Abstract
Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.
Highlights
From the $Department of Biochemistry, The universityof Texas Health Science Center, San Antonio, Texas 78284 and the §Department of Biological Chemistry, Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland21205
The tubulin that binds to the column appears to contain only a and Dl, not When these tubulin fractions are characterized by immunoblotting using the anti-type I1 antibody, the antibody binds only to the 8, band in thebound fraction, not to the @1 band in the unbound fraction
Using polyclonal antibodies generated against the carboxyl-termini of types I, 111, and IV, we demonstrate that the electrophoretic species is comprised of isotypes I, 11, and IV, whereas the & variant is comprised exclusively of type 111 8
Summary
&. gels intothree electrophoretic classes, designated a,&, and It has not been possible hitherto to resolve the different isotypes in a functional form. To this end,we have isolated a monoclonal antibody, using as an immunogena chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type 11) found in the ,&-tubulin electrophoretic fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, 111, and IV, we demonstrate that the electrophoretic species is comprised of isotypes I, 11, and IV, whereas the & variant is comprised exclusively of type 111 8-. Our results suggest that the approach which we are using is likely to be a productive one for obtainingpreparativeamounts of purefunctional isotypes of tubulin
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