Biochemistry and Physiology of Microtubules

Snyder, Judith A.; McIntosh, J. Richard

doi:10.1146/annurev.bi.45.070176.003411

Cited by 331 publications

(104 citation statements)

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“…Microtubules are ubiquitous organelles of virtually all eukaryotic cells and have been implicated in various cell functions, including motility, mitosis, secretion, cell shape, and modulation of surface receptors; therefore, the regulation of their formation has become an area of active interest (1). Tubulin, the subunit protein of microtubules, is'a 6S dimer of 110,000 daltons.…”

mentioning

confidence: 99%

Stoichiometry of GTP hydrolysis and tubulin polymerization.

Maccioni¹,

Seeds²

1977

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

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Microtubule formation from lamb brain tubulin'isolated by affinity chromatography and freed of exchangeable nucleotide requires GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethyleniediphosphate and guanylylimidodiphosphate fail to replace GTP; in addition, neither the presence of mierotubule associated proteins nor 5 M glycerol relieves the GTP requirement. The relation of GTP concentration and microtubule formation shows an association constant K = 1 X 104 M-1; furthermore, GDP and guanylylimidodiphosphate are competitive inhibitors of GTP for polymerization.'Using a rapid filter assay for microtubule formation that allows the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of two molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules has been found. (7) and sedimentation (12) assays, respectively. Using this kinetic assay it is now possible to determine the stoichiometry of GTP hydrolysis and tubulin polymerization. MATERIALS AND METHODSMaterials.['y-32P]GTP (30 Ci/mmol) was purchased from New England Nuclear (Boston). Nonradioactive GTP (Types II and IV), ATP, and cyclic 3':5'-GMP (cGMP) were purchased from Sigma Chemical (St. Louis); other nucleotides [GMP, GDP, GMP-P(NH)P and GMP-P(CH2)P] were obtained from P-L Biochemicals (Milwaukee). All other reagents were of the most pure analytical grade available.Preparation of Tubulin. Microtubules were isolated by two cycles of polymerization from lamb brain homogenates as described (9). The crude microtubule pellets were stored at -750 without resuspension until further use. Tubulin was isolated from the microtubule pellet by deacetylcolchicinic acid (DAC)-affinity chromatography as described (9, 13). The purified tubulin was desalted by Sephadex G-25 column chromatography and concentrated in an Amicon PM-10 ultrafiltration apparatus. The concentrated tubulin was centrifuged at 14,000 X g to remove aggregates and used immediately. The protein concentration was determined by the procedure of Lowry et al. (14).Nucleotide Removal. Tubulin preparations were routinely freed of unbound nucleotide or nucleotide bound to the exchangeable site by two successive extractions with 0.5 ml of a 1 mM EDTA, charcoal (prewashed in imidazole-glycerol buffer containing 2% serum albumin, followed by a wash in only imidazole-glycerol buffer) solution per ml of tubulin sample.The residual guanine nucleotide retained by these char-

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mentioning

confidence: 99%

Stoichiometry of GTP hydrolysis and tubulin polymerization.

Maccioni¹,

Seeds²

1977

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

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“…It is important to note that, because in vitro tubulin polymerization into microtubules requires a minimal critical tubulin concentration above which the free tubulin assembles into microtubules [2], it is necessary to control the tubulin concentration during the homogenization step and the disassembly steps very carefully. Once too much homogenization buffer or disassembly buffer is used, the free tubulin concentration may be lowered to the extent that only a small fraction of tubulin is recovered as microtubules.…”

Section: Tubulinmentioning

confidence: 99%

“…Electron microscopy has shown that microfilaments and microtubules are fibrous structures common to all eukaryotic cells (for recent reviews see [1,2]). Microfilaments contain actin as major structural protein as revealed by heavy meromyosin decoration in electron microscopy [3] and actin antibody decoration in immunofluorescence microscopy [4].…”

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confidence: 99%

“…Microfilaments contain actin as major structural protein as revealed by heavy meromyosin decoration in electron microscopy [3] and actin antibody decoration in immunofluorescence microscopy [4]. The major structural protein of microtubules is the globular tubulin molecule (molecular weight 110 000), which contains two different polypeptide chains of similar molecular weight (for a review see [2]). …”

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confidence: 99%

“…Immunological [5 -81 as well as biochemical studies (for a review see [1,2]) have indicated that the structures of both actin and tubulin have been highly conserved during eukaryotic evolution. Recently partial amino-acid-sequence data and fingerprint analysis have shown a limited number of amino acid exchanges between muscle and brain actins indicating the possibility of several different actin genes in higher organisms [9 -111.…”

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The Isolation of Tubulin and Actin from Mouse 3T3 Cells Transformed by Simian Virus 40 (SV3T3 Cells), an Established Cell Line Growing in Culture

et al. 1977

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Tubulin can be purified from mouse SV3T3 cells (3T3 cells transformed by SV40 virus) by several cycles of temperature-dependent polymerization and depolymerization. Electron microscopical analysis of the final product reveals morphologically normal microtubules. Homogeneous actin can be isolated as a byproduct of the purification procedure. Mouse SV3T3 actin and skeletal muscle actin were compared by fingerprint analysis of the tryptic peptides obtained from performic-acid-oxidized protein. The two actins show a high degree of homology although apparently five of the twenty-five spots visualized by fluorescamine show a difference in chromatographic mobility. The purification procedure described allows the rapid isolation of both actin and tubulin from tissue culture cells in sufficient amounts for comparative biochemicals studies.

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