scholarly journals Functional interrelationship between calponin and caldesmon

1991 ◽  
Vol 280 (1) ◽  
pp. 33-38 ◽  
Author(s):  
R Makuch ◽  
K Birukov ◽  
V Shirinsky ◽  
R Dabrowska
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Smooth Muscle Contraction ◽  
The Other ◽  
High Concentration ◽  
Thin Filaments ◽  
Actomyosin Atpase ◽  
Molar Excess ◽  
Low Concentrations

Calponin and caldesmon, constituents of smooth-muscle thin filaments, are considered to be potential modulators of smooth-muscle contraction. Both of them interact with actin and inhibit ATPase activity of smooth- and skeletal-muscle actomyosin. Here we show that calponin and caldesmon could bind simultaneously to F-actin when used in subsaturating amounts, whereas each one used in excess caused displacement of the other from the complex with F-actin. Calponin was more effective than caldesmon in this competition: when F-actin was saturated with calponin the binding of caldesmon was eliminated almost completely, whereas even at high molar excess of caldesmon one-third of calponin (relative to the saturation level) always remained bound to actin. The inhibitory effects of low concentrations of calponin and caldesmon on skeletal-muscle actomyosin ATPase were additive, whereas the maximum inhibition of the ATPase attained at high concentration of each of them was practically unaffected by the other one. These data suggest that calponin and caldesmon cannot operate on the same thin filaments. CA(2+)-calmodulin competed with actin for calponin binding, and at high molar excess dissociated the calponin-actin complex and reversed the calponin-induced inhibition of actomyosin ATPase activity.

scholarly journals Calcium ion-regulated thin filaments from vascular smooth muscle

1980 ◽  
Vol 185 (2) ◽  
pp. 355-365 ◽  
Author(s):  
S B Marston ◽  
R M Trevett ◽  
M Walters
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin C ◽  
Myosin Atpase ◽  
Protein G ◽  
Thin Filaments ◽  
Myosin Atpase Activity

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 × 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.

Modulation of arterial Na+-K+-ATPase-induced [Ca2+]i reduction and relaxation by norepinephrine, ET-1, and PMA

1999 ◽  
Vol 276 (2) ◽  
pp. H651-H657 ◽  
Author(s):  
Francisco Pérez-Vizcaíno ◽  
Angel Cogolludo ◽  
Juan Tamargo
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Membrane Potential ◽  
Atpase Activity ◽  
Vascular Tone ◽  
Smooth Muscle Contraction ◽  
Mesenteric Arteries ◽  
Free Solution ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

Na+-K+-ATPase plays a major role in regulating membrane potential and vascular tone. We analyzed the modulation by norepinephrine (NE), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) of Na+-K+-ATPase-induced cytoplasmic free Ca2+concentration ([Ca2+]i) reduction and relaxation in isolated endothelium-denuded piglet mesenteric arteries. KCl (0.2–8.8 mM)-induced [Ca2+]ireduction and relaxation in arteries incubated in K+-free solution were used as functional indicators of Na+-K+-ATPase activity. KCl-induced relaxations after exposure to K+-free solution were associated with a reduction in [Ca2+]i, as measured by fura 2 fluorescence. However, KCl reduced [Ca2+]ibelow resting values, whereas force was reduced to near resting values. NE, ET-1, and PMA inhibited the relaxant effects of KCl, and this effect was attenuated by the protein kinase C inhibitor staurosporine but not by the phospholipase A2inhibitor quinacrine. However, ET-1 and PMA potentiated the [Ca2+]i-reducing effect of KCl. In conclusion, ET-1, PMA, and NE are functional inhibitors of Na+-K+-ATPase activity in endothelium-denuded piglet mesenteric arteries, even when the direct effect on the enzyme activity may be stimulatory rather than inhibitory. This can be explained because ET-1, PMA, and NE induce Ca2+ sensitization for smooth muscle contraction, and therefore relaxations do not parallel the reductions in [Ca2+]iafter the activation of Na+-K+-ATPase.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

scholarly journals Calcium-Dependent Myosin from Insect Flight Muscles

1974 ◽  
Vol 63 (5) ◽  
pp. 553-563 ◽  
Author(s):  
William Lehman ◽  
Belinda Bullard ◽  
Kathleen Hammond
Keyword(s):  
Atpase Activity ◽  
Insect Flight ◽  
Calcium Regulation ◽  
Regulatory Proteins ◽  
Myosin Molecule ◽  
Thin Filaments ◽  
Actomyosin Atpase ◽  
Calcium Dependent ◽  
Regulatory Systems ◽  
Flight Muscles

Calcium regulation of the insect actomyosin ATPase is associated with the thin filaments as in vertebrate muscles, and also with the myosin molecule as in mollusks. This dual regulation is demonstrated using combinations of locust thin filaments with rabbit myosin and locust myosin with rabbit actin; in each case the ATPase of the hybrid actomyosin is calcium dependent. The two regulatory systems are synergistic, the calcium dependency of the locust actomyosin ATPase being at least 10 times that of the hybrid actomyosins described above. Likewise Lethocerus myosin also contains regulatory proteins. The ATPase activity of Lethocerus myosin is labile and is stabilized by the presence of rabbit actin. Tropomyosin activates the ATPase of insect actomyosin and the activation occurs irrespective of whether the myosin is calcium dependent or rendered independent of calcium.

The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses

2003 ◽  
Vol 95 (1) ◽  
pp. 413-425 ◽  
Author(s):  
Susan J. Gunst ◽  
Jeffrey J. Fredberg
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Smooth Muscle Contraction ◽  
Mechanical Function ◽  
Thin Filaments ◽  
Cross Bridge ◽  
Contractile Activation ◽  
Contraction Cycle ◽  
Soft Glasses

Smooth muscle exhibits biophysical characteristics and physiological behaviors that are not readily explained by present paradigms of cytoskeletal and cross-bridge mechanics. There is increasing evidence that contractile activation of the smooth muscle cell involves an array of cytoskeletal processes that extend beyond cross-bridge cycling and the sliding of thick and thin filaments. We review here the evidence suggesting that the biophysical and mechanical properties of the smooth muscle cell reflect the integrated interactions of an array of highly dynamic cytoskeletal processes that both react to and transform the dynamics of cross-bridge interactions over the course of the contraction cycle. The activation of the smooth muscle cell is proposed to trigger dynamic remodeling of the actin filament lattice within cellular microdomains in response to local mechanical and pharmacological events, enabling the cell to adapt to its external environment. As the contraction progresses, the cytoskeletal lattice stabilizes, solidifies, and forms a rigid structure well suited for transmission of tension generated by the interaction of myosin and actin. The integrated molecular transitions that occur within the contractile cycle are interpreted in the context of microscale agitation mechanisms and resulting remodeling events within the intracellular microenvironment. Such an interpretation suggests that the cytoskeleton may behave as a glassy substance whose mechanical function is governed by an effective temperature.

scholarly journals Ethanol has different effects on Ca2+-transport ATPases of muscle, brain and blood platelets

1995 ◽  
Vol 312 (3) ◽  
pp. 733-737 ◽  
Author(s):  
F Mitidieri ◽  
L de Meis
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Blood Platelets ◽  
Activity Increase ◽  
Low Concentrations ◽  
Biphasic Effect ◽  
Transport Atpases

The effects of ethanol on different sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCAs) were studied. In sarcoplasmic reticulum vesicles, ethanol concentrations varying from 5 to 20% promoted a progressive inhibition of Ca2+ uptake, enhancement of Ca2+ efflux, activation of the ATPase activity, increase of the enzyme phosphorylation by ATP and inhibition of enzyme phosphorylation by P1. The effects of ethanol on Ca2+ uptake and Ca2+ efflux were antagonized by Mg2+, P(i) and spermine. The increased efflux promoted by ethanol was antagonized by Ca2+ and thapsigargin. In brain and platelet vesicles a biphasic effect of ethanol was observed, so that activation occurred at low concentrations (5-10%) and inhibition at higher concentrations. The activation was not observed with the use of n-propanol and n-butanol. Different from the situation in sarcoplasmic reticulum, the decrease of the Ca2+ uptake in brain and platelet vesicles was associated with an inhibition of the ATPase activity. Mg2+ and P(i) antagonized the enhancement of Ca2+ efflux and the inhibition of Ca2+ uptake promoted by ethanol. However, thapsigargin and Ca2+ did not arrest the Ca2+ efflux promoted by ethanol in brain and platelet preparations. These results suggest that, in sarcoplasmic reticulum vesicles, ethanol uncouples the pump, promoting its activity as a Ca2+ channel. The SERCA isoform found in skeletal muscle has different properties from the isoforms found in brain and blood platelets.

Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig

2010 ◽  
Vol 298 (5) ◽  
pp. C1118-C1126 ◽  
Author(s):  
Masaru Watanabe ◽  
Masatoshi Yumoto ◽  
Hideyuki Tanaka ◽  
Hon Hui Wang ◽  
Takeshi Katayama ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Thin Filaments ◽  
Tension Development

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca2+-induced tension development at any given Ca2+ concentration but had little effects on the Ca2+-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM Mg2+-induced, “myosin light chain phosphorylation-independent” tension development at more than 10 μM. Furthermore, blebbistatin induced conformational change of smooth muscle myosin (SMM) and disrupted arrangement of SMM and thin filaments, resulting in inhibition of actin-SMM interaction irrespective of activation with Ca2+. In addition, blebbistatin partially inhibited Mg2+-ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 μM. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent.

scholarly journals Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon

1995 ◽  
Vol 108 (10) ◽  
pp. 3253-3265 ◽  
Author(s):  
M.F. Pittenger ◽  
A. Kistler ◽  
D.M. Helfman
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Insect Cell ◽  
The Other ◽  
Actin Binding ◽  
Gene Products ◽  
Free Concentration ◽  
Alternatively Spliced ◽  
Beta Gene

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.

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