Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.
Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.
Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.
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Birchfield
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Casida
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002215:Captan; D005420:Flavoproteins; D055768:Halogenated Diphenyl Ethers; D006540:Herbicides / Q000737:chemistry* / Q000633:toxicity*; D008297:Male; D051379:Mice; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000201:enzymology*; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000187:drug effects; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010647:Phenyl Ethers / Q000494:pharmacology; D010797:Phthalimides / Q000737:chemistry*; D050768:Protoporphyrinogen Oxidase; D011869:Radioligand Assay; D014316:Tritium
D005420:Flavoproteins; D055768:Halogenated Diphenyl Ethers; D006540:Herbicides; D024101:Mitochondrial Proteins; D010647:Phenyl Ethers; D010797:Phthalimides; D014316:Tritium; C009417:tetrahydrophthalimide; C016027:oxyfluorofen; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493979:Ppox protein, mouse; D050768:Protoporphyrinogen Oxidase; D002215:Captan