Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1).ATP complex.

In our model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1.ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca2+, S-1.ATP should not turn on the regulated actin filament. In the present study, we tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM X S-1) so that functionally it acts like S-1.ATP, although it does not hydrolyze ATP. We found that, in the absence of Ca2+, neither S-1.ATP nor pPDM X S-1.ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto X S-1). In contrast, in the presence of Ca2+, pPDM X S-1.ATP binding almost completely turns on the regulated acto.S-1 ATPase activity. These results can be explained by our original cooperativity model, with pPDM X S-1.ATP binding only approximately equal to 2-fold more strongly to the turned-on form than to the turned-off form of regulated actin. However, our results are not consistent with our alternative model, which predicts that if pPDM X S-1.ATP binds to actin in the absence of Ca2+ but does not turn on the ATPase activity, then it should also not turn on the ATPase activity in the presence of Ca2+.     

Structure of the actin-myosin complex in the presence of ATP.

The structure of the complex between actin and myosin subfragment 1 (S1), designated the acto-S1 complex, in the presence of ATP was examined by electron microscopy. This was accomplished by using negative staining to study a complex of S1 covalently crosslinked to actin by the zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide. Two levels of S1 binding were studied, with a molar ratio of crosslinked S1 to total actin of either 20% or 50%. The lower percentage was used to observe individual S1 molecules attached to actin, while the higher percentage was used to look at the overall pattern of S1 decoration of the actin filament. In the absence of ATP, the appearances of both the 20% and 50% crosslinked filaments closely resembled the rigor appearances obtained with noncrosslinked proteins. The arrowheads observed had the conventional structure, and individual S1 molecules were elongated and curved and appeared to make an angle of 45 degrees with the thin filament. Addition of ATP to the crosslinked acto-S1 complex caused a radical change in the structure of the cross-bridges. At both 20 and 170 mM ionic strengths, individual S1 molecules appeared to be attached at variable angles which, in contrast to rigor, did not center on 45 degrees. In addition, the S1 molecules often appeared shorter and fatter than in rigor. The 50% crosslinked acto-S1 preparation no longer showed the arrowhead pattern of S1 decoration but instead appeared to be disordered with little obvious polarity. Control experiments with ADP suggest that these effects were not due simply to a weakening of the binding of S1 to actin in the presence of nucleotide but most likely were ATP-specific. The crosslinked acto-S1 complex, which hydrolyzes ATP at about the same rate as the maximal actin-activated ATPase of S1 (Vmax), is composed of a mixture of states A X M X ATP and A X M X ADP X Pi (in which A = actin and M = myosin), with more than 50% of the crosslinked S-1 occurring in state A X M X ATP. Therefore, it appears that both states A X M X ATP and A X M X ADP X Pi have a very different conformation from the classic arrowhead conformation of the A X M state.     

Formation of a ternary complex: Actin, 5′-adenylyl imidodiphosphate, and the subfragments of myosin

The formation of the ternary complex composed of actin, 5'-adenylyl imidodiphosphate [AMP-P(NH)P], and myosin subfragment 1 (S-1) was studied using the analytical ultracentrifuge with UV optics, which enabled the direct determination of the extent of dissociation of actin.S-1 (acto.S-1) by AMP-P(NH)P. In contrast to the reaction with ATP, at saturating levels of AMP-P(NH)P (1.5 mM), extensive formation of the ternary acto.S-1.AMP-P(NH)P complex occurs at 22 degrees . With 40 muM actin present, AMP-P(NH)P causes almost no dissociation of the acto.S-1 complex at 0.04 M ionic strength, while even at 0.22 M ionic strength one-third of the S-1 remains associated with actin and AMP-P(NH)P in a ternary complex. A detailed study of the binding of S-1.AMP-P(NH)P to actin using the Scatchard plot analysis shows that, at saturation, 1 mol of S-1.AMP-P(NH)P binds per mol of actin monomer. There appears to be no cooperativity occurring as the S-1.AMP-P(NH)P binds along the actin filament, with the possible exception of a slight positive cooperativity when most of the sites on the actin filament are saturated. The turbidity of the ternary complex is identical to the turbidity of acto.S-1 alone. Preliminary experiments with the two-headed subfragment of myosin, heavy meromyosin (HMM), show that the binding of HMM.[AMP-P(NH)P](2) to actin is only about twice as strong as the binding of S-1.AMP-P(NH)P to actin, indicating that the second head contributes very little to the free energy of binding.     

Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle.

We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.     

Reciprocal reactivities of specific thiols when actin binds to myosin.

We report measurements of the reactivity (degree of labeling, as mole of ligand per mole of protein, at constant exposure time) of the reactive thiol, "SH1", of a subfragment of myosin (S-1), and of Cys-10 of F-actin under various conditions, using N-iodo-[3H]acetyl-N-(1-sulfo-5-naphthyl)ethylenediamine, a fluorescent radioactive iodoacetamide analog. When either ADP or adenyloyl imidodiphosphate (simulating unhydrolyzed ATP) is bound to the enzymatic site of S-1, the reactivity of "SH1" is slightly enhanced, but when active ATPase is going on, reactivity is reduced by about a third, presumably due to the species, (S-1) ADP,Pi. The reactivity of Cys-10 alone is very low. When the complex, (S-1)-F-actin, is formed, the reactivity of SH1 is strongly decreased, and the reactivity of Cys-10 is strongly increased. The foregoing results explain our further observation (on glycerol-treated rabbit psoas fibers) that when fibers labeled in relaxation solution are compared with fibers labeled in rigor solution, myosin is more reactive and actin is less reactive, in the former case; alpha-actinin and C-protein are also less reactive in the former case.     

Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step.

The Lymn-Taylor model for the actomyosin ATPase suggests that during each cycle of ATP hydrolysis the complex of myosin subfragment 1 (S-1) with actin must dissociate into S-1.ATP plus actin before ATP hydrolysis can occur. In the present study we tested whether such a mandatory detachment step occurs by measuring the effect of actin on the rate and magnitude of the ATP hydrolysis step (initial Pi burst) and on the steady-state ATPase rate. We find that the rate of the initial Pi burst markedly increases at high actin concentration although the Lymn-Taylor model predicts the rate should remain nearly constant or decrease. In addition, at high actin concentration, the magnitude of the initial Pi burst is much larger than is predicted by the Lymn-Taylor model. Finally, at 360 microM actin, at which more than 90% of the S-1.ATP is bound to actin, there is no inhibition of the steady-state ATPase activity although the Lymn-Taylor model predicts that 70% inhibition should occur. We conclude that the acto-S-1 complex is not dissociated by ATP during each cycle of ATP hydrolysis; in fact, the rate of the initial Pi burst appears to be even faster when S-1.ATP is bound to actin than when it is dissociated.     

Cooperative binding of myosin subfragment-1 to the actin-troponin-tropomyosin complex.

The binding of myosin subfragment-1 (S-1) to the F-actin-troponin-tropomyosin complex (regulated F-actin) was examined in the presence of ADP (ionic strength, 0.23 M; 22 degrees C) by using the ultracentrifuge and S-1 blocked at SH1 with iodo[14C]acetamide. S-1 . ADP binds with positive cooperativity to regulated F-actin, both in the presence and absence of calcium; it binds independently to unregulated actin. With and without Ca2+ at very low levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds to the regulated actin with less than 1% of the strength that it binds to unregulated actin, whereas at high levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds about 3-fold more strongly to the regulated actin than it does to unregulated actin. The major difference between the results obtained in the presence and absence of Ca2+ with regulated actin is that, in the absence of Ca2+, the binding of S-1 . ADP remains weak until a higher free S-1 . ADP concentration is reached and the transition to strong binding is much more cooperative. These results are consistent with a model that is basically similar to the cooperative binding model of Hill[Hill, T.L. (1952) J. Chem. Phys. 20, 1259-1273] and of Monod et al. [Monod, J., Wyman, J. & Changeux, J. (1965) J. Mol. Biol. 12, 88-118]: The regulated actin filament can exist in two forms, a weak-binding and a strong-binding form; and Ca2+ and S-1 . ADP, acting as allosteric effectors, shift the equilibrium between the two forms.     

Effects of adenine nucleotides on calcium binding by rat heart sarcoplasmic reticulum.

The purpose of the present study was to investigate the interactions between ATP, ADP and calcium binding by rat heart sarcoplasmic reticulum vesicles (SR), and to re-evaluate the assay method used to study calcium binding. Calcium binding or transport was studied by the Millipore filtration method. Rat heart SR has an unusually high Mg2+ stimulated ATPase activity (1.37 +/- 0.16 mumol Pi per min per mg at 25 degrees C) so that previous incubation with ATP in calcium binding studies releases ADP and Pi. By maintaining ATP at high and ADP at low concentrations with an ATP-regenerating system (phosphoenolpyruvate and pyruvate kinase), calcium binding capacity was increased by two to three times that of a non-ATP-regenerating system and there was a direct relationship between the amount of Ca-binding and SR protein concentration. When Ca2+ and Mg2+ concentrations were controlled and ATP and ADP concentrations were varied independently the initial rate of Ca-binding was inhibited 25% by 1 mmol.litre-1 ADP and 48% by 3mmol.litre-1 ADP. ATP limited the initial rate of Ca-binding only at ATP levels below 2mmol.litre-1. At low ATP concentrations Ca-release was observed. However, in the presence of an ATP-regenerating system no Ca-release was observed, even at low concentrations of ATP. This study shows that ADP is an inhibitor of Ca-binding by rat heart SR. However, the possibility that high ADP concentrations in the presence of Pi from ATP hydrolysis, could facilitate calcium release cannot be excluded. In addition to the possible physiological importance, these effects must be regarded when assaying rat cardiac SR calcium binding.     

Evidence that the Mg-dependent low-affinity binding site for ATP and Pi demonstrated on the isolated beta subunit of the F0.F1 ATP synthase is a catalytic site.

Binding sites for one Pi and two ATP or ADP molecules have been identified on the isolated, reconstitutively active beta subunit from the Rhodospirillum rubrum F0.F1 ATP synthase. Chemical modification of this beta subunit by the histidine reagent diethyl pyrocarbonate or by the carboxyl group reagent Woodword's reagent K results in complete inhibition of Pi binding to beta. The same reagents inhibit the binding of ATP to a Mg-dependent low-affinity site but not to a Mg-independent high-affinity site on this beta subunit. The binding stoichiometry of ADP to either site is not affected by these modifications. The beta subunit modified by either one of these reagents retains its capacity to rebind to beta-less chromatophores but not its ability to restore their photo-phosphorylation. These results indicate that the low-affinity Pi binding site on beta is located at the binding site of the gamma-phosphate group of ATP in the Mg-dependent low-affinity nucleotide binding site. This site contains histidine and carboxyl group residues, both of which are required for the binding of Pi and of the gamma-phosphate group of ATP. The same residues must also be involved in the capacity of the isolated beta subunit to restore the catalytic activity of the beta-less ATP synthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated beta subunit of the R. rubrum F0.F1 ATP synthase is the catalytic site of this enzyme complex.     

GTP is not required for calmodulin stimulation of bovine brain adenylate cyclase.

The importance of guanyl nucleotides for calmodulin stimulation of bovine cerebral cortex adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was examined by using a partially purified calmodulin-sensitive adenylate cyclase that was resolved from calmodulin-insensitive forms of the enzyme. By using 5'-adenylyl imidodiphosphate as a substrate, in the absence of an ATP-regenerating system, it was determined that GTP is not required for calmodulin stimulation of the enzyme. Maximal activation by 5'-guanylyl imidodiphosphate (p[NH]ppG) was 5.3-fold, whereas the combination of p[NH]ppG and calmodulin stimulated the enzyme 27-fold. Although GDP inhibited p[NH]ppG stimulation of the calmodulin-sensitive adenylate cyclase, it did not affect calmodulin stimulation. In addition, calmodulin did not alter the kinetics for activation of the enzyme by p[NH]ppG. It is concluded that GTP is not required for calmodulin stimulation of brain adenylate cyclase and that calmodulin regulation of this enzyme is probably not due to effects of calmodulin on the affinity of the guanyl nucleotide regulatory complex for guanyl nucleotides.     

Affinity of myosin S-1 for F-actin, measured by time-resolved fluorescence anisotropy.

The association constant for myosin subfragment-1 (S-1) and actin was measured, using a new application of fluorescence depolarization which capitalizes on the fact that S-1 has high rotational mobility while F-actin does not. Uncoupling of the time dependences of the anisotropy decay and the association/dissociation phenomena allowed the experimentally determined anisotropy decay curve to be fitted by a sum of two terms weighted by the mole fractions of the free and bound S-1. At 4 degrees C, ionic strength 0.16 M, and pH 7.0, the association constant Ka is (1.73 +/- 0.35) X 10(6) M-1 at infinite dilution. This makes the -deltaG degrees of binding of F-actin to S-1 similar to the -deltaG degrees of binding of ATP to S-1, and the possible physiological relevance of the similarity to muscle contraction is discussed.     

Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding.

A cloned yeast TOP2 gene was modified to produce yeast DNA topoisomerase II (EC 5.99.1.3) epitopically labeled at its amino or carboxyl terminus. Limited digestion with SV8 endoprotease shows three distinct protease-sensitive sites in each polypeptide of the dimeric enzyme. These sites were mapped by immunostaining of the end-labeled proteolytic fragments resolved by SDS/polyacrylamide gel electrophoresis; two of the mapped locations were confirmed by sequencing the amino ends of two unlabeled peptic fragments. Proteolytic cleavage by SV8 endoprotease at a pair of sites corresponding to the carboxyl sides of Glu-411 and Glu-680 is modulated by the binding of the nonhydrolyzable ATP analogs adenosine 5'-[beta, gamma-imido]triphosphate (5'-adenylyl imidodiphosphate) and adenosine 5'-[gamma-thio]triphosphate: in their absence cleavage occurs predominantly at Glu-411; in the presence of either analog, cleavage occurs predominantly at Glu-680. These results are interpreted in terms of allosteric interdomainal movements in the type II DNA topoisomerase following the binding of ATP.     

Modulation of L-type calcium channels by sodium ions.

It is universally believed that the removal of external sodium ions is without effect on calcium current. We now report that in enzymatically isolated guinea pig ventricular cells, the replacement of external sodium ions with certain other cations causes a 3- to 6-fold increase in peak L-type calcium current. The increase in current is reversibly blocked by L-type calcium-channel antagonists, not mediated by changes in internal calcium, and is inhibited by intracellular 5'-adenylyl imidodiphosphate, a nonhydrolyzable ATP analogue. The effects of sodium removal (and isoproterenol) were almost completely blocked by intracellular application of a specific (peptide) inhibitor of cAMP-dependent protein kinase. These experiments demonstrate a previously unknown effect of sodium ions to modulate calcium-channel phosphorylation via cAMP-dependent protein kinase.     

Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.

Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site--the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product--in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl- for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of Cys-707, or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.     

Bioenergetic aspects and polymer length distribution in steady-state head-to-tail polymerization of actin or microtubules.

Wegner's theory of steady-state head-to-tail polymerization of actin (or microtubules) is extended somewhat in order to show the explicit role of the ATP (or GTP) free energy of hydrolysis (X) in the steady-state kinetics. The monomer flux and the ATP flux can both be expressed in terms of X and rate constants of the model. Both fluxes approach zero as X leads to 0 (by variation of the concentrations of ATP, ADP, and Pi); this limit corresponds to ATP equilibrium. The dependence of rate constants on these concentrations is examined. Free energy levels of the monomer kinetic cycle and the rate of free energy dissipation are discussed. The steady-state polymer length distribution is derived for a special case.     

The protein-folding activity of chaperonins correlates with the symmetric GroEL14(GroES7)2 heterooligomer.

Chaperonins GroEL and GroES form, in the presence of ATP, two types of heterooligomers in solution: an asymmetric GroEL14GroES7 "bullet"-shaped particle and a symmetric GroEL14(GroES7)2 "football"-shaped particle. Under limiting concentrations of ATP or GroES, excess ADP, or in the presence of 5'-adenylyl imidodiphosphate, a correlation is seen between protein folding and the amount of symmetric GroEL14(GroES7)2 particles in a chaperonin solution, as detected by electron microscopy or by chemical crosslinking. Kinetic analysis suggests that protein folding is more efficient when carried out by a chaperonin solution populated with a majority of symmetric GroEL14(GroES7)2 particles than by a majority of asymmetric GroEL14GroES7 particles. The symmetric heterooligomer behaves as a highly efficient intermediate of the chaperonin protein folding cycle in vitro.     

Purification and properties of the uvrA protein from Escherichia coli.

The uvrA+ gene product from Escherichia coli was purified to apparent homogeneity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 molecular weight DNA-binding protein having higher affinity for single-stranded than double-stranded DNA. It does not introduce single-strand breaks or alkali-labile bonds in native or UV-irradiated DNA, but it catalyzes hydrolysis of ATP to ADP and Pi. The ATPase activity is not DNA dependent and has a Km of 0.23 mM, which corresponds to the Km for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+, and uvrC+ gene products. ADP and adenosine 5'-[gamma-thio]triphosphate both inhibit the uvrA ATPase as well as the uvrABC endonuclease and also prevent specific binding of the uvrA proteins to UV-irradiated DNA. These results indicate that both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity and that the ATP requirement of the endonuclease reaction is determined by uvrA ATPase.     

In vitro stabilization of the unoccupied glucocorticoid receptor by adenosine 5'-diphosphate

The addition of ATP to rat liver cytosol slows the rate of heat inactivation of the unoccupied glucocorticoid receptor (25 C) and stimulates the rate of activation of the preformed glucocorticoid-receptor complex (15 C). Dose-response curves and kinetic studies show that ADP is as effective as ATP in stabilizing the unoccupied glucocorticoid receptor against heat inactivation. ATP can also be replaced by analogs with a hydrolysis-resistant alpha, beta-pyrophosphate linkage (5'-adenylyl methylenephosphonophosphate or 5'-adenylyl methylenephosphonate); however, the hydrolysis-resistant beta, gamma analog (5'-adenylyl methylenediphosophonate) is ineffective. The addition of creatine phosphate plus creatine kinase, a condition favoring ATP formation, stimulates the rate of inactivation of the unoccupied glucocorticoid receptor, and the effect is only partially overcome by ADP. A condition that favors ADP formation, the addition of creatine plus creatine kinase, has no effect on the rate of inactivation of the unoccupied receptor and does not decrease the protective effect afforded by ATP alone. Collectively, these results suggest that ATP stabilization of the steroid-binding site in vitro is due to ADP generated from the triphosphate by endogenous enzymes and is not due to phosphorylation or adenylation of the receptor by ATP. Unlike ATP stabilization of the steroid-binding site, the ATP-stimulated increase in the rate of activation of the preformed glucocorticoid-receptor complex (15 C) does not require hydrolysis of the beta, gamma-pyrophosphate bond. Dose-response curves show that both ATP and 5'-adenylyl methylenediphosophonate stimulate the rate of activation of the glucocorticoid-receptor complex. Quantitation of nucleotide levels in unfractionated rat liver cytosol by high performance liquid chromatography shows that the effective concentration of added ATP that produces an optimal response is within the physiological range reported for intact cells.     

Cardioprotective effects of mioflazine during 1 h normothermic global ischaemia in the canine heart

The cardioprotective effects of mioflazine, a recently developed cardiovascular drug, were investigated in 41 anaesthetised open chest Beagle dogs subjected to 1 h normothermic global myocardial ischaemia. The severity of the model is evidenced by the finding that only one out of 20 control dogs could be weaned from extracorporeal bypass. Oral pretreatment with mioflazine (2.5 mg X kg-1) resulted in complete functional recovery in 17 out of 20 animals. Biochemical analysis of left ventricular biopsies taken before, during and after aortic cross clamping showed a preservation of purines and a better recovery of ATP, ATP/ADP X Pi ratio and energy charge (p less than 0.05) in the pretreated animals. Morphological and cytochemical assessment of the myocardium demonstrated that the ultrastructure of the sarcolemma and its calcium binding capacity is remarkably well preserved in the drug treated animals. These results indicate a strong cardioprotective effect of mioflazine. The biochemical, cytochemical and ultrastructural findings suggest an interaction of the drug with the sarcolemma.     

On the molecular basis for chemomechanical energy transduction in muscle.

Herein it is developed that energy transduction in muscle is an activity of myosin S-1 and its ligands, actin (A) and nucleotide (N). S-1 shares with other molecular particles (e.g., hemoglobin) the property that binding events at one of its sites, the N-site, influences binding events at a remote site, the A site (specifically, influences both the actin affinity and actin attachment angle at the A site). However, there is a crucial difference between S-1 and the better-known systems. Because the N site is enzymatic, it has a temporal sequence of occupants; this imposes a temporal sequence of actin attitudes--i.e., a sequence of mechanical events.