Effects of reductive methylation on microtubule assembly. Evidence for an essential amino group in the alpha-chain.

Abstract

Microtubule protein from bovine brain was reacted at pH 6.7 with formaldehyde and NaCNBH3. These reagents react specifically with protein amino groups, causing their conversion to mono- and dimethylated forms (Jentoft, N., and Dearborn, D. (1979) J. Biol. Chem. 254, 4359-4365). Reductive methylation both inhibited microtubule assembly and induced extensive depolymerization in assembled microtubules. These effects occurred at low levels of methylation (10%) and appeared to arise from an alteration in tubulin which rendered tubulin assembly incompetent. This alteration had no significant effect on colchicine or GTP binding or on the critical tubulin concentration required for assembly. Comparative methylation studies over a range of formaldehyde concentrations involving microtubule polymer, microtubule protein, and several test proteins suggested that assembly inhibition results from the methylation of one or two highly reactive amino groups in the alpha-chain. The reactivities of these amino group(s) were reduced in the polymerized and denatured states.