Species-dependent immunological differences between vertebrate brain tubulins.

Abstract

The antigenic similarities and differences between highly purified brain tubulins from lamb, mouse, and chick embryo have been examined using rabbit antisera prepared against each of these tubulins. These antisera are capable of binding l25I-labeled tubulin in homologous or heterologous combinations, demonstrating immunological similarity between the tubulins. However ences between tubulins from different classes of microtubules and species differences between flagellar outer doublet tubulins.The present study has quantitatively analyzed the purified cytoplasmic brain tubulins from three different vertebrate species by radioimmunoassay. The results demonstrate that although heterologous combinations of tubulin and antisera will interact, there are clear quantitative differences in the ability of the various radiolabeled tubulins to bind to the antibodies and in the ability of the various unlabeled tubulins to compete with the different labeled tubulins for antibody binding sites. MATERIALS AND METHODSTubulin Purification. Tubulin was isolated and purified from lamb brain, mouse brain, and 14-day embryo chick brain by two cycles of polymerization in 0.1 M 1,4-piperazinediethanesulfonic acid (Pipes) (pH 6.8), containing 1 mM GTP, 1 mM MgCl2, 0.5 mM ethylene glycol bis(f3-aminoethyl ether)-N,N'-tetraacetate (EGTA), and 50% glycerol, followed by chromatography on a column of phosphocellulose (14, 15). Purity was assessed by electrophoresis on sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels (16).Immunization and Antigen Todination. Antisera to Iamb, mouse, and chick brain tubulins were produced by immunizing rabbits with purified antigen that had been partially crosslinked with glutaraldehyde (17). Rabbits were injected subcutaneously with 2 mg of antigen in complete Freund's adjuvant, followed at 2-week intervals by boosters in incomplete Freund's adjuvant. Each antiserum discussed in this paper was collected from a single rabbit.Purified tubulins were radiolabeled with 125I through the iodinated imidoester, methyl-3,5-diiodo-p-hydroxybenzimidate, by a modification of the technique of Wood et al. (18). Ten microliters of a 20 mM solution of methyl-p-hydroxybenzimidate-HCl in 50 mM sodium borate (pH 8.5) was mixed with 10 ,l of a 40mM chloramine T solution, 10 1.l of a 40mM NaI solution, and 10 0A of 125I (-1 mCi) and allowed to react at room temperature for 15 min. The reaction was stopped by the addition of 1 ,l of 1 M 2-mercaptoethanol, and the iodinated imidoester was precipitated by neutralizing with 1 M acetic acid (-5 ,l). The iodinated imidoester was collected by centrifugation at 10,000 X g for 5 min, and the pellet was dissolved in 25 til of 50 mM sodium borate (pH 8.5). Approximately 25 ,4g of purified tubulin was added to the iodinated imidoester, and the mixture was reacted for 24 hr at room temperature. Unbound iodinated imidoester was then removed from the solution by dialysis against borate-buffered saline (pH 8.0), and the iodinated protein was diluted to 10 ml with borate-buffered sali...

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

“…Binding of 1 2 5 1 -t~b~l i n to the antiserum was progressively inhibited by increasing amounts of unlabelled tubulin. Half-maximal inhibition (Iso) was obtained with 3.5 p g of tubulin, which is comparable to the radioimmunoassay of tubulin developed by Morgan et al (1978) and Bulinski et al (1980), but 70 times less sensitive than the assay described by Le Guern et al (1977), probably because of our mild iodination conditions. The sensitivity of the assay was approximately 0.4 pg, while the upper limit of the assay was approximately 25 pg.…”

Distribution of tubulin and actin in neurites and growth cones of differentiating nerve cells

“…The primary antibodies used in this study were generated in rabbits against actin and tubulin purified from chick embryo brain. The methods of antigen purification, rabbit immunication, and antiserum characterization have been previously reported [Morgan et al, 1978a[Morgan et al, , b, 1980Spooner et al, 19791. Briefly, chick brain tubulin was purified by two cycles of polymerization, followed by chromatography on phosphocellulose, while chick brain actin was purified from acetone powders by extraction, chromatography on Sephadex G-150 (F'harmacia), and two cycles of polymerization.…”

Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

“…Morgan et al (1978) examined the antigenic similarities between tubulin of brain from lamb, mouse and chick embryo, using rabbit antisera prepared against each of these tubulins. They found quantitative immunological differences between the tubulins, which implied different densities of shared antigenic determinants on all tubulins and a unique determinant on the chick tubulin molecule.…”