| 1425694 |
Matringe M, Mornet R, Scalla R: Characterization of [3H] acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen. Eur J Biochem. 1992 Nov 1;209(3):861-8.
It is now generally accepted that protoporphyrinogen oxidase is the target-enzyme for diphenyl-ether-type herbicides. Recent studies [Camadro, J-M., Matringe M., Scalla, R. & Labbe, P. (1991) Biochem. J. 277, 17-21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX. In this study, we show that, in purified pea etioplast, [3H] acifluorfen specifically binds to a single class of high-affinity binding sites with an apparent dissociation constant of 6.2 +/- 1.3 nM and a maximum density of 29 +/- 5 nmol/g protein. [3H] Acifluorfen binding reaches equilibrium in about 1 min at 30 degrees C. Half dissociation occurs in less than 30 s, indicating that the binding is fully reversible. The specificity of [3H] acifluorfen binding to protoporphyrinogen oxidase is examined. [3H] Acifluorfen binding is inhibited by all the peroxidizing molecules tested. The phthalimide derivative, N-(4-chloro-2-fluoro-5-isopropoxy) phenyl-3,4,5,6-tetra hydrophthalimide, exerts a mixed-competitive inhibition on this binding. The effects of all these molecules on the binding of [3H] acifluorfen are tightly linked to their capacity to inhibit pea etioplast protoporphyrinogen oxidase activity. Furthermore, protoporphyrinogen IX, the substrate of the reaction catalyzed by protoporphyrinogen oxidase, was able to competitively inhibit the binding of [3H] acifluorfen. In contrast, protoporphyrin IX, the product of the reaction, did not inhibit this binding. All these results provide clear evidence that in pea etioplasts, [3H] acifluorfen exclusively binds to protoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme. |
407(5,5,5,7) |