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Plant Physiol, September 2000, Vol. 124, pp. 115-124
Covalent Binding of the Benzamide RH-4032 to Tubulin in
Suspension-Cultured Tobacco Cells and Its Application in a Cell-Based
Competitive-Binding Assay
David H.
Young* and
Veronica T.
Lewandowski
Rohm and Haas Company, Research Laboratories, Spring House,
Pennsylvania 19477
The benzamide, RH-4032, was found to be a potent antimicrotubule
agent in tobacco (Nicotiana tabacum) cells. It strongly
inhibited root growth and produced swollen club-shaped roots, an
accumulation of cells in arrested metaphase, and loss of microtubules.
RH-4032 inhibited the in vitro assembly of bovine tubulin into
microtubules, with inhibition requiring a relatively long incubation
period. Treatment of tobacco suspension-cultured cells or isolated
bovine tubulin with [14C]RH-4032, and analysis of
radiolabeled protein revealed a highly specific covalent attachment to
 -tubulin. Binding of [3H]RH-4032 in tobacco
suspension-cultured cells was shown to be saturable and to be
influenced by pre-incubation of the cells with various antimicrotubule
agents: Binding of [3H]RH-4032 was inhibited by the
benzamides, pronamide and zarilamide, the
N-phenylcarbamate, chlorpropham, and the
microtubule-stabilizing drug, paclitaxel, whereas trifluralin and
amiprophosmethyl were not inhibitory. A common characteristic of agents
that cause microtubule disassembly was a slight enhancement of
[3H]RH-4032 binding at low concentrations, which did not
occur with the microtubule-stabilizing agent paclitaxel. For structural
analogs of RH-4032 and various N-phenylcarbamates, it
was shown that the ability to inhibit binding of
[3H]RH-4032 was correlated with the ability to inhibit
tobacco root elongation. The results suggest a common binding site on
-tubulin for RH-4032, pronamide, zarilamide, and chlorpropham, which
is distinct from the binding site(s) for trifluralin and
amiprophosmethyl. RH-4032 provides a unique approach to studying
effects of antimicrotubule agents on plant cells by allowing
competitive tubulin binding assays to be conducted in whole cells.
*
Corresponding author; e-mail rsadhy{at}rohmhaas.com; fax
215-619-1617.
© 2000 American Society of Plant Physiologists
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