A dynein‐like protein associated with neurotubules

Gaskin, Felicia; Kramer, Sara B.; Cantor, Charles R.; Adelstein, Robert S.; Shelanski, Michael L.

doi:10.1016/0014-5793(74)80244-9

Cited by 179 publications

(98 citation statements)

References 17 publications

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“…The purified material has a colchicine-binding specific activity 3.5 times greater than the remaining proteins in the soluble phase after tubulin removal. The preparation also contains 2 or 3 minor polypeptides of high molecular weight, which may represent the dynein-like components seen in preparation of cilia and flagella tubulins and recently reported present in brain tubulin preparations isolated by in vitro polymerisation [32,33]. The isolation from human platelets of a colchicinebinding protein, presumed to be microtubular protein, has been previously reported using ammonium sulphate fractionation and ion-exchange chromatography [34].…”

Section: Discussionmentioning

confidence: 85%

Isolation of tubulin from pig platelets

Castle¹,

Crawford²

1975

FEBS Letters

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Section: Discussionmentioning

confidence: 85%

Isolation of tubulin from pig platelets

Castle¹,

Crawford²

1975

FEBS Letters

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“…Above a critical concentration, at equilibrium the tubulin dimer concentration is equal to this critical concentration and is independent of total tubulin concentration (15). Under these conditions, the GTPase activity was proportional to the quantity of tubulin incorporated into microtubules.…”

Section: Discussionmentioning

confidence: 94%

Guanosinetriphosphatase activity of tubulin associated with microtubule assembly.

David‐Pfeuty¹,

Erickson²,

Pantaloni³

1977

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

170

105

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Tubulin, purified by cycles of assembly followed by phosphocellulose chromatography, exhibits a characteristic GTPase activity that is polymerization dependent and can be attributed to the tubulin itself. This activity has been observed, in a standard reassembly buffer containing low Mg2+, under three conditions that induce microtubule assembly: in the presence of microtubule-associated proteins, in the presence of DEAE-dextran, or after addition of high Mg2+ and glycerol. The phosphocellulose-purified tubulin showed no GTPase activity under the following nonpolymerizing conditions: in buffer with low Mg2+ in the absence of microtubule-associated proteins or DEAE-dextran, in buffer with high Mg2+ and glycerol at tubulin concentrations below the critical concentration, or when microtubule assembly was inhibited by vinblastine. Colchicine, on the other hand, while blocking microtubule assembly, induced a significant GTPase activity in the phosphocellulose-purified tubulin. During the process of assembly, GTP appears to be hydrolyzed as a free tubulin dimer polymerizes into a microtubule. A constant GTPase activity when polymerization equilibrium is reached apparently reflects the cyclic polymerization-depolymerization of tubulin dimers at the ends of the microtubules.Microtubules can be reversibly assembled in vitro from brain homogenates. Polymerization occurs at 370 in the presence of GTP, Mg2+, and an appropriate buffer, and the microtubules are rapidly depolymerized at 4°(1, 2). Microtubules purified from the brain homogenates carry with them a number of microtubule-associated proteins (MAPs), which can be separated from the tubulin by ion-exchange chromatography (3, 4). After separation, isolated tubulin is unable to reassemble into microtubules unless MAPs are added back (3, 4). However, the self-assembly property of highly purified tubulin can be restored by at least two other procedures: addition of 5-15 mM of Mg2+ and 3.4 M glycerol to the assembly buffer (5) or addition of a polycation such as DEAE-dextran (6).The stable subunit of microtubules, the 110,000 molecular weight tubulin dimer, possesses two high-affinity binding sites for the guanine nucleotides (7). On the "E" site, the nucleotide is readily exchangeable, while on the "N" site it exchanges very slowly (8), if at all. Previous reports indicate that GTP binding to the "E" site is necessary for the polymerization reaction to occur while, after polymerization, the "E" site on microtubules contains essentially GDP (8-10). Therefore, hydrolysis of "E" site GTP apparently takes place during polymerization.The hydrolysis of GTP during polymerization has been studied by assaying directly the release of inorganic phosphate (11). In this study a polymerization-linked GTP hydrolysis has been claimed to be detected by subtracting a high-background GTPase activity observed in the presence of colchicine, which inhibits microtubule formation. A serious objection to this studyThe costs of publication of this article were defrayed in part by the paym...

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“…It will be interesting to learn more about the structure of this putative erythrocyte MAP 2 or of any other non-neuronal MAP 2 yet to be purified, in order to assess their homology with brain MAP 2. Cytoplasmic brain dynein There were early hypotheses (Burns & Pollard, 1974;Gaskin et al, 1974) that high-Mr MAPs copurifying with microtubules polymerized in vitro from brain were cytoplasmic counterparts of dynein, the force-generating enzyme for microtubule motility in cilia and flagella (Gibbons, 1965) . [As discussed above, this protein has to be distinguished from the MAP 1 C described as a major component of microtubules polymerized from hog brain by temperature-dependent polymerization (Herrmann et al, 1985).]…”

Section: Mapmentioning

confidence: 99%