Reaction intermediates of myosin ATPase from scallop adductor muscles: nonidentical two-headed structure of striated adductor muscle myosin

To determine whether or not the two heads of myosin from striated adductor muscles of scallop are nonidentical and the main intermediate of the ATPase reaction, MADPP, is produced only on one of the two heads, the Pi-burst size, the amount of total bound nucleotides and the amount of bound ADP during the ATPase reaction were measured in this study. The Pi-burst size was 1 mol per mol in the presence of 0.1-5 mM Mg2+ ions. The amount of total nucleotides bound to myosin was 2 mol per mol. Both the amounts of bound ADP and ATP at sufficiently high ATP concentrations were 1 mol per mol of striated adductor myosin, and the affinity for ADP binding was higher than that for ATP binding. These findings indicate that MADPP or MATP is produced on each of the two heads of striated adductor myosin on its interaction with ATP. The fluorescence intensity at 340 nm of striated adductor myosin was enhanced by about 7% upon addition of ATP. The time for the half maximum fluorescence enhancement, tau 1/2, at 5 microM ATP was 0.25 s, which was almost equal to the tau 1/2 values for the Pi-burst and for the dissociation of actomyosin reconstituted from striated adductor myosin and skeletal muscle F-actin. The dependences on ATP concentration of the extent of the fluorescence enhancement and the dissociation of actomyosin could be explained by assuming that these changes are associated with the formation of MADPP on one of the two heads of myosin. The Pi-burst size and the amount of bound ADP of smooth adductor myosin were slightly but significantly larger than 1 mol per mol. Both ATPase reactions of striated and smooth adductor myofibrils showed the substrate inhibition. The extent of substrate inhibition of ATPase of smooth adductor myofibrils was less than that of striated adductor myofibrils. All the present findings support the view that the nonidentical two-headed structure is required for substrate inhibition of the actomyosin ATPase reaction.     

Troponin from Akazara scallop striated adductor muscles

Troponin was isolated from striated adductor muscles of the "Akazara" scallop (Chlamys nipponensis akazara), and purified in an active form by DEAE-cellulose (Whatman DE52) column chromatography and subsequent gel filtration on Sephacryl S-300. According to sodium dodecyl sulfate-gel electrophoresis and densitometry, Akazara troponin is composed of three components having molecular weights of 52,000, 40,000, and 20,000 in a molar ratio of 1:1:1. The three components were separated from each other by column chromatography in the presence of 6 M urea and 1 mM EDTA on SP-Sephadex C-50 and DEAE-cellulose. The Mr 20,000 component was regarded as troponin C according to the Ca2+-binding properties, which was found to bind 0.7 mol of Ca2+/mol at 0.1 mM Ca2+. The association constant of Ca2+ to troponin C was estimated to be 5 X 10(5) M-1, and was not affected by the addition of 2 mM MgCl2. The Mr 52,000 component appeared to be troponin I, since it inhibited, together with Akazara tropomyosin, both Mg-ATPase and superprecipitation activities of actomyosin reconstituted from rabbit myosin and actin, and the inhibition of the ATPase activity was diminished by the addition of Akazara troponin C. Finally, the Mr 40,000 component appeared to be troponin T, since it co-precipitated with actin-tropomyosin filament and was indispensable with Akazara troponin C and the Mr 52,000 component (troponin I) for conferring the Ca2+ sensitivity to reconstituted actomyosin.     

A binary complex of troponin-I and troponin-T from Akazara scallop striated adductor muscle.

A binary complex consisting of Mr 19,000 and Mr 40,000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca(2+)-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the Mr 19,000 and Mr 40,000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The Mr 19,000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the Mr 40,000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the Mr 19,000 and Mr 40,000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the Mr 40,000 component and troponin-T were almost identical, and those of the Mr 19,000 component and Mr 17,000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the Mr 19,000-40,000 binary complex is the troponin-I-troponin-T complex.     

Biochemical characteristics of the Mr 52,000 component of Akazara scallop troponin

Some biochemical properties of the Mr 52,000 component of Akazara scallop striated adductor troponin, which had been tentatively identified as troponin-I, were compared with those of rabbit troponin-I. Both the Mr 52,000 component and rabbit troponin-I together with rabbit tropomyosin inhibited the Mg-ATPase activity of rabbit reconstituted actomyosin to 1/10 of the original activity. The inhibition was neutralized by the addition of Akazara scallop and rabbit troponin-C or Patinopecten scallop calmodulin. The Mr 52,000 component and rabbit troponin-I were insoluble below 0.15 M KCl, but were solubilized by complexing with an equimolar amount of troponin-C or calmodulin. On alkaline urea-polyacrylamide gel electrophoresis, the Mr 52,000 component as well as rabbit troponin-I was found to form a stable complex with troponin-C or calmodulin in the presence of Ca2+.     

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.     

Akazara scallop myosins hybridized with DTNB-light chains of skeletal myosin and with regulatory light chains of gizzard myosin

Two different hybrid myosins were obtained by combining "desensitized" myosin (DM) of Akazara scallop striated adductor with rabbit skeletal DTNB-light chains (DTNB-LC) and with chicken gizzard regulatory light chains (GR-LC). Using the two hybrid myosins, the following were found: (a) DTNB-LC has an inhibitory effect on the Mg-ATPase activities of Akazara DM and acto-DM both in the absence of calcium and in its presence. (b) DTNB-LC also has an enhancing effect on the superprecipitation activity of acto-DM. (c) The Mg-ATPase activities of DM and acto-DM are made sensitive to calcium by GR-LC, regardless of whether GR-LC is phosphorylated or unphosphorylated. (d) However, the Mg-ATPase activity of acto-myosin hybridized with phosphorylated GR-LC is definitely higher than that of acto-myosin hybridized with unphosphorylated GR-LC.     

Cyanogen bromide fragments of Akazara scallop Mr 52,000 troponin-I

Akazara scallop troponin-I of Mr 52,000 (52K) was cleaved into two fragments of 17K and 35K with cyanogen bromide. The 17K fragment, along with tropomyosin, inhibited weakly the rabbit actomyosin Mg-ATPase activity, however, the 35K fragment did not affect it at all. In the presence of Akazara scallop TnT (40K component), the 17K fragment, in turn, strongly inhibited the activity, while the 35K fragment did not. The amino acid composition and partial amino acid sequence suggested that the 17K and 35K fragments were derived from C- and N-terminal regions of the TnI, respectively, and that structural similarity to TnIs from other animals is present in the 17K region.     

Myosin from striated adductor muscle of Chlamys nipponensis akazara.

Myosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant (s 20,2 0 W) and the intrinsic viscosity, [eta] of Akazara myosin thus purified were estimated to be 6.6 S and 2.10 dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of light-chain component (17,000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206,000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1 : 1 : 1. (2) EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin. Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum.     

Reversible changes in the ATPase activity and in the regulatory light chain content upon heat (30 degrees C)-treatment of "Akazara" striated adductor myosin

Myosin from striated adductor muscle of "Akazara" scallop was incubated at 30 degrees C for 5 min in a medium containing 2 mM MgCl2 and various concentrations of Ca2+ ions. It was observed that the 30 degrees C-treatment resulted in a decrease in the Ca2+-sensitivity of myosin-ATPase as well as in the release of the regulatory light chain (EDTA-LC) of myosin. The 30 degrees C-treated myosin was then subjected to a cooling treatment, being kept for 18 h at 0 degrees C. It was found that EDTA-LC recombined with myosin and that Ca2+-sensitivity of myosin-ATPase was restored. It was also found that Ca2+ alone was about 70 times more effective than Mg2+ alone in preventing the heat-induced release of EDTA-LC from occurring and also in recombination of EDTA-LC with the heat-treated myosin.     

Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

Hybrid with rabbit skeletal DTNB-light chains of heavy meromyosin, myosin, and glycerinated fibers from Akazara scallop adductor

It was shown in our previous report (Ojima et al. (1983) J. Biochem. 94, 307-310) that hybridization of Akazara scallop "desensitized" myosin with rabbit skeletal DTNB-light chains led to inhibition of the Mg-ATPase activity of acto-desensitized myosin but to enhancement of its superprecipitation activity. The following are now found: Development of tension in desensitized glycerinated fibers of Akazara adductor is significantly improved when DTNB-light chains are added to the fiber bath. The actin-affinity of desensitized heavy meromyosin in the presence of ATP but in the absence of Ca2+ is decreased by hybridization with chicken gizzard 20K dalton-light chains but significantly increased by that with DTNB-light chains. It is therefore suggested that the increase in actin-binding may account for the enhancing effect of DTNB-light chains on the superprecipitation and on the tension development.     

N-terminal modification and its effect on the biochemical characteristics of Akazara scallop tropomyosins expressed in Escherichia coli

Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar alpha-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca(2+)-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca(2+)-regulation together with troponin.     

Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon

The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.     

Regulation of embryonic smooth muscle myosin by protein kinase C

Phosphorylation of the 20-kDa light chain regulates adult smooth muscle myosin; phosphorylation by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase stimulates the actomyosin ATPase activity of adult smooth muscle myosin; the simultaneous phosphorylation of a separate site on the 20-kDa light chain by the Ca2+/phospholipid-dependent enzyme protein kinase C attenuates the myosin light chain kinase-induced increase in the actomyosin ATPase activity of adult myosin. Fetal smooth muscle myosin, purified from 12-day-old fertilized chicken eggs, is structurally different from adult smooth muscle myosin. Nevertheless, phosphorylation of a single site on the 20-kDa light chain of fetal myosin by myosin light chain kinase results in stimulation of the actomyosin ATPase activity of this myosin. Protein kinase C, in contrast, phosphorylates three sites on the fetal myosin 20-kDa light chain including a serine or threonine residue on the same peptide phosphorylated by myosin light chain kinase. Interestingly, phosphorylation by protein kinase C stimulates the actomyosin ATPase activity of fetal myosin. Moreover, unlike adult myosin, there is no attenuation of the actomyosin ATPase activity when fetal myosin is simultaneously phosphorylated by myosin light chain kinase and protein kinase C. These data demonstrate, for the first time, the in vitro activation of a smooth muscle myosin by another enzyme besides myosin light chain kinase and raise the possibility of alternate pathways for regulating smooth muscle myosin in vivo.     

Effect of nitric oxide on ATPase activity of smooth muscle actomyosin

The effects of nitric oxide donor sodium nitroprusside (SNP) on ATPase activities of smooth muscle actomyosin and myosin were investigated. The effect of SNP on actomyosin ATPase activity was biphasic: the low concentration of this reagent increased the actomyosin ATPase activity while the high concentration exerted opposite effect. These effects were similar to those induced by the specific thiol-alkylating agent N-ethylmaleimide. These data demonstrate that nitric oxide exert the direct effect on smooth muscle contractile proteins. Such effect may be involved in physiological action of NO on smooth muscle.     

Actomyosin isolated from bovine tracheal smooth muscle.

A contractile protein (actomyosin) was isolated from bovine tracheal smooth muscle by the use of "classical" procedures. The protein was considered to be actomyosin because it demonstrated: ATPase activity; superprecipitation upon the addition of ATP, and the solubility and extraction characteristics of actomyosin. The ATPase and superprecipitation reactions were not inhibited by EGTA, and did not require calcium. Lack of an effect of either calcium or EGTA could not be reversed by the addition of active bovine skeletal muscle troponin and tropomyosin. No troponin-tropomyosin like activities could be demonstrated in various tracheal muscle fractions.     

Role of myosin light chain kinase in muscle contraction

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

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.     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.