PROTEIN COMPLEXES CONTAINING GAMMA-TUBULIN ARE PRESENT IN MAMMALIAN BRAIN MICROTUBULE PROTEIN PREPARATIONS

The presence of gamma-tubulin in microtubule preparations, obtained by disassembly/assembly cycles at 0 degrees C/37 degrees C from the brai n of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast gamma-tubulin was absent from pure tubulin obtained by chroma tography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the gamma-tubul in was present in cold stable material remaining after microtubule dis assembly at 0 degrees C and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly pr ocedure used for alpha/beta-tubulin heterodimers. Brain gamma-tubulin was purified by affinity chromatography with gamma-tubulin antibodies raised against its carboxyl terminal region. Purified gamma-tubulin co nsisted of at least two polypeptides present in equal quantities and e xhibiting a pi of 6.5 and 6.6, respectively. It was associated with th e alpha/beta-tubulin heterodimer and with at least five other polypept ides 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 gamma-tub ulin complexes isolated from Xenopus eggs. But, in contrast with Xenop us egg complexes, brain complexes exhibited a considerable heterogenei ty of their apparent masses and composition in sucrose gradient centri fugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, gamma-tubulin comple xes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain gamma-tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively us ed for studying both microtubule dynamics and the activity of microtub ule poisons. (C) 1997 Wiley-Liss, Inc.