Studies on the distribution and properties of microtubular proteins in nervous tissue

Abstract

The work reported in this thesis concerns investigation of the subcellular distribution of colchicine-binding activity (of presumed microtubular origin) in mammalian brain, and studies on the bio-chemical properties of colchicine-binding protein ('tubulin'). The results led to the following conclusions:

About half the CB-activity present in sucrose-containing buffered homogenates of brain was consistently recovered in the particulate fraction obtained after high-speed centrifugation, and further investigations showed that CB-activity was widely distributed amongst the various subfractions obtained after gradient centrifugation of this pelleted material. The majority of the recovered particulate CB-activity was found to be associated with the fractions rich in synaptic membranes. Tubulin from this source appeared to represent a 'stabilised' form of CB-protein compared to that obtained from the high-speed soluble fraction, as judged from the results of experiments in which CB-activity was subjected to thermal decay under a variety of conditions. In addition, it was also shown that the tubulin isolated in particulate preparations was very tightly bound to membrane material, since it could not be readily solubilised by treatment with the detergent Triton X-100 or by sonication.

It was found, using partly purified tubulin preparations, that tubulin could act as a substrate for a cAMP-stimulated protein kinase that was closely associated with the protein, and that it bound cAMP. In addition, the antimitotic agent vincristine, which was found to stimulate CB-activity, also showed the remarkable property of almost selectively precipitating CB-protein from soluble extracts of brain, in the cold.

Finally, the previously inferred relation between neurotubular protein and CB-protein was proved when attempts made at isolating intact micro-tubules from brain homogenates prepared in various stabilising media were successful, and it was shown that these preparations were enriched in CB-activity.