Role of microsomal cytochrome P-450 in the formation of ecdysterone in larval house fly.

1. Six cytochrome P-450 species have been purified to varying extents from microsomes obtained from ecdysone-induced house fly larvae by the use of octylamino Sepharose-4B, Synchropak AX-300, Synchropak CM-300 and TSK-DEAE-5 PW column chromatography. 2. One of the fractions apparently corresponded to a mixture of low- and high-spin cytochrome P-450 as judged by spectral characteristics. 3. Molecular weights of the cytochrome P-450 species ranged from 50,000 to 57,000. 4. In a reconstituted system, all the microsomal species hydroxylated ecdysone at rates within the range of microsomal suspensions, as it occurs with mitochondrial fractions 1, 2, 3, 5, and 6 (Srivatsan et al., 1990, Biochem, biophys. Res. Commun. 166, 1372-1377); whereas, mitochondrial fraction 4 hydroxylates ecdysone at significantly higher rates. 5. It is postulated that the 20-monooxygenation of ecdysone is a mitochondrial event which requires the induction of a low-Km cytochrome P-450 species by ecdysone. 6. Microsomal hydroxylation of ecdysone may not be of physiological significance, as Km values for the reaction are above the normal concentrations of the hormone and the activity is not inducible by ecdysone (Agosin et al., 1988, Arch. Insect Biochem. Physiol. 9, 107-117).