The Journal of Cell Biology, Vol 101, 1782-1789, Copyright © 1985 by The Rockefeller University Press

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Analysis of the microtubule-binding domain of MAP-2

RA Gottlieb and DB Murphy

We examined the microtubule-binding domain of the microtubule- associated protein (MAP), MAP-2, using rabbit antibodies that specifically bind to the microtubule-binding region ("stub") and the projection portion ("arm") of MAP-2. We found that (a) microtubules decorated with arm antibody look similar to those labeled with whole unfractionated MAP antibody, though microtubules are not labeled with stub antibody; (b) incubation of depolymerized microtubule protein with stub antibody prior to assembly partially inhibits the rate of microtubule elongation, presumably because MAPs that are complexed with antibody cannot bind to microtubules and stabilize elongating polymers; (c) the rate of appearance and amounts of 36- and 40-kD microtubule- binding peptides produced by digestion with chymotrypsin are distinct for MAPs associated with microtubules vs. MAPs free in solution. The enhanced stability of the 40-kD peptide when associated with microtubules suggests that this domain of the protein is closely associated with, or partially buried in, the microtubule surface; (d) MAP-2 is a slender, elongate molecule as determined by unidirectional platinum shadowing (90 +/- 30 nm), which is in approximate agreement with previous observations. Stub antibody labels MAP-2 in the terminal one-quarter of the extended protein, indicating an intrinsic asymmetry in the molecule.
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