Studies on the Specificity and Site of Action of α-Cyclopropyl-α-[p-methoxyphenyl]-5-pyrimidine Methyl Alcohol (Ancymidol), a Plant Growth Regulator

Abstract

a-Cyclopropyl-a-Ip-methoxyphenyll-5-pyrimidine methyl alcohol (ancymidol) is an inhibitor of ent-kaur-16-ene oxidation in microsomal preparations from the liquid endosperm of immature Marab macrocarpus seeds. The Ki for this inhibitor is about 2 x 10-9 M. Ancymidol also blocks entkaur-16-en-19-ol and ent-kaur-16-en-19-al oxidation by the same preparations with a similar efficiency, but does not significantly inhibit ent-kaur-16-en-19-oic acid oxidation. Ancymidol appears to be specific for this series of oxidations in higher plant tissues. It does not inhibit the oxidation of kaurene nor kaurenoic acid in rat lver microsomes and has no significant effect on the oxidation of cinnamic acid in microsomal preparations from Sorghum bicolor seedlings. Ancymidol Ancymidol' is a substituted pyrimidine with potent growth regulatory activity in higher plants (1,10,19,22,23). The inhibition of normal growth by ancymidol can be overcome by applications of gibberellic acid (GA3) (1,10,19,23 other oxidative reactions to determine if ancymidol is a general mixed function oxidase inhibitor, or if it has some degree of specificity for the reactions of this pathway. This paper describes the effects of ancymidol on the oxidation of kaurene and some of its closely related metabolic derivatives in microsomal preparations from immature seed of Marah macrocarpus, the fungus Fusarium moniliforme, and rat liver. The effects of ancymidol on the hydroxylation ofcinnamic acid in microsomal preparations from sorghum seedlings are also reported. In addition, some evidence is presented which indicates that ancymidol interacts directly with Cyt P-450. MATERIALS AND METHODSEnzyme Sources. Immature fruits of M. macrocarpus (Greene) Greene, wild cucumber, were collected in the spring of 1976 in the Santa Monica Mountains. Immature seeds were removed, rinsed, and stored at -20 C until used. Liquid endosperm was removed from the seeds, ground in a Teflon to glass homogenizer (Thomas), and centrifuged 15 min at 10,000g. The resulting supernatant was centrifuged 90 min at 150,000g in a Beckman model Ti-60 rotor. The 150,000g pellet was then resuspended in 0.1 M Tris-HCl buffer (pH 7.5) containing 25% (v/v) glycerol. The soluble and microsomal preparations were used either directly as an enzyme source or frozen and stored in liquid N2 for future use.Sorghum bicolor (Northrup King) seeds were disinfected with 1% (v/v) NaOCl for 1 hr, thoroughly rinsed, and germinated in moist Vermiculite in the dark. The top 2 cm were harvested on the 5th day after planting and used to prepare microsomes. Approximately 20 g of leaf tissue were ground in a chilled mortar and pestle with 2 volumes of 0.15 M K-phosphate (pH 8) containing 0.35 M NaCl and 1 g of insoluble PVP (Polyclar AT). The homogenate was filtered through Miracloth and centrifuged as above for M. macrocarpus microsomes. The microsomal pellets were resuspended in 4 ml of 75 mm Tricine (pH 8), frozen, and stored in liquid N2 until used. F. moniliforme (ACC 917, M419) stock cultures were maintai...

Gibberellic Acid Regulates Cell Wall Extensibility in Wheat (Triticum aestivum L.)

“…Similar treatment of the gibberellin insensitive double-dwarf mutants had no significant effect. Ancymidol, a potent inhibitor of gibberellin biosynthesis (5), decreased plastic extensibility of both the tall and semidwarf genotypes, by 71 and 69%, respectively, but had no significant effect on the double dwarf genotypes.…”

Arabidopsis ent-Kaurene Oxidase Catalyzes Three Steps of Gibberellin Biosynthesis

“…Other data also support the proposal that the oxidation of ent-kaurene to ent-kaurenoic acid is catalyzed by a single enzyme. Coolbaugh et al (1978) showed that in wild cucumber (Marah macrocarpus) the inhibition of each of the three steps from ent-kaurene to ent-kaurenoic acid by ancymidol had the same kinetics, whereas inhibition of oxidation of ent-kaurenoic acid to ent-7␣-hydroxykaurenoic acid was greater, suggesting that it is catalyzed by a different enzyme.…”

Chemical Regulators of Gibberellin Status and Their Application in Plant Production

“…The structural feature common to all these inhibitors of ent-kaurene oxidation is a lone electron pair on the sp 2 -hybridised nitrogen of their heterocyclic ring. In each case, this electron pair is located at the periphery of the molecule (Rademacher et al, 1987) and it appears likely that it displaces oxygen from its binding site at the protoheme iron (Coolbaugh et al, 1978). Evidence for such a type of interaction has been presented for ancymidol in microsome preparations of Marah macrocarpus (Coolbaugh and Hamilton, 1976;Coolbaugh et al, 1978) and for BAS 111..W …”