scholarly journals Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase

1989 ◽  
Vol 258 (3) ◽  
pp. 831-836 ◽  
Author(s):  
D H Heeley ◽  
L B Smillie ◽  
E M Lohmeier-Vogel
Keyword(s):  
Ionic Strength ◽  
Binding Model ◽  
Thin Filaments ◽  
System A ◽  
Atpase Assay ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Low Ionic Strength ◽  
Atpase Inhibition

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.

Contractile proteins from the tomato

1980 ◽  
Vol 58 (7) ◽  
pp. 797-801 ◽  
Author(s):  
Maryanne Vahey ◽  
Stylianos P. Scordilis
Keyword(s):  
Ionic Strength ◽  
Ethylenediaminetetraacetic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Sds Page ◽  
Myosin Subfragment 1 ◽  
Parenchymal Cells ◽  
Low Ionic Strength

Proteins exhibiting all of the basic structural and biochemical characteristics of actin and myosin have been isolated from the parenchymal cells of the fruit of the tomato, Lycopersicon esculentum. Crude cytoplasmic extracts of these cells contain filaments that can be decorated by rabbit skeletal muscle myosin subfragment-1 (S-1). Polymerized tomato actin activates the Mg2+–ATPase of both skeletal and tomato myosin at physiological ionic strength. Tomato actin comigrates with skeletal actin on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) indicating an apparent molecular weight of 45 000. High ionic strength extracts of tomato contain a myosin whose ATPase activity in 0.5 M KCl is maximal in the presence of K+-ethylenediaminetetraacetic acid (K+-EDTA) and is inhibited by Mg2+. Tomato myosin interacts with skeletal F-actin to form an actomyosin complex that can be dissociated by ATP. At low ionic strength the Mg2+–ATPase of the myosin can be activated by actin.

scholarly journals Affinity chromatography of immobilized actin and myosin

1975 ◽  
Vol 149 (2) ◽  
pp. 365-379 ◽  
Author(s):  
R C Bottomley ◽  
I P Trayer
Keyword(s):  
Ionic Strength ◽  
Salt Concentration ◽  
Free Solution ◽  
Heavy Meromyosin ◽  
Chemical Modifications ◽  
Low Concentrations ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
One Step ◽  
Low Ionic Strength

Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.

Binding of adenylosuccinate synthetase to contractile proteins of muscle

1989 ◽  
Vol 257 (1) ◽  
pp. C29-C35 ◽  
Author(s):  
J. P. Manfredi ◽  
R. Marquetant ◽  
A. D. Magid ◽  
E. W. Holmes
Keyword(s):  
Ionic Strength ◽  
Dissociation Constant ◽  
Thin Filament ◽  
Contractile Proteins ◽  
Purine Nucleotide ◽  
Apparent Dissociation Constant ◽  
Thin Filaments ◽  
Adenylosuccinate Synthetase ◽  
Purine Nucleotide Cycle ◽  
Low Ionic Strength

The muscle isozyme of adenylosuccinate synthetase (AdSS), an enzyme of the purine nucleotide cycle, has previously been shown to bind to purified F-actin in buffers of low ionic strength and pH (Ogawa et al. Eur. J. Biochem. 85: 331-338, 1978). We have extended these observations by measuring the association of both crude and purified AdSS with the contractile proteins of muscle in buffers of physiological ionic strength and pH. Under these conditions, the enzyme binds to F-actin, actin-tropomyosin complexes, reconstructed thin filaments, and myofibrils but not to myosin. The apparent dissociation constant of 1.2 microM and binding maximum of 2.6 nmol enzyme/mg myofibrils indicate that binding of AdSS to myofibrils can be physiologically significant. The results suggest that AdSS in muscle may be associated with the thin filament of myofibrils.

scholarly journals The Role of Magnesium in Binding of the Nucleotide Polyphosphate Chain to the Active Site of Myosin Subfragment-1

1983 ◽  
Vol 134 (2) ◽  
pp. 197-204 ◽  
Author(s):  
Marcus C. SCHAUB ◽  
John G. WATTERSON ◽  
Klaus LOTH ◽  
Daniela FOLETTA
Keyword(s):  
Active Site ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Gastropod predation sites: the role of predator and prey in chemical attraction of the hermit crab Clibanarius vittatus

1990 ◽  
Vol 70 (3) ◽  
pp. 583-596 ◽  
Author(s):  
D. Rittschof ◽  
C.M. Kratt ◽  
A.S. Clare
Keyword(s):  
Salivary Gland ◽  
Ionic Strength ◽  
Salivary Glands ◽  
Hermit Crab ◽  
Sea Water ◽  
Hermit Crabs ◽  
Muscle Proteins ◽  
Clibanarius Vittatus ◽  
Low Ionic Strength

Gastropod shells are essential to most hermit crabs. Shell availability limits hermit crab populations. Shells provide protection and the degree of shell-fit controls crab growth and fecundity. Crabs locate new gastropod shells from a distance under water by molecules released from gastropod flesh during predation events. Here we test the hypothesis that the salivary glands of the predatory gastropod are the source of enzymes that digest muscle proteins and release peptide attractants. We describe the anatomy of both the acinous salivary glands and the tubular accessory salivary glands of Busycon contrarium which are similar to those of B. carica. The salivary gland ducts empty at the mouth, suggesting a role in the primary digestion of food. We show that gastropod muscle proteins, extracted by salt solutions with the ionic strength of sea water and purified by precipitation in low ionic strength can be digested by gastropod salivary gland enzymes to generate peptides attractive to the hermit crab, Clibanarius vittatus, in field assays.

scholarly journals Regulation of the acto·myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto·myosin subfragment 1 states

1991 ◽  
Vol 279 (3) ◽  
pp. 711-718 ◽  
Author(s):  
D F A McKillop ◽  
M A Geeves
Keyword(s):  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Thin Filaments ◽  
Closed State ◽  
Myosin Subfragment ◽  
Actomyosin Interaction ◽  
Binding Isotherms ◽  
Myosin Subfragment 1 ◽  
Site Binding

The co-operative binding of myosin subfragment 1 (S1) to reconstituted skeletal-muscle thin filaments has been examined by monitoring the fluorescence of a pyrene probe on Cys-374 of actin. The degree of co-operativity differs when phosphate, sulphate or ADP are bound to the S1 active site. Binding isotherms have been analysed according to the Geeves & Halsall [(1987) Biophys. J. 52, 215-220] model, which proposed that troponin and tropomyosin effected regulation of the actomyosin interaction by controlling an isomerization of the actomyosin complex. The data support the proposal that seven actin monomers associated with a single tropomyosin molecule act as a co-operative unit that can be in one of two states. In the ‘closed’ state myosin can bind to actin, but the subsequent isomerization is prevented. The isomerization is only allowed after the seven-actin unit is in the ‘open’ form. Ca2+ controls the proportion of actin filaments in the ‘closed’ and ‘open’ forms in the absence of myosin heads. The ratio of ‘closed’ to ‘open’ forms is approx. 50:1 in the absence of Ca2+ and 5:1 in its presence.

scholarly journals Correlation between calponin and myosin subfragment 1 binding to F-actin and ATPase inhibition

1997 ◽  
Vol 321 (2) ◽  
pp. 519-523 ◽  
Author(s):  
Pawel T. SZYMANSKI ◽  
Zenon GRABAREK ◽  
Terence TAO
Keyword(s):  
Troponin I ◽  
Sequence Similarity ◽  
Actin Binding ◽  
Amino Acid Sequence Similarity ◽  
Smooth Muscle Contractility ◽  
Skeletal Muscle Myosin ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Muscle Myosin ◽  
Atpase Inhibition

Calponin is a thin-filament-associated protein that has been implicated in the regulation of smooth-muscle contractility. It binds to F-actin and inhibits the MgATPase activity of actomyosin. In the present work we have examined the effect of recombinant chicken gizzard α-calponin (RαCaP) on the binding of rabbit skeletal-muscle myosin subfragment 1 (S1) to F-actin and on the inhibition of its actin-activated MgATPase. We have found that binding of one RαCaP molecule to every three to four actin monomers is sufficient for maximal inhibition of actoŐS1 ATPase. At this RαCaP/actin ratio RαCaP does not interfere with S1 binding to F-actin. At higher concentrations, RαCaP displaces S1 from F-actin and a 1:1 RαCaPŐactin monomer complex is formed. RαCaP is also able to displace troponin I from its complex with F-actin which may reflect the amino acid sequence similarity between RαCaP and troponin I in their actin-binding regions.

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