Presence of oligomers at subcritical actin concentrations

Newman, Jay; Estes, James E.; Selden, Lynn A.; Gershman, Lewis C.

doi:10.1021/bi00327a037

Cited by 55 publications

(35 citation statements)

References 26 publications

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“…(Here, monomeric actin is operationally defined as actin at concentrations less than the critical concentration. We recognize that dimeric and oligomeric actin may be present and contribute to the observed ATP hydrolysis (Newman et al, 1985).) This monomeric actin ATPase activity is markedly higher with Mg 2+ rather than Ca 2+ bound to the high affinity site .…”

Section: Influence Of the Bound Cation On Monomer Actin Atpase Activitymentioning

confidence: 85%

“…The enhancement of spontaneous actin nucleation and the generation of short polymers (oligomerization) by low concentrations of MgC12 has been repeatedly observed (Maruyama, 1981;Tobacman & Kom, 1983;Mozo-Villarias & Ware, 1985;Newman et al, 1985). This observation can be accounted for by the formation of Mg-actin in the actin population, and the facilitated formation of nuclei or oligomers by Mg-actin is attributed to a lower free energy of nucleation for Mg-actin than for Ca-actin (Oosawa & Asakura, 1975;Oosawa, 1983;Tobacman & Kom, 1983;Newman et al, 1985).…”

Section: Nucleationmentioning

confidence: 86%

“…This observation can be accounted for by the formation of Mg-actin in the actin population, and the facilitated formation of nuclei or oligomers by Mg-actin is attributed to a lower free energy of nucleation for Mg-actin than for Ca-actin (Oosawa & Asakura, 1975;Oosawa, 1983;Tobacman & Kom, 1983;Newman et al, 1985). Several other reports of dimer formation have been reported and all have implicated the requirement for MgCI2 (Millonig et al, 1985;Goddette et al, 1986;Matsudaira et al, 1987).…”

Section: Nucleationmentioning

confidence: 98%

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Tightly-bound divalent cation of actin

Estes

Selden

Kinosian

et al. 1992

J Muscle Res Cell Motil

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134

135

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Actin is known to undergo reversible monomer-polymer transitions that coincide with various cell activities such as cell shape changes, locomotion, endocytosis and exocytosis. This dynamic state of actin filament self-assembly and disassembly is thought to be regulated by the properties of the monomeric actin molecule and in vivo by the influence of actin-associated proteins. Of major importance to the properties of the monomeric actin molecule are the presence of one tightly-bound ATP and one tightly-bound divalent cation per molecule. In vivo the divalent cation is thought to be Mg2+ (Mg-actin) but in vitro standard purification procedures result in the preparation of Ca-actin. The affinity of actin for a divalent cation at the tight binding site is in the nanomolar range, much higher than earlier thought. The binding kinetics of Mg2+ and Ca2+ at the high affinity site on actin are considered in terms of a simple competitive binding mechanism. This model adequately describes the published observations regarding divalent cation exchange on actin. The effects of the tightly-bound cation, Mg2+ or Ca2+, on nucleotide binding and exchange on actin, actin ATP hydrolysis activity and nucleation and polymerization of actin are discussed. From the characteristics that are reviewed, it is apparent that the nature of the bound divalent cation has a significant effect on the properties of actin.

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Section: Influence Of the Bound Cation On Monomer Actin Atpase Activitymentioning

confidence: 85%

Section: Nucleationmentioning

confidence: 86%

Section: Nucleationmentioning

confidence: 98%

See 1 more Smart Citation

Tightly-bound divalent cation of actin

Estes

Selden

Kinosian

et al. 1992

J Muscle Res Cell Motil

Self Cite

134

135

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“…Mg-actin assumes a F-monomer-like conformation so that it has higher ATPase activity than Ca-actin. However, there are oligomers in Mg-G-actin solutions even at subcritical concentrations, although no filaments are detectable (27,28). Thus, the higher ATPase activity of Mg-G-actin may result from transient oligomer formation from Mg-G-actin rather than from a more active monomer.…”

Section: Discussionmentioning

confidence: 97%

The Effects of Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop on the Dynamics and Stability of Yeast G-actin

Kuang

Rubenstein

1997

Journal of Biological Chemistry

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The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33°C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.

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“…Although the x-ray patterns show that the G-actin solution contained a homogeneous population of monomers, we would not have been able to detect small qligomqrs in our solutions if they had constituted less than 5% of the-species. Since quasielastic light scattering (39,40,45) has demonstrated that gel-filtered actin always contains trace amounts of oligomers, these nuclei could rapidly (within 50 msec) convert a near homogeneous solution of actin monomers into a large population of short oligomers. However, two pieces of evidence suggest that this explanation is not correct.…”

Section: Methodsmentioning

confidence: 99%

Synchrotron x-ray diffraction studies of actin structure during polymerization.

Matsudaira¹,

Bordas²,

Koch³

1987

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

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Synchrotron x-ray diffraction was used to identify the oligomers that formed during the earliest stages of actin polymerization. Solution diffraction patterns from Gactin (monomer) and from F-actin (polymer) contain information about the size and shape of actin monomers and the length, width, and subunit organization of filaments. Comparison of patterns collected during polymerization reveals an increase in scatter at spacings >9.0 nm; formation of scattering bands at 5.4, 4.9, and 3.4 nm; formation of a scattering minimum at 6.5 nm; and the presence of an isosbestic point at 9.0 nm. These scattering bands arise from the formation of, and organization of subunits in, framents. At various actin concentrations (0.37-5 mg/ml), the change in scatter in these regions follows simple exponential kinetics with no detectable lag. Our analysis of the x-ray patterns shows that by 0.4 sec after mixing, most of the actin has formed dimers, which then rapidly incorporate into oligomers. At 4 mg/ml the early oligomers increase in length to >30.0 nm within 10 sec. These results suggest that under our conditions actin molecules condense into filaments without the rate-limiting formation of nuclei.Studies on the mechanism of actin polymerization in vitro have influenced our ideas on the regulation of actin function in vivo. Central to these studies, and based partly on the kinetics of helical polymerization, is the proposal by Oosawa and colleagues (1-3) that actin polymerization is a nucleation-condensation process. During the initial phase of assembly, three to four actin monomers cooperatively associate into a short oligomer that nucleates rapid filament elongation. Nucleation is the rate-limiting step in assembly and is detected in kinetic studies as a lag in the time course of polymerization. The existence and size of nuclei is deduced from modeling the kinetics of the elongation phase, assuming that nucleation is an obligatory step in the reaction, and from the presence of a critical concentration for assembly. Detailed analysis using sophisticated computer programs have modified the model for actin assembly to include (i) a first-order Mg2" activation step, (ii) nuclei composed of actin trimers, (iii) different rates of assembly at the ends of the filament, and (iv) enhanced nucleation by spontaneous filament breakage. Although a variety of methods, including viscometry (4,5), flow birefringence (6), light scattering (7-11), and fluorimetry (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21), have provided indirect evidence that nucleation is an important feature of actin assembly in vitro, no structural study has directly identified the existence or the structure of the nucleating species.Time-resolved x-ray diffraction using high-intensity synchrotron radiation sources has proved a powerful technique for describing the structure and assembly of proteins into multisubunit complexes. Studies on microtubule and collagen fiber assembly have shown that time-resolved x-ray scattering methods can identify the structures forme...

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Presence of oligomers at subcritical actin concentrations

Cited by 55 publications

References 26 publications

Tightly-bound divalent cation of actin

Tightly-bound divalent cation of actin

The Effects of Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop on the Dynamics and Stability of Yeast G-actin

Synchrotron x-ray diffraction studies of actin structure during polymerization.

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