Developmental and Biochemical Analysis of Chick Brain Tubulin Heterogeneity

Abstract

Tubulin, isolated from brain tissue of chicks at different stages during late embryonic and early post-hatched development by ion-exchange chromatography and by in vitro microtubule reassembly, was analyzed by high-resolution isoelectric focusing and by two-dimensional polyacrylamide gel electrophoresis. Similar results were obtained with tubulins purified by the two methods. Sixteen isoelectric species of tubulin that differ in apparent net charge under denaturing conditions were detected by isoelectric focusing. By two-dimensional polyacrylamide gel electrophoresis, the chick brain tubulins were resolved into at least seven forms of alpha and 10 forms of beta tubulin. The number and relative proportions of the multiple brain tubulins were modulated during development. Since there are only four alpha tubulin and four beta tubulin genes in chickens, posttranslational modification of the tubulins must play a prominent role in the heterogeneity. Analysis of isotubulin distributions through cycles of microtubule assembly and disassembly indicated that the tubulins differ very little, if at all, in their capacity to assemble into microtubules. Therefore, the chemical differences that distinguish the multiple tubulins have very little structural impact on the protein surface areas involved in microtubule formation. Partial fractionation of the multiple tubulins during ion-exchange chromatography was observed, suggesting that it may be possible to isolate individual native tubulin variants for biochemical studies.

Indibulin, a Novel Microtubule Inhibitor, Discriminates between Mature Neuronal and Nonneuronal Tubulin

“…The posttranslational modifications of the different isotypes include acetylation, phosphorylation, polyglutamylation, detyrosination, and polyglycylation (1,2). This tubulin microheterogeneity is much higher in neuronal cells than in other cells (5)(6)(7)(8)(9).…”

Regulation of Tubulin Expression in Brain

“…The isoelectric variants of tubulin in the chick brain include seven aisoforms and 10 l3-isoforms; most are present in the 13-day embryo, but their relative levels are developmentally modulated (Sullivan and Wilson, 1984). As noted in the mouse and rat, changes in the levels of a number of the chick tubulin isoforms can be temporally correlated with developmental events occurring in the embryo and in the chick, after hatching (Sullivan and Wilson, 1984).…”

“…As noted in the mouse and rat, changes in the levels of a number of the chick tubulin isoforms can be temporally correlated with developmental events occurring in the embryo and in the chick, after hatching (Sullivan and Wilson, 1984).…”

“…Although multiple a-tubulin isoforms are resolved when total brain tubulin is separated on high-resolution isoelectric focusing gels (Sullivan and Wilson, 1984;Field et at., 1984), only two a-tubulin in vitro translation products have been resolved (Gozes et at., 1980). Separation of in vitro translation products of mRNAs isolated from mouse neuroblastoma cells and chick, rat, and human brain by two-dimensional gel electrophoresis resolves two u-tubulin isoforms with the same isoelectric points but different molecular weights (Morrison et at., 1979Bryan et at., 1978;Marotta et at., 1979b;.…”

“…They differ in isoelectric points, 132 being more acidic than 131 (Marotta et at., 1978;. High-resolution, one-dimensional isoelectric focusing separates between 9 and 21 brain 01.-and f3-tubulin isoforms (Gozes and Littauer, 1978;Forgue and Dahl, 1979;George et at., 1981;Stromska, 1982;Wolff et at., 1982;Sullivan and Wilson, 1984;Field et at., 1984). These isoforms are highly conserved (Field et at., 1984).…”