Turnover of tubulin and the N site GTP in chinese hamster ovary cells
| Autor: | Marc W. Kirschner, Bruce M. Spiegelman, Stephen M. Penningroth |
|---|---|
| Rok vydání: | 1977 |
| Předmět: |
GTP'
Swine Nerve Tissue Proteins macromolecular substances Microtubules General Biochemistry Genetics and Molecular Biology Cell Line Cricetulus Tubulin Microtubule Cricetinae Mole Animals Nucleotide Glycoproteins Gel electrophoresis chemistry.chemical_classification biology Hydrolysis Chinese hamster ovary cell Ovary Brain Molecular biology Biochemistry chemistry biology.protein Female Guanosine Triphosphate Intracellular |
| Zdroj: | Cell. 12:587-600 |
| ISSN: | 0092-8674 |
| Popis: | Radioactively labeled tubulin from Chinese hamster ovary (CHO) cells can be isolated by copolymerization with nonradioactive porcine brain microtubule protein. 75% of the soluble tubulin in CHO extracts co-polymerizes with the porcine protein through several cycles, without preferential loss of either CHO or porcine subunits. After phosphocellulose chromatography of the co-polymerized microtubules, the CHO tubulin is radiochemically homogeneous, as judged by SDS-polyacrylamide gel electrophoresis. CHO tubulin purified in this way has 1 mole of nucleotide per mole of protein noncovalently bound at the non-exchangeable or N site. Thin-layer chromatography indicates that the N site nucleotide is entirely ribo-GTP. Label and chase experiments show that the N site GTP exchanges intracellularly with a half-time of 33 hr in growing cells which have a generation time of 17 hr, while the tubulin poly-peptides are degraded with a half-time of 48 hr. Intracellular hydrolysis of the γ-phosphate of the N site nucleotide can be detected but occurs very slowly, with a half-time of 24 hr. These results suggest that the N site nucleotide may function in vivo as a stable structural co-factor of the tubulin molecule and render improbable the possibility that it has a regulatory role in microtubule assembly. |
| Databáze: | OpenAIRE |
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