ATPase Activity Of Subfragment-1 Research Articles

Two Drosophila Myosin Transducer Mutants with Distinct Cardiomyopathies Have Divergent ADP and Actin Affinities

Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila.

Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3 microM SIN-1 for 8.7 microM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca(2+)-dependent and K(+)/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys(707) and Cys(697)), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5'-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys(707) labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP.V(1) to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

C-terminal truncation of cardiac troponin I causes divergent effects on ATPase and force: implications for the pathophysiology of myocardial stunning.

Myocardial stunning is a form of reversible myocardial ischemia/reperfusion injury associated with systolic and diastolic contractile dysfunction. In the isolated rat heart model, myocardial stunning is characterized by specific C-terminal proteolysis of the myofilament protein, troponin I (cTnI) that yields cTnI1-193. To determine the effect of this particular C-terminal truncation of cTnI, without the confounding factor of other stunning-induced protein modifications, a series of solution biochemical assays has been undertaken using the human homologue of mouse/rat cTnI1-193, cTnI1-192. Affinity chromatography and actin sedimentation experiments detected little, or no, difference between the binding of cTnI (cTnI1-209) and cTnI1-192 to actin-tropomyosin, troponin T, or troponin C. Both cTnI and cTnI1-192 inhibit the actin-tropomyosin-activated ATPase activity of myosin subfragment 1 (S1), and this inhibition is released by troponin C in the presence of Ca2+. However, cTnI1-192, when reconstituted as part of the troponin complex (cTn1-192), caused a 54+/-11% increase in the maximum Ca2+-activated actin-tropomyosin-S1 ATPase activity, compared with troponin reconstituted with cTnI (cTn). Furthermore, cTn1-192 increased Ca2+ sensitivity of both the actin-tropomyosin-activated S1 ATPase activity and the Ca2+-dependent sliding velocity of reconstituted thin filaments, in an in vitro motility assay, compared with cTn. In an in vitro force assay, the actin-tropomyosin filaments bearing cTn1-192 developed only 76+/-4% (P<0.001) of the force obtained with filaments composed of reconstituted cTn. We suggest that cTnI proteolysis may contribute to the pathophysiology of myocardial stunning by altering the Ca2+-sensing and chemomechanical properties of the myofilaments.

Conformation and dynamics of the SH1-SH2 helix in scallop myosin.

Atomic structures of scallop myosin subfragment 1(S1) with the bound MgADP, MgAMPPNP, and MgADP.BeF(x) provide crystallographic evidence for a destabilization of the helix containing reactive thiols SH1 (Cys703) and SH2 (Cys693). A destabilization of this helix was not observed in previous structures of S1 (from chicken skeletal, Dictyostelium discoideum, and smooth muscle myosins), including complexes for which solution experiments indicated such a destabilization. In this study, the factors that influence the SH1-SH2 helix in scallop S1 were examined using monofunctional and bifunctional thiol reagents. The rate of monofunctional labeling of scallop S1 was increased in the presence of MgADP and MgATPgammaS but was inhibited by MgADP.V(i) and actin. The resulting changes in ATPase activities of S1 were symptomatic of SH2 and not SH1 modification, which was confirmed by mass spectrometry analysis. With bifunctional reagents of various lengths, cross-linking did not occur on a short time scale in the absence of nucleotides. In the presence of MgADP, cross-linking was greatly enhanced for all of the reagents. These reactions, as well as the formation of a disulfide bond between SH1 and SH2, were much faster in scallop S1.ADP than in rabbit skeletal S1.ADP and were rate-limited by the initial attachment of the reagent to scallop S1. The cross-linking sites were mapped by mass spectrometry to SH1 and SH2. These results reveal isoform-specific differences in the conformation and dynamics of the SH1-SH2 helix, providing a possible explanation for destabilization of this helix in some scallop S1 but not in other S1 isoform structures.

Alterations in Thin Filament Regulation Induced by a Human Cardiac Troponin T Mutant That Causes Dilated Cardiomyopathy Are Distinct from Those Induced by Troponin T Mutants That Cause Hypertrophic Cardiomyopathy

We have compared the in vitro regulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the DeltaLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing DeltaLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase at pCa4.5. The Ca(2+) sensitivity of ATPase activation was increased (DeltapCa(50) = +0.2 pCa units) and cooperativity of Ca(2+) activation was virtually abolished. Equimolar mixtures of wild type and DeltaLys-210 troponin T gave a lower Ca(2+) sensitivity than with wild type, while maintaining the diminished ATPase activation at pCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca(2+) sensitivity was increased but there was no change in cooperativity. In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the DeltaLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli.

Structural basis for the higher Ca2+-activation of the regulated actin-activated myosin ATPase observed with Dictyostelium/Tetrahymena actin chimeras

Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca2+-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of ATPase activation in the presence of tropomyosin-troponin and Ca2+ to that without tropomyosin-troponin becomes about four times as large as the ratio for the wild-type actin. To understand the structural basis of this higher Ca2+-activation, we have determined the crystal structures of the 1:1 complex of Dictyostelium mutant actins (half chimera-1 and full chimera) with gelsolin segment-1 to 2.0 Å and 2.4 Å resolution, respectively, together with the structure of wild-type actin as a control. Although there were local changes on the surface of the subdomain 4 and the phenolic side-chain of Tyr230 displaced the side-chain of Leu236 from a non-polar pocket to a more solvent-accessible position, the structures of the actin chimeras showed that the mutations in the 228-232 region did not introduce large changes in the overall actin structure. This suggests that residues near position 230 formed part of the tropomyosin binding site on actin in actively contracting muscle. The higher Ca2+-activation observed with A230Y-containing mutants can be understood in terms of a three-state model for thin filament regulation in which, in the presence of both Ca2+ and myosin heads, the local changes of actin generated by the mutation (especially its phenolic side-chain) facilitate the transition of thin filaments from a “closed” state to an “open” state. Between 394 and 469 water molecules were identified in the different structures and it was found that actin recognizes hydrated forms of the adenine base and the Ca ion in the nucleotide binding site.

Fluorescent Phalloidin Enables Visualization of Actin Without Effects on Myosin's Actin Filament Sliding Velocity and Hydrolytic Propertiesin vitro

Recent reports have demonstrated an activating effect of phalloidin in striated muscle. Furthermore, modeling of X-ray diffraction and crystallographic data suggests that phalloidin binding may induce conformational changes in actin. To determine whether phalloidin affects the mechanics of the actomyosin interaction, the velocity of actin filaments variably labeled with rhodamine-phalloidin was measured. In addition, solution actin-activated myosin subfragment-1 ATPase activity with phalloidin-labeled actin was compared to unlabeled actin. Here we found that phalloidin does not significantly effect actin filament velocity or parameters of ATPase, namely Vmaxand Km. Possible differences between muscle strip data and thesein vitroresults are discussed.

Fluorescence probing of yeast actin subdomain 3/4 hydrophobic loop 262-274. Actin-actin and actin-myosin interactions in actin filaments.

Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys374 or Cys265. These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was approximately 50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 A of one another. Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.

Mapping myosin-binding sites on actin probed by peptides that inhibit actomyosin interaction.

A 2-kDa peptide (2K peptide) which was derived from the neck region of porcine aorta smooth muscle myosin heavy chain binds to actin competitively with skeletal myosin subfragment 1 (S1) in the absence of ATP and inhibits acto-S1 ATPase activity [Katoh, T. and Morita, F. (1993) J. Biol. Chem. 268, 2380-2388]. Using this and other peptides, myosin-binding sites on actin were mapped and their functions were studied. The 2K peptide inhibited the acto-S1 ATPase activity without inhibiting the binding of S1 to actin in the presence of ATP. On the other hand, the dansylated 2K peptide (DNS-2K peptide) inhibited not only the acto-S1 ATPase activity but also the binding of S1 to actin in the presence of ATP. Then, DNS-2K peptide was crosslinked to actin with 1-ethyl-3[3-(dimethylamino)propyl] carbodiimide. Amino acid composition and sequencing analyses of the fluorescent lysylendopeptidase-peptides of the crosslinked product indicated that DNS-2K peptide was crosslinked to acidic residues within residues 1-18 (Asp1, Glu2, Asp3, Glu4, and/or Asp11), 19-50 (Asp25), and 85-113 (Glu99 or Glu100) of actin. A competition experiment for the crosslinking with unlabeled 2K peptide showed that the crosslinking to residues 85-113 of actin was specific for DNS-2K peptide. In addition, isolated actin peptide 85-113 was found to show the competitive inhibition of actin-activated ATPase activity of S1 with respect to actin. These results suggest that the site within residues 1-28 of actin participates in the actin-activation of myosin ATPase activity, and the site within residues 85-113 of actin participates in the weak binding of myosin to actin in the presence of ATP.

Effect of divalent cations on the formation and stability of myosin subfragment 1-ADP-phosphate analog complexes.

Myosin belongs to the family of motor proteins. Its interaction with actin coupled with hydrolysis of ATP is the molecular basis of muscle contraction. The head segment of myosin, called subfragment 1 (S1), contains the distinct binding sites for ATP and actin and responsible for the ATPase activity. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1-MgADP-Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate (Vi) or beryllium fluoride (BeF(x)), highly stabilizes the complex. We studied the role of the divalent cations in the ATPase activity and in the formation and decomposition of PA-containing stable complexes by substituting Mg2+ with Fe2+, Mn2+, Ni2+, Co2+, and Ca2+. These metal ions supported the actin activation of S1 ATPase and affected the obtained kinetic parameters, Km and V(max). The ATPase activity of S1 in the absence of actin increased with the increasing ionic radius of the metal (Me) ions. These ions also substituted for Mg2+ in the formation of the stable ternary S1-MeADP-PA complexes, which cannot be generated in the absence of divalent cations. Upon formation of stable ternary complexes, S1 reversibly loses its ability to catalyze the hydrolysis of ATP. The formation of the complexes can be followed by monitoring the disappearance of the ATPase activity. The rate of the complex formation depends on the divalent cation present and decreases in the order Mn > Fe > Ni > Co > Mg and Ca > Mn > Fe > Mg > Co in the Vi- and BeF(x)-containing complexes, respectively. The ATPase activity of S1 is recovered upon addition of actin, which causes the decomposition of the complex. The spontaneous decomposition of the complexes was studied in the presence of ethylenediaminetetraacetic acid (EDTA), which chelates the metal divalent cations released from the complex and prevents its reformation. The rate of decomposition was assessed by monitoring the recovery of the ATPase activity of S1 in the presence of EDTA. The rate of decomposition of the Vi- and BeF(x)-containing complexes follows the order Mn > Fe > Co > Mg > Ni and Ca >> Mn > Fe > Co > Mg, respectively. The rate of decomposition increases with the increasing ionic radius of the metal ions, similarly as observed in the case of ionic radius dependence of the ATPase activity. On the basis of this similarity, it is assumed that the decomposition of the complexes consists of two steps, the first step being the very slow release of PA followed by a rapid dissociation of MeADP from S1. The stability of the complexes has been calculated from the formation and decomposition rates. Except in the case of Mg, the stabilities of the BeF(x) complexes are higher than those containing Vi. The results indicate that the metal cations have a significant role in maintaining the proper structure of the transient state complex in the myosin-catalyzed ATP hydrolysis.

Immunochemical probing of the functional role of the 238-246 and 567-574 sequences of myosin heavy chain.

Polyclonal site-directed peptide antibodies were raised against the 567-574 and 238-246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation-sensitive since they specifically reacted with S1 in solid-phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567-574 sequence, Ab567-574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly-attached state, while Ab238-246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca- and K(EDTA)-activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567-574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin.

Inhibition of acto-myosin subfragment-1 ATPase activity by peptides corresponding to various segments of the 20-kDa domain of myosin heavy chain.

As reported previously, the synthetic heptapeptide having the amino acid sequence around the reactive Cys (SH1) of myosin heavy chain, IRICRKG-NH2, inhibited acto-myosin subfragment-1 (S-1) ATPase activity and half inhibition (K1/2) was observed at a peptide concentration of 0.06 mM. The inhibitory ability of the peptide was found to be decreased to one-fifth by acetylation of its N-terminal alpha-amino group. A similar effect of N-acetylation was observed with a nonapeptide, EGIRICRKG-NH2, and an undecapeptide, VLEGIRICRKG-NH2. These results indicate that N-terminal-free synthetic peptides do not act as proper analogs of the corresponding segment of S-1 heavy chain against F-actin. We isolated a longer peptide extending from Thr682 to Lys709 in S-1 heavy chain, with two Cys residues corresponding to SH1 and SH2. This peptide, having 28 residues (28peptide), inhibited acto-S-1 ATPase activity with a K1/2 of 0.23 mM. A cosedimentation binding assay indicated that the 28peptide completely dissociated acto-S-1 in the presence of ATP. This behavior is different from that observed with the N-terminal-free synthetic heptapeptide, and thus the 28peptide might be an analog of the corresponding segment. There is a possibility that the region corresponding to the 28peptide in S-1 heavy chain may bind directly with F-actin and may be involved in determining the acto-S-1 link during the steady state of the acto-S-1 ATPase reaction.

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.

Changes of carp myosin subfragment-1 induced by temperature acclimation

Heavy meromyosin subfragment-1 (S1) was prepared by α-chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca2+- and K+ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 μmol Pi·min-1·mg-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (KD) of S1 from cold-acclimated carp was 32.1x10-4· s-1, compared to 13.2x10-4·s-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg2+-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.

A comparison of the effect of vanadate on the binding of myosin‐subfragment‐1 · ADP to actin and on actomyosin subfragment 1 ATPase activity

Equilibrium binding studies were used to determine the binding constant of vanadate ion (Vi), to the complex of actomyosin subfragment 1 (S1) with ADP and Vi and of actin to the myosin S1.ADP.Vi complex. The proteins used were obtained from rabbit skeletal muscle. Pre-steady-state measurements were also performed to determine the rates of Vi association and dissociation from the actomyosin S1.ADP.Vi complex. Using these measured values in a simple model, the steady-state actomyosin S1 ATPase activity was predicted over a range of Vi concentrations. This model predicted that Vi would have little effect on the actomyosin S1 ATPase activity. In agreement with this prediction, the measured ATPase activity of actomyosin S1 was not greatly inhibited by Vi, except at high concentrations at which polymeric species of Vi may occur (greater than 900 microM).

Evidence for the Existence of Two Equilibrium Conformations of the Ternary Complex of Myosin Subfragment-1, ADP, and Orthovanadate

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.

Kaempferol inhibits myosin light chain kinase

Kaempferol, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, was found to inhibit bovine aorta myosin light chain kinase with a Ki of 0.3−0.5 uM . It was found to be competitive with ATP and non-competitive with isolated myosin light chains. The specificity of this inhibitor was studied relative to protein kinase C and cAMP dependent protein kinase (IC50 = 15 uM and 150 uM , respectively). It appears not to interact strongly with calmodulin binding proteins, such as Ca 2+-calmodulin dependent phosphodiesterase (IC50 - 45 uM ), and had little effect on actin-activated myosin subfragment-1 ATPase activity (IC50 > 100 uM ) or smooth muscle phosphatase activities (IC50 > 100 uM ).

The modeling of the actomyosin subfragment-1 ATPase activity

In summary the four state model can at least qualitatively account for all of the available steady state and presteady state data. In order to account for the ATPase data, the four state model predicts a linear plot at low actin concentrations that extrapolates to a considerably higher Vmax and a weaker Kapp than measured experimentally. The inhibition term then dominates wrapping the predicted curve around the data. However, the four state model can account for the ATPase data within an acceptable margin of error. Controversy exists over how well the four state model can account for the burst magnitude. Stein et al., working under conditions where a four- to eightfold difference exists between KATPase and Kbinding, found that the four state model was inadequate for accounting for their quench flow burst data. On the other hand, Rosenfeld and Taylor, working under conditions where the ratio of Kbinding to KATPase was of the order of three, found a somewhat better fit to the quench flow burst data. Both laboratories, however, found that the fluorescence burst appeared larger than could be explained by the four state model. Of interest, Stein et al. found that their burst measurements were somewhat smaller than those predicted by the six state model. However, unlike the ATPase activity, the magnitude measurements are quite difficult, and therefore the results must be viewed as less conclusive. Finally, the four state model can qualitatively account for the rise in the fluorescence rate as a function of the actin concentration, but has difficulty accounting for it quantitatively. However, because of turbidity transients that may be occurring during the measurement, these data are also not as conclusive as they might be. On the other hand the six state model clearly fits the data better, but this is not surprising considering the increased degrees of freedom afforded this model. Controversy continues concerning the interpretation of the kinetics of the crosslinked actoS-1, especially the kinetics of the 18O exchange that appears on the surface to support a four state model in the case of skeletal S-1, but to oppose a four state model in the case of cardiac S-1. Further work will be necessary to determine the adequacy of the models discussed.

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.