| Autor: |
Minotti, A M, Barlow, S B, Cabral, F |
| Zdroj: |
Journal of Biological Chemistry; February 1991, Vol. 266 Issue: 6 p3987-3994, 8p |
| Abstrakt: |
A sensitive and reproducible method to measure relative levels of polymerized and soluble tubulin in cells has been developed. This method involves metabolically labeling cells with radioactive amino acids followed by lysis in a microtubule-stabilizing buffer, centrifugation to separate soluble from polymerized tubulin, resolution of the proteins in each fraction by two-dimensional gel electrophoresis, and quantitation of the tubulin by liquid scintillation counting of spots excised from the gel. Several buffers were evaluated for their reproducibility and efficacy in preserving the state of in vivo microtubule assembly at the time of cell lysis, and the ability of the technique to measure drug-induced changes in tubulin polymerization was determined. Results using this method indicate that Chinese hamster ovary cells maintain approximately 40% of the cellular tubulin in an assembled form. Dose-dependent decreases in tubulin polymerization could be measured in Colcemid-treated cells, while dose-dependent increases in assembly were measured in taxol-treated cells. The results with taxol indicate that, following the increase in microtubule polymerization, there is a time-dependent bundling of microtubules that occurs without further increases in the extent of tubulin assembly. Examination of drug-resistant Chinese hamster ovary cells reveals that Colcemid-resistant mutants maintain more tubulin in the polymerized state (approximately 50%), while taxol-resistant mutants maintain less assembled tubulin (about 28%). Similar changes occur regardless of whether the mutant cells have an alteration in alpha- or in beta-tubulin. A model to explain these results is discussed. |
| Databáze: |
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