ORYZALIN BINDS TO PLANT TUBULIN ISOLATED FROM FLAGELLA OF THE ALGA CHLAMYDOMONAS
Abstract
Dinitroaniline herbicides such as oryzalin (3,5-dinitro-N('4),N('4)-dipropylsulfanilamide) disrupt mitosis in the meristematic cells of seedling plants by inhibiting the formation of microtubules. For further understanding of the biochemical mechanism of action of oryzalin, laboratory analyses with isolated plant tubulin must be employed. Plant tubulin from flagella of the alga Chlamydomonas was isolated and purified. This tubulin was incubated with {('14)C}oryzalin, and free oryzalin was separated from oryzalin bound to plant tubulin by miniature DEAE-cellulose chromatography. Under optimal conditions of incubation (30 min, 25(DEGREES)C, pH 6.9), the molar ratio of oryzalin bound to plant tubulin is 0.98 (+OR-) 0.06. The association constant for the oryzalin-tubulin complex is 2.08 (+OR-) 0.08 x 10('5) M('-1) at 25(DEGREES)C. The thermodynamic values for the formation of the oryzalin-tubulin complex at 25(DEGREES)C are (DELTA)G(DEGREES) = -7.25 (+OR-) 0.02 kcal mol('-1), (DELTA)H(DEGREES) = 6.5 (+OR-) 0.2 kcal mol('-1), and (DELTA)S(DEGREES) = 46 (+OR-) 2 cal mol('-1) deg('-1) (mean (+OR-) standard error). Oryzalin has little or no affinity for intact microtubules, previously denatured plant tubulin, actin, bovine serum albumin, calmodulin, ferredoxin, trypsin, or urease, indicating oryzalin is specific for the biologically active conformation of plant tubulin. Oryzalin probably binds to free plant tubulin to form a complex that is incapable of polymerizing into microtubules. Oryzalin was chosen for these studies because this compound maintains a constant solubility at concentrations up to 14 (mu)M. This concentration is approximately three times the concentration lethal to Chlamydomonas. Also, this herbicide does not adsorb to glass as other commercially-available dinitroaniline compounds do.
Degree
Ph.D.
Subject Area
Botany
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