Triazole Plant Growth Regulator Binding to Native and Detergent-Solubilized Plant Microsomal Cytochrome P450

BAS 111..W, a triazole which retards plant growth, is an inhibitor of cytochrome P450 monooxygenase enzymes in the gibberellic acid biosynthetic pathway. Microsomal membranes isolated from endosperm of immature pumpkin (Cucurbita maxima L. cv. Big Max) seeds at a developmental stage corresponding to 15-45% cotyledon fill were found to be enriched in cytochrome P450 and exhibited spectral characteristics that indicate binding of BAS 111..W to cytochrome P450. Magnesium-dependent density shifts on equilibrium sucrose gradients demonstrated that NADPH:cytochrome c reductase, total dithionite-reducible cytochrome P450, and BAS 111..W-binding cytochrome P450 were localized exclusively in the endoplasmic reticulum in endosperm cells of developing C. maxima seed. Binding competition experiments between ent-kaurene and BAS 111..W were not conclusive, but suggested that the substrate and inhibitor compete for binding at the same site on the ent-kaurene oxidase cytochrome P450. We used the detergent (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) to solubilize the BAS 111..W-binding cytochrome P450 from seed endosperm microsomes. Detergent-solubilized C. maxima microsomal cytochrome P450 retained high-affinity triazole-binding characteristics observed with microsomes, but had reduced affinity for ent-kaurene.