Biochemical properties of microtubular protein in brain subcellular fractions

Abstract

Chick brain was early shown to be a rich source of microtubular protein, but had not been previously employed to investigate its sub-cellular distribution.

During the course of this work, subcellular fractions of 1-3 day old chick brain were prepared using different procedures based on floatation-sedimentation centrifugation techniques. The subcellular distribution of microtubular protein was determined by assaying the colchicine-binding (CB) activity in the various fractions obtained.

It was shown that both the soluble and synaptic plasma membrane-containing fractions were enriched in CB activity. A membrane fraction enriched in myelin was also found to bind colchicine, but the CB in this case was atypical since it was not stabilized by vinblastine.

The distribution of vinblastine-binding activity was also investigated. The myelin fraction showed the highest binding activity for this alkaloid.

Furthermore, the polymerization-competence of tubulin present in soluble extracts of synaptosomes, prepared from chick brain according to two different methods, was investigated. Preliminary attempts failed to demonstrate the polymerization of tubulin in such synaptosol preparations incubated under typical microtubule assembly conditions, although SDS-PAGE analysis showed the presence of both alpha- and beta-like protein bands.

However, incorporation of glycerol and DMSO, as microtubule stabilizing agents, during extraction of synaptosomes carried out at room temperature was found to yield a sedimentable form of CB-protein which apparently represented stabilized micro tubular structures and accounted for ca. 10% of the total tubulin (CB-protein) content of synaptosomes. Such structures were readily solubilized in the cold and could be reassembled into filamentous elements under standard microtubule assembly conditions. The non-sedimentable form of tubulin (ca. 40% of total synaptosomal tubulin) prepared under these conditions was not polymerization-competent and probably represents the free tubulin subunit pool of synaptosomes. The remainder (ca. 50%) CB-protein present in synaptosomes was firmly associated with membrane structures.

Filaments reassembled from synaptosome extracts appeared to be enriched in an anomalous slower form of alpha-tubulin as judged by SDS-PAGE analysis. In addition, these filaments were enriched in a faster-migrating protein component which migrated slightly ahead of actin. Possible reasons for the failure to reassemble typical microtubules from synaptosome extracts are discussed.