In vivo and in vitro synthesis of rat brain α- and β-tubulins

Abstract

The in vivo synthesis of brain tubulin was studied in 8 and 15 day old rats. The rats were injected intracranially with [ 3S]methionine. Soluble protein from the brains and purified tubulin were fractionated by electrophoresis in urea-SDS-polyacrylamide gels, and by a two-dimensional system employing isoelectric focusing in the first dimension, and electrophoresis in the second dimension. The two-dimensional system resolves the α- and β-tubulins in both dimensions. The β-polypeptide, which migrates faster in the urea-SDS-polyacrylamide gels, has an isoelectric point more acidic than the α-polypeptide, and seems to be composed of two distinct molecular species with slightly different pIs. Storage of tubulin at − 20 °C results in the production of artifactual charge heterogeneity of both α- and β-tubulins which can be detected by isoelectric focusing. Rat brain RNA was translated in vitro in wheat germ extracts, and in micrococcal nuclease-treated reticulocyte lysates. Both cell-free systems, synthesize polypeptides which have the same isoelectric points, and the same migration in urea-SDS-polyacrylamide gels as authentic α- and β-tubulins. β-Tubulin synthesized in vitro also seems to be composed of two distinct molecular species with different pIs. The mRNAs coding for α- and β-tubulins co-sediment with 18S rRNA in sucrose-formamide gradients, and therefore they must be around 2 000 nucleotides long.