A model for the myosin molecule

Kielley, W. Wayne; Harrington, William F.

doi:10.1016/0006-3002(60)90037-8

Cited by 492 publications

(140 citation statements)

References 37 publications

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“…Other major contractile proteins were identified on two-dimensional gels (13) by comigration of labeled proteins with human contractile proteins purified directly from adult human muscle (14). The distribution of acetylcholine receptors was assayed by autoradiographic analysis of the binding of '"I-labeled-a-bungarotoxin to intact myotubes in culture (15,16).…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of human muscle cells.

Blau¹,

Webster²

1981

Proc. Natl. Acad. Sci. U.S.A.

351

211

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We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations ofmuscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, aactin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.

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Section: Methodsmentioning

confidence: 99%

Isolation and characterization of human muscle cells.

Blau¹,

Webster²

1981

Proc. Natl. Acad. Sci. U.S.A.

351

211

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“…Frozen stocks were 2-4-fold less active than freshly prepared MmCP. Myosin from rabbit skeletal muscle (19) was treated with N-ethylmaleimide (Sigma) (20) and stored at Ϫ20°C in 10 mM imidazole, pH 7, 0.5 M KCl, 1 mM DTT, and 50% glycerol.…”

Section: Proteinsmentioning

confidence: 99%

Single Molecule Kinetic Analysis of Actin Filament Capping

Kuhn

Pollard

2007

Journal of Biological Chemistry

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We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 ؋ 10 6 M ؊1 s ؊1 and dissociation of 0.0003 s ؊1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ), PI(4,5)P 2 , and PI(3,4,5)P 3 , prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 ؋ 10 5 M ؊1 s ؊1 ) and a faster dissociation rate constant (0.004 s ؊1 ). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.The motility of animal cells depends on the coordinated assembly of actin filaments at the leading edge. Arp2/3 complex promotes actin polymerization by nucleating filaments that grow as branches from a mother filament (1, 2). Capping protein (CP, 3 called CapZ in muscle) appears to play an important role in generating the zone of short, highly branched actin filaments at the leading edge. CP is a heterodimer of structurally similar ␣ and ␤ subunits that form a mushroom-like structure with one flexible and one immobile COOH-terminal "tentacle" that bind the two terminal subunits of an actin filament (3). CP binds the barbed end of actin filaments tightly with a K d of 0.1-1 nM. Given an association rate constant of 3-4 M Ϫ1 s Ϫ1 and a cytoplasmic concentration of 1 M (4), CP should terminate the growth of actin filaments with a half-time of about 0.2 s after their nucleation. Terminating growth quickly keeps the filaments short, so that they do not buckle under the force of polymerization as they push the membrane forward.Vertebrate CP dissociates from barbed ends with a half-time of 30 min (4), far too slow for simple, spontaneous dissociation to be involved with the conversion of the network of short, branched filaments at the leading edge to longer, unbranched filaments toward the cell body. As CP prevents both dissociation of subunits from the barbed end and end-to-end filament annealing, some mechanism must accelerate the removal of CP from filament ends.Both lipids and proteins, such as Ena/VASP (5-7) and CARMIL (8), can inhibit the interaction of CP with actin filaments. Polyphosphatidylinositides (PPIs) bind CP and block its interaction with filaments both in vivo (9) and in vitro (10). Addition of PIP 2 to mixtures of capped filaments, CP, and actin monomers in vi...

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“…The apparent partial specific volume, 6, was then calculated from the solution density by (Kielley & Harrington, 1960;Casassa & Eisenberg, 1961, 1964 where p is the density of the solution in grams per milliliter, p o is that of the solvent, and c is the protein concentration in grams per milliliter. The densities of protein solutions were measured at 20 "C at conditions such that the molality of the solvent composition and the chemical potential were kept, in turn, identical in the solvent and in the protein solution.…”

Section: Density Measurementsmentioning

confidence: 99%

On the role of surface tension in the stabilization of globular proteins

1996

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The stabilization of proteins by a variety of co-solvents can be related to their property of increasing the surface tension of water. It is demonstrated that, during the thermal unfolding of proteins, this increase of the surface tension can be overcome by the increase in the temperature of the solution at the midpoint of the transition, T,, and the weak binding of co-solvent molecules. Three such co-solvents were studied: trehalose, lysine hydrochloride (LysHCl), and arginine hydrochloride (ArgHC1). Trehalose and LysHCl increase the midpoint of T,. The increase of the surface tension by addition of trehalose is completely compensated by its decrease due to the increase in T,. However, for LysHC1, the increase of the surface tension by the co-solvent is partly reduced by its binding to the protein. Later studies on the preferential interaction2 of proteins with a variety of co-solvents have identified the surface tension ef-'The term preferential interaction refers to the net interaction between the protein and the solvent components, water and the co-solvent, as measured by an equilibrium thermodynamic approach, such as dialysis equilibrium. When the interactions are weak, as is the case with co-solvents, such as sugars, glycerol, amino acids, or urea, the measured quantity is the net balance between the weak interactions (binding) of water and co-solvent molecules to the protein over its entire surface. If, on average, the affinity of protein surface loci for the co-solvent is greater than that for water, the dialysis equilibrium experiment will result in an excess of co-solvent at the protein surface over its concentration in the bulk solvent, and the measured binding stoichiometry will be positive, which means that co-solvent is preferentially bound relative to water. In that case, the "preferential binding" will be positive. In the converse case, in which, on average, the affinity of the protein surface loci is greater for water than for co-solvent molecules, there will be an excess of water at the protein surface over its concentration in the 372

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A model for the myosin molecule

Cited by 492 publications

References 37 publications

Isolation and characterization of human muscle cells.

Isolation and characterization of human muscle cells.

Single Molecule Kinetic Analysis of Actin Filament Capping

On the role of surface tension in the stabilization of globular proteins

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