JBC, Vol. 252, Issue 6, 2101-2105, Mar, 1977
Microtubular protein catalytic interactions with nucleotides
B. Zeeberg, A. Hassid and M. Caplow
Purified tubulin prepared from pig brain in the absence of added guanine
nucleotides contains 1 mol each of GDP and GTP/mol of tubulin. Incubation
of a tubulin preparation with inorganic [32P]phosphate results in the
incorporation of 32P into tubulin-associated GDP and GTP. Typically, in a
5-h incubation, 0.45 mM 32Pi reacts with 22 muM tubulin to form 3.3 muM
[32P]GDP, and 0.3 muM [32P]GTP. The yield of labeled nucleotide is
decreased as the result of a hydrolase activity associated with the
preparation. The [32P]GTP is exclusively beta-labeled and is therefore
formed by phosphorylation of [32P]GDP by a phosphate donor other than
inorganic phosphate, most likely by nonradioactive GTP. Added GDP
significantly decreases the yield of labeled GTP, but increases the yield
of labeled GDP in a manner that is consistent with a partial protection
against [32P]GDP hydrolysis, but not consistent with significant additional
[32P]GDP formation. Added GMP is an inhibitor of both labeling activities
associated with the preparation, although it has no effect on the hydrolase
activity. Added ADP (0.4 MM) does not form labeled ADP or ATP and does not
influence the labeling of GDP or GTP. The formation of [32P]GDP was shown
to occur by an exchange mechanism rather than through net synthesis from
GMP and Pi. These results provide evidence for a reversible guanidylation
of the protein. The labeling activity is always specifically associated
with highly purified tubulin preparations. Microtubular protein
preparations are found to catalyze an exchange of oxygen from H218O into
inorganic phosphate. Thus, there are two distinct catalytic properties
associated with tubulin.
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