J. Ste~'oid Biochem. Mole¢. Biol. Vol. 46, No. 5, pp. 565--572, 1993 0960-0760/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd CYTOCHROME P450c17 FROM PORCINE AND BOVINE ADRENAL CATALYSES THE FORMATION OF 5,16- ANDROSTADIEN-3fl-OL FROM PREGNENOLONE IN THE PRESENCE OF CYTOCHROME b5 W. J. MEADUS, 1 J. I. MASON 2 and E. J. SQUIRES l* IDepartment of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada NIG 2Wl and 2Green Center for Reproductive Biology, Southwestern Medical Center, University of Texas, Dallas, TX 75235, U.S.A. (Received 13 May 1993; accepted 5 July 1993) Summary--The synthesis of 5,16-androstadien-3fl-ol from pregnenolone occurs via a cyto- chrome P450-dependent reaction (andien-fl synthase) that is analogous to the CI7- hydroxylase/lyase reaction. It is not known whether the andien-fl synthase activity in adult porcine testis involves cytochrome P450c17 or is unique to porcine testis. Andien-fl synthase activity in testis microsomes was inhibited by high pH and concentration of salt, while C17-hydroxylase/lyase activity was stimulated under these conditions. Cytochrome P450c17 purified from adult porcine testis and adrenal glands and bovine adrenal glands had only C17-hydroxylase/lyase activity in the absence of cytochrome bs. However, when cytoehrome b5 isolated from porcine testis was added, andien-fl synthase activity was detected in all three preparations of cytocbrome P450c17, with the highest activity found in the porcine prep- arations. The andien-fl synthase activity was further increased from 2.5 to 6 times when NADH cytochrome b5 reductase was added along with cytochrome bs. Levels of mRNA for cytochrome b5 relative to cytochrome P450c17 mRNA were five times higher in porcine testis than in porcine adrenal. It appears that the andien-fl synthase activity is catalysed by cytochrome P450c 17, which is not unique to the porcine testis and is dependent upon adequate levels of cytochrome b5. INTRODUCTION The androst-16-ene steroids are C19 steroids produced primarily by Leydig cells in porcine testis. They include androstenone, which has a strong musk-like odour and is the primary cause of 'boar taint'. The first step in the synthesis of androst-16-ene steroids from pregnenolone is the formation of 5,16-androstadien-3fl-ol[1]. This reaction is catalysed by the andien-fl syn- thase system in a cytochrome P450-dependent reaction[2-4]. Cytochrome P450c17 cataly- ses the 17~t-hydroxylation of pregnenolone sometimes followed by the C17-1yase (desmo- lase) reaction to produce dehydroepiandro- sterone [5]. Cytochrome P450c17 isolated from neonatal porcine testis has also been shown to have andien-fl synthase activity in the presence of cytochrome b5 [4]. The interaction between cytochrome b5 and P450 is sensitive to in vitro ionic and pH conditions. Generally, conditions which favour Van der Waals forces promote the *To whom correspondenceshould be addressed. transfer of electrons between cytochrome b5 and P450LM2 [6], P450CAM [7], or metmyo- globin [8]. We have therefore examined the sensitivity of the andien-fl synthase and C I 7-hy- droxylase/lyase activities from adult porcine testis to changing pH and ionic conditions in vitro to determine if cytochrome b5 is involved in these reactions. Cytochrome P450 with C17-hydroxyl- ase/lyase activity has been isolated from both porcine adrenal[9] and neonatal porcine testis [2]. These different preparations of cyto- chrome P450 had different molecular weights and amino acid compositions. However, in human and bovine[10--12], the C17-hydroxyl- ase/lyase activity in both testis and adrenal is due to the same cytochrome P450. We have isolated cytochrome P450c17 from adult porcine testis and adrenal, and bovine adrenal to determine if the andien-fl synthase activity is due to this form of cytochrome P450 and if it is unique to porcine testis. We also deter- mined the relative levels of cytochrome b5 and P450c17 mRNA in porcine adrenal and testis to 565 sn 46/~--D