Total Tubulin and Its Aminoacylated and Non-aminoacylated Forms During the Development of Rat Brain

The amount of total tubulin in the soluble fraction of rat brain was measured by a method based on the purification of tubulin previously labeled by incorporation of [14C]tyrosine in the C terminus of its alpha-chain. The tubulin content decreased from 2.01 to 1.30 nmol/mg protein when the animals passed from 4 to 30 days of age and then remained practically constant. The amounts of aminoacylated and non-aminoacylated tubulin present in the soluble brain extracts were determined from the incorporation of [14C]tyrosine into the free acceptor sites of tubulin preparations, that were preincubated without carboxypeptidase A or with this enzyme to eliminate tyrosine and phenylalanine from the C terminus of the alpha-chain of tubulin. The values obtained were corrected for the inactivation of tubulin to accept [14C]tyrosine that occurred during the isolation and incubation of the soluble fractions. The ratio non-aminoacylated/aminoacylated tubulin increased from 1.62 +/- 0.03 in the 4-day-old rats to 2.11 +/- 0.17 in the 120-day-old rats. The aminoacylatable tubulin, that is the sum of aminoacylated plus non-aminoacylated tubulin, decreased from 1.71 to 0.75 nmol/mg protein from 4-day-old to 30-day-old rats respectively and then remained practically constant. The amount of aminoacylatable tubulin is lower than that of total soluble tubulin. Therefore there is a fraction of tubulin that is unable to accept tyrosine. This non-aminoacylatable tubulin fraction increases with the age of the animal so that in the 120-day-old rats this tubulin species accounts for 48% of the total soluble tubulin.