Incorporation of l-Tyrosine, l-Phenylalanine and l-3,4-Dihydroxyphenylalanine as Single Units into Rat Brain Tubulin

Abstract

The product of the incorporation of [¹⁴C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [³H]colchicine-binding method (10.5% of the total protein). The incorporated [¹⁴C]-tyrosine was found in the α-subunit of tubulin. Protein labelled with [³H]colchicine and [¹⁴C]tyrosine was precipitated with vinblastine sulphate and the radioactivity of ³H and that of ¹⁴C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate - polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the ¹⁴C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Binding of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit.