composition, and indicate that isozymic LC1 variation does not alter the steady-state ATPase rate of human cardiac S-1.     

Elementary steps of the actin-activated ATPase reaction of cardiac muscle myosin subfragment-1

The rates of the elementary steps of the actomyosin ATPase reaction were measured using the myosin subfragment-1 of porcine left ventricular muscle. The results could be explained only by the two-route mechanism for actomyosin ATPase (Inoue, Shigekawa, & Tonomura (1973) J. Biochem. 74, 923-934), in which ATP is hydrolyzed via routes with or without accompanying dissociation of actomyosin. The dependence on the F-actin concentration of the rate of the acto-S-1 ATPase reaction in the steady state was measured in 5 mM KCl at 20 degrees C. The maximal rate, Vmax, and the dissociation constant for F-actin of the ATPase, Kd, were 3.0 s-1 and 2.2 mg/ml, respectively. The Kd value was almost the same as that determined from the extent of binding of S-1 with F-actin during the ATPase reaction. The rate of recombination of the S-1-phosphate-ADP complex, S-1ADPP, with F-actin, vr, was lower than that of the ATPase reaction in the steady state. Thus, ATP is mainly hydrolyzed without accompanying dissociation of acto-S-1 into S-1ADPP and F-actin. In the cardiac acto-S-1 ATPase reaction, the rate of the ATPase reaction in the steady state and that of recombination of S-1ADPP with F-actin were about 1/5 those of the skeletal acto-S-1 ATPase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Structural stability of fish myosin subfragment-1

1. Tryptic cleavage of fish myosin subfragment-1 (S-1) revealed its similar substructure of heavy chain to that of rabbit S-1. 2. The structural stability of fish S-1 was studied by thermal denaturation method, and a rapid polymerization of inactivated fish S-1, detected by turbidity increase, was characteristic. 3. The light-chain release and tryptic susceptibility increase upon heating were significant with fish S-1.     

Enzymatic properties of the heavy meromyosin subfragment of cardiac myosin from normal and thyrotoxic rabbits.

Myosin from the hearts of thyrotoxic animals (myosin-T) exhibits elevated Ca2+-ATPase activity. To clarify the physiological significance of this increased activity, we have investigated the steady state kinetics of the interaction of actin and MgATP with the double-headed heavy meromyosin subfragment of cardiac myosin from thyrotoxic rabbits (HMM-T). The enhanced Ca2+-ATPase activity of myosin-T was completely retained in HMM-T. The Vmax for actin-activated MgATP hydrolysis by HMM-T (1.08 +/- 0.10 mumol of Pi/mg/min). Under physiological ionic conditions, the Vmax was 0.14 +/- 0.02 mumol of Pi/mg/min as compared with the normal value of 0.08 +/- 0.01 mumol of Pi/mg/min. Furthermore, the salt dependence of Vmax and Kapp for the actin-activated ATPase of HMM-T differed markedly from normal and resembled that usually associated with the single-headed (S1) cleavage product of myosin. These results suggest that the changes in enzymatic properties of myosin-T are responsible for the increased speed of contraction observed in the hearts of thyrotoxic animals. Also, the alteration in the interaction of HMM-T with actin suggests that a loss of cooperativity between the myosin heads may occur.     

The magnesium-ion-dependent adenosine triphosphatase of bovine cardiac Myosin and its subfragment-1.

The kinetics of the Mg2+-dependent ATPase (adenosine triphosphatase) activity of bovine cardiac myosin and its papain subfragment-1 were studied by using steady-state and pre-steady-state techniques, and results were compared with published values for the corresponding processes in the ATPase mechanism of rabbit skeletal-muscle myosin subfragment-1. The catalytic-centreactivity for cardiac subfragment-1 is 0.019s-1, which is less than one-third of that determined for the rabbit protein. The ATP-induced isomerization process, measured from enhancement of protein fluorescence on substrate binding, is similarly decreased in rate, as is also the isomerization process associated with ADP release. However, the equilibrium constant for ATP cleavage, measured by quenched-flow by using [gamma-32P]ATP, shows little difference in the two species. Other experiments were carried out to investigate the rate of association of actin with subfragment-1 by light-scattering changes and also the rate of dissociation of the complex by ATP. The dissociation rate increases with increasing substrate concentration, to a maximum at high ATP concentrations, with a rate constant of about 2000s-1. It appears that isomerization processes which may involve conformational changes have substantially lower rate constants for the cardiac proteins, whereas equilibrium constants for substrate binding and cleavage are not significantly different. These differences may be related to the functional properties of these myosins in their different muscle types. Kinetic heterogeneity has been detected in both steady-state and transient processes, and this is discussed in relation to the apparent chemical homogeneity of cardiac myosin.     

Binding of magnesium to myosin subfragment-1 ATPase

Tyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylylimidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435 nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of delta A at 435 nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25 X 10(-8) M and 1.24 X 10(-7) with AMPPNP and ADP, respectively. The difference spectrum was also obtained in the presence of ATP. The delta epsilon value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADP(a), was estimated as 6 X 10(-8) M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.     

Binding of myosin subfragment-1 to F-actin.

During a part of the hydrolytic cycle, myosin head (S1) carries no nucleotide and binds strongly to an actin filament forming a rigor bond. At saturating concentration of S1 in rigor, S1 is well known to form 1:1 complex with actin. However, we have provided evidence that under certain conditions S1 could also form a complex with 2 actin monomers in a filament (Andreev, O.A. & Borejdo, J. (1991) Biochem. Biophys. Res. Comm. 177, 350-356). This view was recently challenged by Carlier & Didry (Carlier, M-F. & Didry, D. (1992) Biochem. Biophys. Res. Comm. 183, 970-974) who interpreted our data by suggesting that F-actin underwent a simple depolymerization and implied that, when only actin in the F-form was scored, the real stoichiometry in our experiments was 1:1. We show here that under conditions of our experiments less than 8% of actin was depolymerized. Moreover, we have repeated the experiments in the presence of phalloidin and show that under these conditions too, when S1 was added slowly to a fixed concentration of F-actin, it formed a different complex with F-actin than when it was added quickly. This confirms our original conclusion that S1 can bind actin in two different ways and shows that depolymerization of F-actin is not responsible for this finding.     

Fluorescence energey transfer in myosin subfragment-1

Fluorescent probes have been selectively introduced into skeletal muscle myosin subfragment-1 and the fluorescence emission characteristics of the labeled products studied. The fluorophores employed were the thiol-specific reagents N-[[(iodoacetyl)aminolethyl-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, the spectral properties of which render them a particularly effective donor-acceptor pair in F?rster energy-transfer studies. Alkali 1 light chain, labeled at a single cysteine with either of these probes, was incorporated into chymotryptic subfragment-1 by the exchange procedure of Wagner & Weeds [Wagner, P.D., & Weeds, A.G. (1977) J. Mol. Biol. 109, 455-473]. The resultant, fluorescently labeled subfragment-1 was isolated by ion-exchange chromatography. Determination of the extent of incorporation by extinction and fluorescence indicated that greater than 80% of the subfragment-1 population possessed a fluorescently labeled alkali 1 light chain. The introduction of labeled alkali 1 did not perturb the K+-, Ca2+-, or actin-activated adenosine triphosphatases of subfragment-1. The addition of adenosine triphosphate (ATP), liganded by various cations, to this singly labeled subfragment-1 induced a 6-10% decrease in the fluorescence intensity of the extrinsic chromophore. An intensity decrease of approximately 4% was obtained when the hydrolysis of ATP was complete, and also upon direct addition of adenosine diphosphate. The ATP analogue adenylyl imidodiphosphate induced a decrease of approximately 7% in intensity. The addition of F-actin to the subfragment-1 in the presence of MgATP elicited no further fluorescence intensity change. A second, appropriate fluorophore was introduced into the singly labeled subfragment-1 at the SH1 thiol on the heavy chain. F?rster energy transfer was observed between this labeled site and the fluorophore previously introduced on the alkali 1 light chain. The measured efficiency of energy transfer indicated that the two fluorophores were approximately 40 A apart. The same value was obtained upon reversal of the donor and acceptor attachment sites, suggesting that the uncertainty in the calculated distance introduced by the choice of orientation factor is probably less than 20%. Steady-state observations did not reveal any obvious change in this distance upon the addition of MgATP and then F-actin to the doubly labeled subfragment-1.     

Bundling of myosin subfragment-1-decorated actin filaments

We have reported previously that rabbit skeletal myosin subfragment-1 (S-1) assembles actin filaments into bundles. The rate of this reaction can be estimated roughly from the initial rate (Vo) of the accompanying turbidity increase ("super-opalescence") of the acto-S-1 solution. Vo is a function of the molar ratio (r) of S-1 to actin, with a peak at r = 1/6 to 1/7 and minimum around r = 1. In the present paper we report a different type of opalescence (we call it "hyper-opalescence") of acto-S-1 solutions, which also resulted from bundle formation. Adjacent filaments in the bundles had a distance of approximately 180 A. Hyper-opalescence occurred at r approximately equal to 1 when KCOOCH3 was used instead of KCl. By comparing the effects of ADP, epsilon-ADP, tropomyosin or ionic strength upon the super- and hyper-opalescence, we concluded that the two types of S-1-induced actin bundling had different molecular mechanisms. The hyper-opalescence type of bundling seemed to be induced by S-1, which was not complexed with actin in the manner of conventional rigor binding. The presence of the regulatory light chain did not affect hyper-opalescence (or super-opalescence), since there were no significant differences between papain S-1 and chymotryptic S-1 with respect to these phenomena.     

Betaine protects urea-induced denaturation of myosin subfragment-1

We have demonstrated previously that urea inhibits the activity and alters the tertiary structure of skeletal muscle myosin in a biphasic manner. This was attributed to differential effects on its globular and filamentous portion. The inhibition of catalytic activity was counteracted by methylamines. With the aim of comprehending the effects of urea on the catalytic (globular) portion of myosin, this study examines the effects of urea and the countereffects of betaine on the catalytic activity and structure of myosin subfragment-1. It is shown that urea inactivates subfragment-1 in parallel with its ability to induce exposure of the enzyme hydrophobic domains, as assessed using intrinsic and extrinsic fluorescence. Both effects are counteracted by betaine, which alone does not significantly affect subfragment-1. Urea also enhances the accessibility of thiol groups, promotes aggregation and decreases the alpha-helix content of S1, effects that are also counteracted by betaine. We conclude that urea-induced inactivation of the enzyme is caused by partial unfolding of the myosin catalytic domain.     

Transient kinetic analysis of N-phenylmaleimide-reacted myosin subfragment-1

Alkylation of myosin's Cys-707 (SH1) and Cys-697 (SH2) has profound consequences for myosin's ability to interact with actin and hydrolyze MgATP. Pre-steady-state measurements of myosin-S1 alkylated at SH1 and SH2 by N-phenylmaleimide (NPM) in the presence of ATP were taken to identify the steps of the reaction that are altered. It was found that the rate constant most affected by this modification is the apparent rate of the ATP hydrolysis step. This rate constant is reduced 20000-fold, an effect comparable in magnitude to the effect of the same modification on the binding of MgATP to S1 or acto-S1 [Xie, L., and Schoenberg, M. (1998) Biochemistry 37, 8048]. In contrast, the rate constants of phosphate release and dissociation of acto-S1 by ATP were reduced <20-fold. For unmodified S1, the enhancement of fluorescence seen after addition of ATP had the same rate constant as the ATP hydrolysis step (S1.ATP if S1.ADP.Pi) measured by single-turnover experiments in a quench-flow experiment. This is consistent with results previously observed [Johnson, K. A., and Taylor, E. W. (1978) Biochemistry 17, 3432]. However, NPM-modified S1 exhibited virtually no fluorescence enhancement upon ATP binding. This provides further evidence that M.ATP is the predominant intermediate of NPM-S1-catalyzed ATP hydrolysis.     

Tryptic cleavage and substructure of bovine cardiac myosin subfragment 1

The method of limited tryptic proteolysis has been used to compare and contrast the substructure of bovine cardiac myosin subfragment 1 (S-1) to that of skeletal myosin S-1. While tryptic cleavage of cardiac S-1, like that of skeletal S-1, yields three fragments, the 25K, 50K, and 20K peptides, the digestion of cardiac S-1 proceeds at a 2-fold faster rate. The increased rate of cleavage is due entirely to an order of magnitude faster rate of cleavage at the 25K/50K junction of cardiac S-1 compared to that of skeletal, with approximately equal rates of cleavage at the 50K/20K junctions. Actin inhibits the tryptic attack at this latter junction, but its effect is an order of magnitude smaller for the cardiac than for the skeletal S-1. Furthermore, the tryptic susceptibility of the 50K/20K junction of cardiac S-1 in the acto-S-1 complex is increased in the presence of 2 mM MgADP. This effect is not due to partial dissociation of the cardiac acto-S-1 complex by MgADP. Our results indicate that in analogy to skeletal S-1, the cardiac myosin head is organized into three protease-resistant fragments connected by open linker peptides. However, the much faster rate of tryptic cleavage of the 25K/50K junction and also the greater accessibility of the 50K/20K junction in the cardiac acto-S-1 complex indicate substructural differences between cardiac and skeletal S-1.     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.