Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.
Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.
Camadro
J M
JM
Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, CNRS, Université Paris 7, France.
Matringe
M
M
Thome
F
F
Brouillet
N
N
Mornet
R
R
Labbe
P
P
D000345:Affinity Labels; D001665:Binding Sites / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D055768:Halogenated Diphenyl Ethers; D006540:Herbicides / Q000378:metabolism* / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D009579:Nitrobenzoates / Q000378:metabolism* / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010647:Phenyl Ethers / Q000378:metabolism* / Q000494:pharmacology; D050768:Protoporphyrinogen Oxidase; D011720:Pyrazoles / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D055768:Halogenated Diphenyl Ethers; D006540:Herbicides; D009579:Nitrobenzoates; D010647:Phenyl Ethers; D011720:Pyrazoles; C058904:M&B 39279; C016027:oxyfluorofen; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; C018425:acifluorfen