Comparative biochemistry of non-muscle actins.

Gordon, Debbie; Boyer, James L.; Korn, Edward D.

doi:10.1016/s0021-9258(17)40971-9

Cited by 248 publications

(40 citation statements)

References 30 publications

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“…It is an essential component of muscle and contractile structures and cytoskeletons of nonmuscle cells. Nonmuscle cell extracts prepared under physiological salt conditions contain actin in both monomeric and filamentous forms (1, 7, 10, 14,31,33,43). It is generally agreed that the filamentous actin plays a major role in cell motility; it is also assumed that transformation from the monomeric to filamentous form takes place when cells form contractile and/or cytoskeletal structures.…”

mentioning

confidence: 99%

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

Hosoya¹,

Mabuchi²

1984

The Journal of Cell Biology

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A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase ISepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.

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mentioning

confidence: 99%

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

Hosoya¹,

Mabuchi²

1984

The Journal of Cell Biology

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“…The yield of nonmuscle actin is sufficient for several types of biochemical assays, including kinetic analyses of binding of actin binding proteins [Schafer et al, 1996] and comparable to yields obtained from other methods for purifying nonmuscle actin (Gordon et al, 1977;Weir and Frederiksen, 1982]. Approximately 3 mg of actin was obtained from 200 g of chicken brain, ~ 2 mg of erythrocyte actin was obtained from 6 L of bovine blood, and ~ 1 mg of actin was obtained from 8 g of chick embryo brain; the increased yield of actin/g tissue from chick embryo brain may result because the Tris-Triton extract was run directly on the DNase I affinity column, bypassing the DE-53 column.…”

Section: Resultsmentioning

confidence: 81%

“…Actin isoforms exhibit different biophysical and biochemical properties under some experimental conditions in vitro [Allen et al, 1996;Gordon et al, 1977;Just et al, 1994]. These studies include the findings that some actin binding proteins (i.e., profilin [Ohshima et al, 1989], thymosin β4 [Weber et al, 1992], ezrin [Shuster and Herman, 1995] and plastin [Prassler et al, 1997]) exhibit preferences for binding muscle and nonmuscle actins.…”

Section: Introductionmentioning

confidence: 99%

“…Nonetheless, most studies of actin assembly or actin binding use skeletal muscle α-actin because of the ease of obtaining the actin in high yield [Spudich and Watt, 1971]. Actin from nonmuscle sources is rarely used for biochemical analyses, even though several protocols for purifying nonmuscle actin have been described [Gordon et al, 1977;Puszkin et al, 1978;Schaier, 1992;Segura and Lindberg, 1984;Weir and Frederiksen, 1982]. One reason for this may be that many of the published methods, which are based on the method for purifying actin from skeletal muscle, are not suited for use with tissues where actin is less abundant.…”

Section: Introductionmentioning

confidence: 99%

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Rapid and efficient purification of actin from nonmuscle sources

Schafer

Jennings

Cooper

1998

Cell Motil. Cytoskeleton

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The need for biochemical quantities of nonmuscle actin has been increased by observations that actin isoform composition of a cell influences the cell's motile and structural properties. In addition, the number of actin binding proteins that exhibit different binding interactions with β‐ and γ‐actin compared to α‐actin from skeletal muscle is growing. We report a procedure designed to purify actin from nonmuscle tissues employing extraction of monomeric actin from tissues with high concentrations of Tris, chromatography on DE‐53 cellulose, and affinity chromatography of DNase I‐agarose. The preparation is easy to perform and yields quantities of nonmuscle actin sufficient for biochemical and cell biological assays. Actin from bovine erythrocytes and from brains of adult and embryonic chickens was obtained using this method, which can be readily used with other sources of tissue. Coomassie‐Blue‐stained SDS gels of the purified actin show no contaminants; capping protein, a common contaminant of actin preparations, is absent by immunoblotting. This method for purifying nonmuscle actin will be useful to investigate functional differences in the biology of actin isoforms or their regulating proteins. Cell Motil. Cytoskeleton 39:166–171, 1998. © 1998 Wiley‐Liss, Inc.

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“…Polymerization can be induced in vitro with the addition of hypertonic buffer and ATP, however, the rate of polymerization is particularly sensitive to the presence of divalent cations, particularly Mg 2+ and Ca 2+ (Gordon, Boyer et al 1977;Estes, Selden et al 1992).…”

Section: Protein 3 -β-Actinmentioning

confidence: 99%

The Molecular Pharmacology of Pateamine A

Matthews¹

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<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

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Comparative biochemistry of non-muscle actins.

Cited by 248 publications

References 30 publications

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

Rapid and efficient purification of actin from nonmuscle sources

The Molecular Pharmacology of Pateamine A

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