The relative affinities of nucleotides to G-actin and their effects

Iyengar, M R; Weber, Hans Hermann

doi:10.1016/0304-4165(64)90094-7

Cited by 37 publications

(18 citation statements)

References 18 publications

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“…Thus, the loss of polymerizing capacity of actin precipitated in the absence of ATP (4) or in the presence of UTP or GTP (18) were in agreement. It was reported that UTP or GTP, unlike ATP, was weakly bound to G-actin molecule (25). The acceleration of polymerization of cardiac G-actin by Mg 2+ and deceleration by Ca 2+ (26), the binding of Ca 2+ to G-actin (27,28) and its possible role in polymerization (3) are in agreement with the results obtained using actin from skeletal muscle.…”

Section: Murthy Crevasse Shippsupporting

confidence: 81%

Actin Polymerization and Its Relationship to Adenosine Triphosphatase Activity

Murthy

Crevasse

Shipp

1968

Circulation Research

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F-actin was prepared from pig heart myofibrils without the use of organic solvents. G-actin, prepared from F-actin by sonication, had adenosine triphosphatase activity. Dephosphorylation of added ATP by G-actin had a broad pH optimum between pH 7.5 and pH 9.0, specifically required Mg 2+ , and was activated by EGTA. G-actin prepared from skeletal muscle of rabbits by similar methods also showed ATPase activity. Cardiac G-actin and G-actin from skeletal muscle had equimolar nucleotide bound to them. Polymerization of cardiac G-actin was greatest at a pH of about 6.5 and declined above pH 6.8. KCl was necessary for polymerization, and EGTA inhibited the conversion to F-actin. By selectively acetylating G-actin, using acetic anhydride, a preparation of G-actin was obtained that did not have adenosine triphosphatase activity but did polymerize under optimal conditions. It is concluded that dephosphorylation of ATP and the polymerization process in cardiac G-actin are two different reactions occurring at different sites on the actin molecule.

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Section: Murthy Crevasse Shippsupporting

confidence: 81%

Actin Polymerization and Its Relationship to Adenosine Triphosphatase Activity

Murthy

Crevasse

Shipp

1968

Circulation Research

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“…The ATP requirement for actin is based on two criteria: the isolation of actin from a number of sources with a bound ATP, and the demonstration that there is a 500-fold higher affinity of muscle actin for ATP than for GTP (3). This difference in nucleotide affinity was also observed in polymerization experiments.…”

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confidence: 98%

GTP-Yeast Actin

Wen

Yao

Rubenstein

2002

Journal of Biological Chemistry

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Because of the apparently greater conformational flexibility of yeast versus muscle actin and the ability of other members in the actin protein superfamily to efficiently use both ATP and GTP, we assessed the ability of yeast actin to function with GTP. Etheno-ATP exchange studies showed that the binding of GTP to yeast actin is about 1/9 as tight as that of ATP in contrast to the 1/1,240 ratio for muscle actin. Proteolysis of GTP-bound G-yeast actin suggests that the conformation of subdomain 2 is very much like that of ATP-bound actin, but CD studies show that GTP-bound actin is less thermostable than ATP-bound actin. GTP-actin polymerizes with an apparent critical concentration of 1.5 M, higher than that of ATP-actin (0.3 M) although filament structures observed by electron microscopy were similar. Yeast actin hydrolyzes GTP in a polymerization-dependent manner, and GTP-bound F-actin decorates with the myosin S1. Conversion of Phe 306 in the nucleotide binding site to the Tyr found in muscle actin raised the nucleotide discrimination ratio from the 1/9 of wild-type actin to 1/125. This result agrees with modeling that predicts that removal of the Tyr hydroxyl will create a space for the C2 amino group of the GTP guanine.Two cytoskeletal filament systems in the cell, actin microfilaments and tubulin-containing microtubules, depend on the ability to bind and hydrolyze nucleoside triphosphates for proper function. For microtubules, the nucleotide is GTP, and for the actin filaments, the nucleotide is assumed to be ATP (1). ATP F-actin is more stable than ADP F-actin, and the instability brought about by hydrolysis of the nucleotide is believed to be an important factor in the regulation of the dynamics of the actin cytoskeleton (2).The ATP requirement for actin is based on two criteria: the isolation of actin from a number of sources with a bound ATP, and the demonstration that there is a 500-fold higher affinity of muscle actin for ATP than for GTP (3). This difference in nucleotide affinity was also observed in polymerization experiments. Martonosi and Gouvea (4) first reported that actin, in the presence of 0.1 M KCl, would not polymerize in the presence of a stoichiometric amount of GTP. Iyengar and Weber (3) also generated muscle G-actin free of nucleoside triphosphates and examined the ability of different nucleotides to affect polymerization when added back to the preparation in the presence of salt. Their results showed that whereas 20 M G-actin would completely polymerize in the presence of a stoichiometric amount of ATP, 100 M GTP was required to polymerize the actin. Apparently, the free GTP needed to be well above the nM-M range required for ATP to saturate the high affinity nucleotide binding site of the actin.We have previously used yeast actin as a model system for examining actin structure-function relationships in vitro, and our results have demonstrated significant quantitative differences in the behavior of yeast versus muscle actin. Yeast actin is more susceptible to controlled proteolysis an...

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“…It has been reported that actin could polymerize in the presence of other nucleotides in vitro (31). Nevertheless, actin filament assembly and stability are highly favored in the presence of ATP and ADP (21,31,48). Yeast actin binds to and hydrolyzes GTP, but with much lower binding affinity and hydrolytic rate than ATP (21, 48), presumably because of a more open nucleotide cleft (2).…”

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confidence: 99%

“…Nevertheless, actin filament assembly and stability are highly favored in the presence of ATP and ADP (21,31,48). Yeast actin binds to and hydrolyzes GTP, but with much lower binding affinity and hydrolytic rate than ATP (21,48), presumably because of a more open nucleotide cleft (2). Similarly, members of the actin superfamily, DnaK (27) and hexokinase (35), are known to bind and hydrolyze ATP more effectively than other nucleotides.…”

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confidence: 99%

GTPase Activity, Structure, and Mechanical Properties of Filaments Assembled from Bacterial Cytoskeleton Protein MreB

2006

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MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.Prokaryotic actin homologues MreB/ParM/Mbl are, along with tubulin homologue FtsZ and intermediate-filament homologue crescentin, the major components of what appears to be an extended filamentous cytoskeleton in bacteria (28). Recent studies have demonstrated the importance of these proteins in bacterial functions (1,3,5,6,23,36). Fluorescence microscopy in vivo shows that MreB aggregates into a large filamentous spiral structure that lies underneath the cell membrane and spans the cell length (24). Several studies suggest an essential role for MreB in chromosome segregation (16,25), polar localization of proteins (15, 36), maintenance of cell shape, and resistance to external mechanical stresses. When MreB is depleted, the bacterial cell wall displays gross morphological defects (47): vibrioid-shaped Caulobacter crescentus cells become lemon-shaped (12), and rod-shaped Bacillus subtilis (5) and Escherichia coli cells (47) become rounded. Peptidoglycan cell wall synthesis has been linked to the role of the MreB homolog, Mbl (5); however, the mechanism by which MreB may provide mechanical support directly to the cell or indirectly by affecting peptidoglycan wall integrity remains unclear.In eukaryotic cells, cell stiffness is primarily provided by actin filaments, which organize into orthogonal arrays and ordered bundles that confer extraordinary elasticity to the cell (18). In physiological conditions, actin requires ATP or ADP to stabilize its folding and to polymerize (8). It has been reported that actin could polymerize in the presence of other nucleotides in vitro (31). Nevertheless, actin filament assembly and stability are highly favored in the presence of ATP and ADP (21,31,48). Yeast actin binds to and hydrolyzes GTP, but with much lower binding affinity and hydrolytic rate than ATP ...

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The relative affinities of nucleotides to G-actin and their effects

Cited by 37 publications

References 18 publications

Actin Polymerization and Its Relationship to Adenosine Triphosphatase Activity

Actin Polymerization and Its Relationship to Adenosine Triphosphatase Activity

GTP-Yeast Actin

GTPase Activity, Structure, and Mechanical Properties of Filaments Assembled from Bacterial Cytoskeleton Protein MreB

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