Heterogeneity of vertebrate brain tubulins.

Abstract

We have examined the extent of brain tubulin heterogeneity in six vertebrate species commonly used in tubulin research (rat, calf, pig, chicken, human, and lamb) using isoelectric focusing, two-dimensional electrophoresis, and peptide mapping procedures that provide higher resolution than previously available. The extent of heterogeneity is extremely similar in all of these organisms, as judged by number, range of isoelectric points, and distribution of the isotubulins. A minimum of 6 a and 12 f3 tubulins was resolved from all sources. Even the pattern of spots on two-dimensional peptide maps is remarkably similar. These similarities suggest that the populations of tubulin in all of these brains should have similar overall physical properties. It is particularly interesting that chicken, which has only four or five 8-tubulin genes, contains approximately 12 P tubulins. Thus, post-translational modification must generate at least some of the tubulin heterogeneity. Mammalian species, which contain 15-20 tubulin DNA sequences, do not show any more tubulin protein heterogeneity than does chicken. This suggests that expression of only a small number of the mammalian genes may be required to generate the observed tubulin heterogeneity.Microtubules are protein filaments composed principally of a-and f-tubulin subunits. They are involved in a variety of cellular functions including mitosis, maintenance of cell shape, cell motility, intracellular transport, and secretion (1). It has long been thought that these diverse functions are not all performed by microtubules assembled from a homogeneous pool of identical tubulin subunits (2). Indeed, there is now compelling evidence from electrophoresis (3, 4), chromatography (5, 6), isoelectric focusing (7-15), protein sequencing (16,17), and DNA sequencing (18-20) that a-and f-tubulin subunits are actually populations of heterogeneous proteins.The nature, extent, and significance of tubulin heterogeneity are not fully understood. Substantial progress has been made in determining the nature of the heterogeneity: multiple, nonidentical tubulin genes have been identified in all the higher eukaryotes that have been examined and in several, possibly all, cases more than one gene is expressed (see ref.21 for a recent review). This implies that different gene products probably account for some of the heterogeneity. Posttranslational modification may account for the remaining heterogeneity: detyrosylation (22), aminoacylation (23), phosphorylation (24), glycosylation (25,26), and acetate-metabolite addition (27) have been reported, although it is not known whether these modifications occur in all organisms. The functional significance of the heterogeneity has been more difficult to determine. Many observations suggest a correlation between tubulin differences and particular functions: expression of tubulin genes is tissue dependent (28-33); the occurrence and distribution of tubulin proteins is tissue specific (34-39); specific tubulins are associated with distinct tubulin popu...

Photoinduced partial unfolding of tubulin bound to meso‐tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro

“…The substrate used for adsorption and immobilization of microtubules in the AFM study was mica modified with a layer of 3-aminopropyltriethoxysilane (AP-mica) [40]. This forms a positively charged surface around neutral pH, which is capable of immobilizing the slightly negative MT (the pI being 4.96 for a-tubulin and 4.78 for b-tubulin [41]) to enable AFM imaging [42]. The deposited solution was incubated on the substrate for 5 minutes.…”

Stu2, the Budding Yeast XMAP215/Dis1 Homolog, Promotes Assembly of Yeast Microtubules by Increasing Growth Rate and Decreasing Catastrophe Frequency

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Identification of ?III- and ?IV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): Antibody mapping and cDNA sequencing

“…Isoelectric focusing (IEF) of cod brain tubulin was performed by the method of Field et al [1984] as modified by Detrich and Overton [1986]. IEF slab gels containing 4.2% polyacrylamide, 9.16 M urea, and 2% carrier ampholytes (0.5% servalyt 4-6, 1% servalyt 5-6, 0.5% servalyt 5-7, Serva, Germany), was prefocused (6 W, 1 h, 22°C), and thereafter tubulin was focused at 7 W for 5 h. Two-dimensional electrophoresis was run by the method of Field et al [1984] with some modifications [Detrich et al, 1992;Overton, 1986, 1988] as described previously by Billger et al [1994].…”