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 mic rosomal ecdysone-binding cytochrome P450 protein from locust Malpighian tub ules. This method involved, after prepurification on omega -octylamino-agar ose and hydroxylapatite, binding of cytochrome P450 to an immobilized triaz ole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolate d material showed a typical cytochrome P450 spectrum, a specific heme conte nt of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PA GE. Based on a tryptic undecapeptide sequence the isolated protein may be i dentical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned fr om the same tissue. Ecdysone 20-monooxygenase activity could be partially r econstituted from microsomal detergent extracts, when supplemented with pur ified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, howeve r. Therefore, purification of the locust cytochrome P450 was monitored by e cdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstil bestrol and testosterone 3-thiosemicarbazone, but not with the 17 beta -hem isuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography d escribed here may be generally applicable to the isolation of cytochromes P 450.