Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin.

Gordon, Debbie; Yang, Y Z; Korn, Edward D.

doi:10.1016/s0021-9258(17)32873-9

Cited by 204 publications

(37 citation statements)

References 22 publications

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“…Actin concentration was measured from the absorbance at 290nm, the absorbance of lmg of pure actin/ml (light-path 1 cm) being taken to be 0.62 (Gordon et al, 1976). Molar concentration of G-actin was calculated on the basis of an Mr value of 42000 (Collins & Elzinga, 1975).…”

Section: Methodsmentioning

confidence: 99%

The polymerization of actin. A study of the nucleation reaction

Grazi¹,

Ferri²,

Cino³

1983

Biochemical Journal

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We compared the properties of the nuclei that accumulate in 7.5 mM-KCl in ATP-G-actin solutions and of the oligomers that are formed by sonication of either G-actin or F-actin. We found that the ability of the above species to prime the polymerization of actin decays with different rates. The nuclei are stable in 7.5 mM-KCl (they decay with a rate constant of 1.5 X 10(-3) s -1 at pH 7.8 at 22 degrees C in the absence of KCl). The oligomers formed by sonication of either G-actin or F-actin, once the sonication is stopped, revert to simpler structures or evolve into F-actin, depending on the KCl concentration in which they are kept. In 10.5 mM-KCl at pH 7.8 at 22 degrees C their priming ability decays with a rate constant of 6 X 10(-3) s -1. We propose that the nuclei that form spontaneously in 7.5 mM-KCl are not directly susceptible to elongation. They must first be converted into activated nuclei, which exist in very low concentration at the steady state. The activated nuclei are directly susceptible to elongation, they have a short life and they decay rapidly into the ground state unless the elongation reaction occurs. Sonication displaces the steady-state concentration in favour of the activated state.

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Section: Methodsmentioning

confidence: 99%

The polymerization of actin. A study of the nucleation reaction

Grazi¹,

Ferri²,

Cino³

1983

Biochemical Journal

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“…There is considerable evidence that actin isoforms in vitro can co-polymerize and activate myosin ATPase (23,24). However, isoforms vary in their interactions with some actin regulating proteins (25), in their sensitivities to parameters affecting polymerization, and in their activations of myosin ATPase (4, 23, 24).…”

Section: Isoforms Of Actinmentioning

confidence: 99%

Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes.

McKenna¹,

Meigs²,

Wang³

1985

The Journal of Cell Biology

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We have investigated whether living muscle and nonmuscle cells can discriminate between microinjected muscle and nonmuscle actins. Muscle actin purified from rabbit back and leg muscles and labeled with fluorescein isothiocyanate, and nonmuscle actin purified from lamb brain and labeled with lissamine rhodamine B sulfonyl chloride, were co-injected into chick embryonic cardiac myocytes and fibroblasts. When fluorescence images of the two actins were compared using filter sets selective for either fluorescein isothiocyanate or lissamine rhodamine B sulfonyl chloride, essentially identical patterns of distribution were detected in both muscle and nonmuscle cells. In particular, we found no structure that, at this level of resolution, shows preferential binding of muscle or nonmuscle actin. In fibroblasts, both actins are associated primarily with stress fibers and ruffles. In myocytes, both actins are localized in sarcomeres. In addition, the distribution of structures containing microinjected actins is similar to that of structure containing endogenous F-actin, as revealed by staining with fluorescent phalloidin or phallacidin. Our results suggest that, at least under these experimental conditions, actin-binding sites in muscle and nonmuscle cells do not discriminate among different forms of actins.

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“…The method was modified by incorporation of l mM calcium chloride, 0.1 M sodium chloride, and 0.01% PMSF throughout. Purified DNAase I was calibrated for inhibition, using G-actin, standardized spectrophotometrically (42) and corrected for any denatured fraction in terms of the change in intrinsic fluorescence on denaturation with EDTA, following Lehrer and Kerwar (43).…”

Section: Deoxyribonuclease Assay For Actinmentioning

confidence: 99%

Structural and dynamic states of actin in the erythrocyte

Pinder¹,

Gratzer²

1983

The Journal of Cell Biology

110

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Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons shows it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1 x 10 s and 5 x 10 s for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays for actin on lysed whole cells. The concentration of monomeric actin at 0°C is ~16 #g/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing ("pointed") ends of free actin filaments. This suggests that these ends of the actin "protofilaments" in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to -180/~g/ml of packed cells, which is nearly 70% of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250/~g/ml of cells, compared with 270#g/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion.

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Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin.

Cited by 204 publications

References 22 publications

The polymerization of actin. A study of the nucleation reaction

The polymerization of actin. A study of the nucleation reaction

Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes.

Structural and dynamic states of actin in the erythrocyte

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