A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.
A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450.
Winter
J
J
Syngenta Crop Protection AG, Basel, Switzerland.
Eckerskorn
C
C
Waditschatka
R
R
Kayser
H
H
D000375:Aging / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D017886:Durapatite; D004440:Ecdysone / Q000378:metabolism*; D006110:Grasshoppers / Q000378:metabolism*; D007202:Indicators and Reagents; D008024:Ligands; D008317:Malpighian Tubules / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D012685:Sepharose; D012997:Solvents; D013250:Steroid Hydroxylases / Q000378:metabolism; D014230:Triazoles / Q000737:chemistry
D007202:Indicators and Reagents; D008024:Ligands; D012997:Solvents; D014230:Triazoles; D004440:Ecdysone; D012685:Sepharose; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C020095:ecdysone 20-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase