Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations

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Abstract
The presence of γ-tubulin in microtubule preparations, obtained by disassembly/assembly cycles at 0°C/37°C from the brain of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast γ-tubulin was absent from pure tubulin obtained by chromatography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the γ-tubulin was present in cold stable material remaining after microtubule disassembly at 0°C and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly procedure used for α/β-tubulin heterodimers. Brain γ-tubulin was purified by affinity chromatography with γ-tubulin antibodies raised against its carboxyl terminal region. Purified γ-tubulin consisted of at least two polypeptides present in equal quantities and exhibiting a pI of 6.5 and 6.6, respectively. It was associated with the α/β-tubulin heterodimer and with at least five other polypeptides of 75, 105, 130, 195, and 250 kDa. With the exception of the 250 kDa polypeptide, all of these proteins seem to be present in γ-tubulin complexes isolated from Xenopus eggs. But, in contrast with Xenopus egg complexes, brain complexes exhibited a considerable heterogeneity of their apparent masses and composition in sucrose gradient centrifugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, γ-tubulin complexes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain γ-tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively used for studying both microtubule dynamics and the activity of microtubule poisons. Cell Motil Cytoskeleton 36:179–189, 1997