Colchicine-binding measurements of tubulin in variously sampled and incubated tissues from the mammalian brain

Tissue preparations from the brain of rats and guinea-pigs were examined for their activity in binding isotopically labelled colchicine, as a measure of their content of tubulin. The amount of binding material extracted into a cold buffered solution was unaffected by keeping tissues at 0°C. It diminished by one quarter when tissues were incubated at 37°C in bicarbonate glucose salines for 1–2 h. This diminution increased when glucose was omitted from incubating solutions and was less when tissues were stimulated electrically. It was modified also by the calcium content of the incubation fluids. Of the binding activity lost on incubation only a little was recovered in surrounding fluids. About half the colchicine-binding activity of the tissues was not extracted by the solution employed; this particulate-attached activity changed little, if at all, on normal incubation but diminished when incubating fluids contained a cyclic AMP-fluoride theophylline mixture which was known to modify tubulin assembly.The retention of both categories of colchicine-binding under normal conditions of incubation is consistent with the ability of such tissues to perform microtubule-dependent processes, notably cytoplasmic transport. Examination of the isolated tissues by the methods reported is of value in indicating which of various factors known to affect separated tubulin and microtubule structures, operate in a cell-containing system under chosen experimental conditions.