Embryonic chick nerve cells, from dissociated dorsal root ganglia, were cultured on polylysine substrata and examined for tubulin and actin distribution by indirect immunofluorescence. Antibodies generated against chick brain tubulin produced specific fluorescence in growth cones, neurites, and cell bodies without revealing distribution differences or substructure in the nerve cells. However, at reduced antitubulin concentrations, differences were resolved. Tubulin fluorescence remained uniform and intense in neurites and cell bodies, but exhibited reduced intensity and patterning in growth cones. Nonneuronal cells in the cultures served as controls for typical cytoplasmic tubulin fluorescence distribution. Straining controls demonstrated that fluorescence resulted from tubulin-antitubulin binding. Analogous studies, using antibodies generated against chick brain actin, demonstrated distribution differences at reduced antiactin concentrations, including "hot spots" of intense fluorescence in growth cones and a paucity of fluorescence in neurites.
The antigenic similarities and differences between various actins were explored by use of antisera against purified bovine cardiac actin and chicken embryo brain actin. In double-antibody coprecipitation tests, purified iodinated actins from bovine cardiac muscle, rabbit skeletal muscle, chicken embryo brain, and bovine brain all bound to antiserum against chicken embryo brain actin. This result demonstrates the presence of shared antigenic determinants among these actins. Cardiac actin antiserum, on the other hand, bound cardiac and skeletal actin, but failed to bind significantly either brain actin. In radioimmunoassay, all four unlabeled actins were capable of some degree of inhibition of binding of (125)I-labeled chicken embryo brain actin to homologous antiserum. The results confirm the existence of shared or similar antigenic determinants, but also show that the molecules are not antigenically identical. In the cardiac actin radioimmunoassay, unlabeled cardiac and skeletal muscle actins inhibited the binding of (125)I-labeled cardiac actin to homologous antiserum, but neither brain actin inhibited the binding. Thus, the muscle actins possess at least one antigenic determinant not expressed by the brain actins, in addition to the shared determinants. Furthermore, cardiac actin and skeletal actin generated different inhibition curves in the cardiac actin radioimmunoassay, demonstrating that, although antigenically related, they are not identical. Correlations with existing sequence data imply that substitutions in only a few residues alter the antigenic properties of actin.
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...
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