Turnover of tubulin and the N site GTP in Chinese hamster ovary cells.
Radioactively labeled tubulin from Chinese hamster ovary (CHO) cells can be isolated by co-polymerization 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. This-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 polypeptides are degraded with a half-time of 48 hr. Intracellular hydrolysis of the gamma-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.
D000818:Animals; D001921:Brain; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010053:Ovary; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D014404:Tubulin; D006160:Guanosine Triphosphate