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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.
Insights
Reductive methylation of microtubule protein using formaldehyde and sodium cyanoborohydride inhibits microtubule assembly. This modification alters tubulin structure, rendering it assembly-incompetent without affecting key binding properties.
Area of Science:
- Biochemistry
- Cell Biology
- Structural Biology
Background:
- Microtubules are essential cytoskeletal polymers involved in cell division and transport.
- Tubulin, the protein subunit of microtubules, undergoes post-translational modifications that regulate its function.
- Understanding the chemical modifications affecting tubulin assembly is crucial for deciphering microtubule dynamics.
Purpose of the Study:
- To investigate the effects of reductive methylation on microtubule protein assembly.
- To identify the specific chemical modifications and their impact on tubulin structure and function.
- To determine the molecular basis for the inhibition of microtubule assembly.
Main Methods:
- Bovine brain microtubule protein was subjected to reductive methylation using formaldehyde and sodium cyanoborohydride (NaCNBH3) at pH 6.7.
- Microtubule assembly and depolymerization dynamics were monitored following methylation.
- Colchicine and GTP binding assays were performed to assess the impact on tubulin's functional sites.
- Comparative methylation studies were conducted on microtubule polymer, microtubule protein, and other proteins.
Main Results:
- Reductive methylation significantly inhibited microtubule assembly and induced depolymerization of existing microtubules.
- Low levels of methylation (10%) were sufficient to render tubulin assembly-incompetent.
- The methylation did not significantly affect colchicine or GTP binding affinities or the critical tubulin concentration.
- Methylation of specific, highly reactive amino groups on the alpha-chain of tubulin was identified as the cause of assembly inhibition.
Conclusions:
- Reductive methylation specifically targets and modifies key amino groups in tubulin, disrupting its ability to polymerize.
- The identified modification site is critical for tubulin assembly, but not for ligand binding.
- These findings provide insights into the chemical regulation of microtubule dynamics and the structural requirements for tubulin polymerization.