scholarly journals Pressure-relaxation studies of pyrene-labelled actin and myosin subfragment 1 from rabbit skeletal muscle. Evidence for two states of acto-subfragment 1

1985 ◽  
Vol 232 (2) ◽  
pp. 351-356 ◽  
Author(s):  
J H Coates ◽  
A H Criddle ◽  
M A Geeves
Keyword(s):  
Experimental Data ◽  
Skeletal Muscle ◽  
Ionic Strength ◽  
Pressure Jump ◽  
Pressure Sensitivity ◽  
Pressure Relaxation ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Relaxation Studies ◽  
Significant Conformational Change

We have used actin labelled at Cys-374 with N-(1-pyrenyl)iodoacetamide [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38] to monitor pressure-induced relaxations of acto-myosin subfragment 1. This label greatly increases the sensitivity of measurement of dissociated actin and reveals the presence of two relaxations. The experimental data can be fitted by a model in which actin binds subfragment 1 relatively weakly (K = 5.9 × 10(4) M-1) and then isomerizes to a more tightly bound complex (K = 1.7 × 10(7) M-1). This directly observed isomerization supports the model of Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell. Motil. 5, 351-361]. The rate of the isomerization is too high to be observed in the pressure-jump apparatus (less than 200 microseconds), but analysis of the amplitudes allows estimation of the equilibrium constant of the isomerization as 280 (20 degrees C, 0.1 M-KCl, pH 7). The equilibrium is sensitive to temperature, pressure, ionic strength and the presence of ethylene glycol. The pressure-sensitivity of the isomerization suggests a significant conformational change of the acto-myosin subfragment 1 complex.

scholarly journals A novel pressure-jump apparatus for the microvolume analysis of protein–ligand and protein–protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1

2002 ◽  
Vol 366 (2) ◽  
pp. 643-651 ◽  
Author(s):  
David S. PEARSON ◽  
Georg HOLTERMANN ◽  
Patricia ELLISON ◽  
Christine CREMO ◽  
Michael A. GEEVES
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Protein Interactions ◽  
Muscle Protein ◽  
Smooth Muscles ◽  
High Volume ◽  
Pressure Jump ◽  
Original Design ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50μl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383–1393]. This equipment can apply pressure changes of ±20MPa (maximally) in time periods as short as 80μs and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1min) exchange of samples. In the present study, we show that this system can perturb the binding of 2′(3′)-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2′(3′)-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23° tilt in the tail of S1 bound to actin.

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 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.

Comparison of effects of smooth and skeletal muscle tropomysins on interactions of actin and myosin subfragment 1

Biochemistry ◽  
1984 ◽  
Vol 23 (18) ◽  
pp. 4150-4155 ◽  
Author(s):  
David L. Williams ◽  
Lois E. Greene ◽  
Evan Eisenberg
Keyword(s):  
Skeletal Muscle ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Biochemistry ◽  
1984 ◽  
Vol 23 (21) ◽  
pp. 4885-4889 ◽  
Author(s):  
Joseph M. Chalovich ◽  
Leonard A. Stein ◽  
Lois E. Greene ◽  
Evan Eisenberg
Keyword(s):  
Ionic Strength ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

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.

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle

1986 ◽  
Vol 229 (1254) ◽  
pp. 85-95 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Binding Affinity ◽  
Kinetic Studies ◽  
Rabbit Skeletal Muscle ◽  
Fluorescent Label ◽  
Weakly Bound ◽  
Thin Filaments ◽  
Fluorescence Titration ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

We have used actin labelled in Cys–374 with N -(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin–troponin–tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin–tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin–subfragment 1 complex from a state in which actin is weakly bound ( K a = 5.9 x 10 4 M -1 ) to a more tightly bound complex ( K a = 1.7 x 10 7 M -1 ) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin–tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.

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