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.

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 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.

scholarly journals ELECTRON MICROSCOPIC INVESTIGATIONS OF ACTOMYOSIN AS A FUNCTION OF IONIC STRENGTH

1968 ◽  
Vol 39 (3) ◽  
pp. 620-629 ◽  
Author(s):  
N. Ikemoto ◽  
S. Kitagawa ◽  
A. Nakamura ◽  
J. Gergely
Keyword(s):  
Ionic Strength ◽  
Uranyl Acetate ◽  
Myosin Molecule ◽  
Heavy Meromyosin ◽  
Electron Microscopic ◽  
Low Ionic Strength ◽  
Microscopic Investigations

Natural actomyosin at µ = 0.6 appears in various forms, including the regular arrowhead structures originally reported by Huxley (1), when it has been stained negatively with 1% uranyl acetate. In addition to the arrowheads, thin whiskers, 700–1200 A in length and 20 A in width, attached to the arm of the arrowheads have been demonstrated. The dimensions of the whiskers and arms of the arrowheads are practically the same as those of the light meromyosin (LMM) and the heavy meromyosin (HMM) moieties of the single myosin molecule, respectively. Changes in the electron microscopically distinguishable elements during aggregation of natural actomyosin on reduction of the ionic strength have been observed. At µ = 0.4, partial aggregation of the LMM whiskers begins to result in some parallel alignment of the arrowhead-bearing filaments (acto-HMM). In the range of µ = 0.3–0.1, the LMM whiskers merge into smooth filaments which are arranged alternatingly with arrowhead-bearing filaments. Thus, lateral aggregation of composite actomyosin filaments (acto-HMM + LMM whiskers) results with the LMM moieties as links. This view is supported by the following facts: (a) acto-HMM is devoid of whiskers and does not show lateral aggregation at µ = 0.1; (b) natural actomyosin digested with trypsin at µ = 0.6, which was followed by removal of LMM aggregates at low ionic strength, is essentially the same as acto-HMM at µ = 0.1; and (c) digestion with trypsin of natural actomyosin at µ = 0.2 for varying periods of time leads to a separation of arrowhead-bearing filaments from LMM aggregates.

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 Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

1982 ◽  
Vol 95 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M S Mooseker ◽  
T D Pollard ◽  
K A Wharton
Keyword(s):  
Ionic Strength ◽  
Intestinal Epithelial ◽  
Capping Protein ◽  
The Core ◽  
Nucleated Polymerization ◽  
Myosin Subfragment ◽  
Brush Borders ◽  
Pointed End ◽  
Low Ionic Strength

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

scholarly journals The Passive Permeability of the Red Blood Cell to Cations

1966 ◽  
Vol 50 (1) ◽  
pp. 171-188 ◽  
Author(s):  
Paul L. LaCelle ◽  
Aser Rothstein
Keyword(s):  
Ionic Strength ◽  
Inflection Point ◽  
Ph Effect ◽  
Human Red Blood Cells ◽  
Low Concentrations ◽  
Goldman Equation ◽  
Low Ionic Strength ◽  
Straight Lines ◽  
The Relationship ◽  
Determining Factor

The efflux of salt from human red blood cells suspended in isotonic sucrose plus low concentrations of salt, was measured under steady-state conditions. The relationship between the efflux and the log of the salt concentration can be fitted by two straight lines with a sharp inflection point, the steeper slope occurring at concentrations below 0.2 mM NaCl. The determining factor in the rate of efflux is the ionic strength rather than the specific monovalent cations or anions and the effects are completely reversible. With an increase in temperature, the effects of reduced ionic strength are more pronounced and the inflection point is shifted toward higher salt concentrations. An increase in pH leads to an increased efflux at a given ionic strength, but the size of the pH effect is small at low ionic strength. At a given pH, the data can be fitted by a simplified form of the Goldman equation suggesting that with reduction in ionic strength, the permeability remains constant until the inflection point is reached. At that ionic strength, a sharp reversible transition to a new permeability state occurs. The permeability increases with an increase in the external but not the internal pH.

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