Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.1 (myosin ATPase)
1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca2+. Ultracentrifugation in the presence of 10-20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca(2+)-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca2+ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca(2+)-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.
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PMID:Filamin and gelsolin influence Ca(2+)-sensitivity of smooth muscle thin filaments. 770 23

The binding of chicken gizzard caldesmon to actin was studied both in the presence and the absence of caltropin using Airfuge centrifugation experiments, disulfide cross-linking studies, and the fluorescent probe acrylodan (6-acryloyl-2-(dimethylamino)naphthalene). In co-sedimentation studies most of the caldesmon pelleted along with actin. However, when caldesmon in the presence of caltropin was mixed with actin, caldesmon did not pellet along with actin following high speed centrifugation, suggesting that caltropin has significantly weakened its binding to actin. The caltropin effect was noticed even when tropomyosin was included in the reaction mixture. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. The addition of actin produced a nearly 70% increase in fluorescent intensity, accompanied by a blue shift in the emission maximum (i.e. lambda em (max) = 505 +/- 2 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of labeled caldesmon with actin indicated a strong affinity (K alpha = approximately 6 x 10(7) M-1). When actin was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered (K alpha = approximately 2 x 10(7) M-1). Caltropin, which is very effective in reversing caldesmon's inhibition of the actin-activated myosin ATPase (Mani, R. S., McCubbin, W. D., and Kay, C. M. (1992) Biochemistry 31, 11896-11901), is shown in the present study to have a pronounced effect on its binding to actin, suggesting a major role for caltropin in regulating caldesmon in smooth muscle.
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PMID:Effect of caltropin on caldesmon-actin interaction. 789 6

The steric model of muscle regulation holds that tropomyosin strands running along thin filaments move away from myosin-binding sites on actin when muscle is activated. Exposing these sites would permit actomyosin interaction and contraction to proceed. This compelling and widely cited model is based on changes observed in X-ray diffraction patterns of skeletal muscle following activation. Although analysis of X-ray patterns can suggest models of filament structure, unambiguous interpretation is not possible. In contrast, three-dimensional reconstruction of thin-filament electron micrographs could, in principle, offer direct confirmation of the predicted tropomyosin movement, but so far tropomyosin in skeletal muscle has been resolved definitively only in the 'on' state but not in the 'off' state. Thin filaments from the arthropod Limulus have a similar composition to those from vertebrate skeletal muscle, and troponin-tropomyosin is distributed in both species with the same characteristic 38-nm periodicity. Limulus thin filaments activate skeletal muscle myosin ATPase at micromolar Ca2+ concentrations and confer a high calcium dependence on the enzyme. Arthropod and vertebrate troponin subunits form functional hybrids in vitro and the respective tropomyosins are functionally interchangeable, arguing for a common mechanism of thin-filament-linked regulation in the two phyla. Here we report that tropomyosin is readily resolved in native filaments of troponin-regulated Limulus muscle in both the 'on' and 'off' states, and demonstrate tropomyosin movement, providing support for the importance of steric effects in muscle activation.
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PMID:Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. 810 84

We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity-purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin-activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7-9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thin-filament-like system in erythrocyte.
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PMID:Purification and characterization of erythrocyte caldesmon. Hypothesis for an actin-linked regulation of a contractile activity in the red blood cell membrane. 830 18

The increasing interest in the metal ion aluminum fluoride and beryllium fluoride complexes as phosphate analogs in the myosin ATPase reaction and in muscle fiber studies prompted the examination of their interactions with the regulatory system of troponin and tropomyosin. In this work, the effects of these metal ion analogs on the spectral properties of the Ca(2+)-binding subunit of troponin, troponin C (TnC), were examined. In contrast to beryllium fluoride which did not change the spectral properties of TnC, aluminum fluoride binding induced an increase in both the alpha-helicity and the tyrosine fluorescence of TnC and exposed a hydrophobic region on this protein for fluorescent probe binding. Aluminum fluoride also reduced the Ca2+ and/or Mg(2+)-induced changes on TnC. These results indicate a direct interaction of aluminum fluoride with TnC and merit consideration in designing muscle fiber experiments with this phosphate analog.
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PMID:Aluminum fluoride interactions with troponin C. 831 88

We expressed the following fragments of chicken gizzard caldesmon in the pMW 172/BL21 (DE3) system at 0.4-2.2 mg of pure protein/liter of culture: full-length smooth muscle caldesmon (CDh) (amino acids 1-756), nonmuscle caldesmon (CDl), amino acids 1-128 (N128), 1-578 (N578), 230-419, 606-756 (606C), and 658-756 (658C). CDh bound tropomyosin with a Kd of 1.5 microM; N578, 230-419, and 606C bound with affinities at least 2-5 fold lower; N128 bound weakly; and 658C did not bind. Only N128 and N578 bound to smooth muscle myosin, both about 10-fold weaker than CDh and CDl. Only 606C and 658C bound to actin-tropomyosin with affinities CDh = 606C > 658C. The binding to actin-tropomyosin was biphasic, whereas the binding to actin was monophasic, corresponding to the weak binding component found in the presence of tropomyosin. Calmodulin bound only to the C-terminal fragments with the same affinity as CDh. CDh, 606C, and 658C inhibited actin-tropomyosin-activated myosin ATPase, with maximal inhibition correlated with 1 caldesmon bound/14 actins, and inhibition was reversed by Ca(2+)-calmodulin. Thus, the actomyosin ATPase regulatory function, calmodulin binding, and most actin binding is located within the C-terminal 99 amino acids, whereas tropomyosin binding is distributed throughout the rest of the molecule.
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PMID:Binding and regulatory properties of expressed functional domains of chicken gizzard smooth muscle caldesmon. 849 61

The smooth muscle tropomyosin isoforms beta and gamma were isolated in pure form and labeled with N-(1-pyrenyl)iodoacetamide (PIA) on the cysteine residues at either the N- or the C-terminal region (Cys-36 and Cys-190 of beta- and gamma-isoforms, respectively). The effect of caldesmon (CaD) on local conformational changes in different regions of the tropomyosin molecule was determined on the basis of changes in the excimer fluorescence (excited dimer of pyrene) formed in homodimers of tropomyosin isoforms. In the absence of actin, excimer fluorescence from the pyrene at Cys-190 of gamma-tropomyosin homodimer decreased in a simple manner on the addition of CaD, whereas the excimer from the Cys-36 of beta-tropomyosin homodimer exhibited a biphasic change, suggesting that additional weak binding sites exist near Cys-36. In the presence of actin, CaD-induced changes in the excimer fluorescence of pyrene-tropomyosin were observed only with Cys-36, and this change was associated with an inhibition of actin-activated myosin ATPase. A competition study with unlabeled tropomyosin isoforms indicated that the different excimer changes exhibited by beta- and gamma-tropomyosin in the presence of CaD were due to conformational changes in different regions of the tropomyosin molecule and not to differences in their affinities for CaD. Experiments with recombinant CaD mutants derived using the baculovirus expression system showed that the inhibition of tropomyosin potentiation of actomyosin ATPase by CaD requires the regions between residues 728-756 and 718-727 on the CaD molecule, although the latter region was sufficient for direct interaction with tropomyosin.
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PMID:Inhibition of smooth muscle actomyosin ATPase by caldesmon is associated with caldesmon-induced conformational changes in tropomyosin bound to actin. 852 57

A series of C-terminal deletion mutants of chicken gizzard smooth muscle caldesmon (CaD) were made using a polymerase chain reaction cloning strategy and a baculovirus expression system, and the precise locations of the functional domains of CaD involved in the regulation of actomyosin ATPase and the binding of actin, tropomyosin, and calmodulin were analyzed. Our results reveal a high affinity calmodulin-binding domain that consists of at least three calmodulin-binding determinants localized in residues 690-717, 658-689, and 628-657. The residues between positions 718 and 756 and positions 598 and 627 have no detectable calmodulin-binding site. A high affinity tropomyosin-binding domain is located between residues 718 and 756. The 159 residues at the C terminus of CaD contain multiple actin-binding determinants; the major ones are localized in the regions between residues 718 and 756 and residues 690 and 717. The amino acid residues between positions 718 and 756 contain the major determinant involved in the inhibition of the actin activation of smooth muscle myosin ATPase since CaD-(1-717) caused only 30% of the inhibition produced by the full-length CaD. Further deletion between residues 690 and 717 (CaD-(1-689) revealed a low level (10% of that seen for full-length CaD) of inhibition of the actomyosin ATPase. These data clearly demonstrate that the region of the last 66 amino acid residues at the CaD C terminus contains two or more major actin-binding motifs, one tropomyosin-binding domain, one high affinity calmodulin-binding determinant, and the domain that is responsible for the inhibition of the actin-activated ATPase of myosin.
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PMID:Characterization of the functional domains on the C-terminal region of caldesmon using full-length and mutant caldesmon molecules. 856 84

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy. For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1-->V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats.
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PMID:Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy. 856 62

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.
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PMID:Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin. 868 82


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