Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In eukaryotes, both chromosome segregation and the determination of the cell division cleavage plane depend on the mitotic spindle apparatus. Spindle malfunctioning can lead to chromosome mis-segregation and cytokinesis defects and hence result in aneuploidy. Thus, the understanding of the structure and function of mitotic spindles is of interest not only from the perspective of basic science, but has implications also for human health and disease. Until recently, this complex microtubule-based structure was studied mainly by cell biological techniques in mammalian cells, by biochemical assays in Xenopus egg extracts, and by genetic approaches in genetically tractable organisms such as yeast, flies, and nematodes. With the rapid development of mass spectrometry and its increasing application to biological problems, it has become possible to subject highly complex structures, such as the mitotic spindle apparatus, to proteomics approaches. Such studies require the isolation of the mitotic spindle, or its substructures, in sufficient amounts and free of excessive contaminants. A number of methods for the isolation of mitotic spindles from mammalian tissue culture cells have been developed in the past. We have compared these methods and found that protocols based on the stabilization of microtubules by taxol were most efficient and reproducible. Here, we describe the further optimization of a taxol-based method, originally developed by Zieve and Solomon [Cell 28 (1982) 233-242], and its application to the isolation of human mitotic spindles at a scale suitable for mass spectrometric analysis [G. Sauer, R. Korner, A. Hanisch, A. Ries, E.A. Nigg, H.H.W. Sillje, Mol. Cell. Proteomics 4 (2005) 35-43].
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PMID:Purification of mitotic spindles from cultured human cells. 1634 35

Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements focus on the distance between the G' region of EF-G and the N-terminal region of L11 (L11-NTD), located within the GTPase activation center of the ribosome. We demonstrate that single-turnover ribosome-dependent EF-G GTPase proceeds according to a kinetic scheme in which rapid G' to L11-NTD movement requires prior GTP hydrolysis and, via branching pathways, either precedes P(i) release (major pathway) or occurs simultaneously with it (minor pathway). Such movement retards P(i) release, with the result that P(i) release is essentially rate-determining in single-turnover GTPase. This is the most significant difference between the EF-G.GTPase activities of vacant and translocating ribosomes [Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) Mol. Cell 11, 1517-1523], which are otherwise quite similar. Both the G' to L11-NTD movement and P(i) release are strongly inhibited by thiostrepton but not by FA. Contrary to the standard view that FA permits only a single round of GTP hydrolysis [Bodley, J. W., Zieve, F. J., and Lin, L. (1970) J. Biol. Chem. 245, 5662-5667], we find that FA functions rather as a slow inhibitor of EF-G.GTPase, permitting a number of GTPase turnovers prior to complete inhibition while inducing a closer approach of EF-G to the GAC than is seen during normal turnover.
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PMID:EF-G-dependent GTPase on the ribosome. conformational change and fusidic acid inhibition. 1648 43