Purification of assembly-competent tubulin from Saccharomyces cerevisiae.

We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure.

Complete separation of tyrosinated, detyrosinated, and nontyrosinatable brain tubulin subpopulations using affinity chromatography.

The maximum achievable tyrosination level of neurotubulin, in vitro, is about 50%. We have developed a method to obtain a complete separation of the tyrosinatable and nontyrosinatable species. We use an immunoaffinity column, with coupled YL 1/2 monoclonal antibody (anti-Tyr-tubulin) and rapid desalting methods.

Cortical ablation fails to influence striatal dopamine target cell supersensitivity induced by nigrostriatal denervation in the rat.

The possible influence of the corticostriatal (glutamatergic) pathway on denervation-induced striatal dopamine target cell supersensitivity has been investigated in the rat by measuring the changes in striatal acetylcholine levels induced by the dopamine agonist pergolide and the basal dopamine D2-receptor density after combined 6-hydroxydopamine-induced lesion of the substantia nigra and cortical ablation. Lesion of the nigrostriatal dopaminergic pathway alone enhanced the ability of pergolide (0.06-1 mg/kg i.