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A dualaffinity method was established to purify, for the first time, a microsomal ecdysonebinding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on ωoctylaminoagarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazolebased general P450 inhibitor (typeII ligand) followed by elution with the substrate ecdysone (typeI 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 SDSPAGE. 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 detergentextracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysoneinduced typeI difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrixbound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17β hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dualaffinity chromatography described here may be generally applicable to the isolation of cytochromes P450.

A Microsomal Ecdysone-Binding Cytochrome P450 from the Insect Locusta migratoria Purified by Sequential Use of Type-II and Type-I Ligands