Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

A Banerjee,M.C Roach,P Trcka,R.F Luduena

doi:10.1016/s0021-9258(18)42811-6

A Banerjee, M.C Roach + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)42811-6

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Abstract

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.

Highlights

More assembly was noted with afin and abInthan with a@IvW. hen assembly is measured in the presence of taxol (10 p ~ )li,ttle difference is seen among the isotypically purified dimerosr between them and unfractionated tubulin
Comparison of the sequences of the different a and /3 isotypes shows that the differences among the as are fewer and more conservative than are the differences among the j3s (Little and Seehaus, 1988); this suggests that a fruitful approach in exploring the issue of the functional significance of tubulin isotypes would be to separate the tubulin dimers accordingto their p isotypes
We have previously reported preparing monoclonal antibodies specific for theand isotypes and using them to remove, by immunoaffinity chromatography under nondenaturing conditions, the corresponding tubulin dimers from a preparation of bovine brain tubulin (Banerjee et al, 1988, 1990)

Summary

TUBULINDIMERS FROM BOVINE BRAIN*

We have previously reported preparing monoclonal antibodies specific for theand isotypes and using them to remove, by immunoaffinity chromatography under nondenaturing conditions, the corresponding tubulin dimers from a preparation of bovine brain tubulin (Banerjee et al., 1988, 1990). We here report the preparation of a monoclonal antibody specific for the & isotype and describe the use of the antibodies against&I, PIII,and PIV to prepare highly pure a&I, a&I, and aBIv tubulin dimers in a functional state. In the presence of 10 p~ taxol, the dimers assemble to microtubules at about the same rate These results have been reported in preliminary form elsewhere (Roach et al, 1988;Banerjee et al, 1989)

EXPERIMENTAL PROCEDURES

RESULTS

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