title: Some enzymatic vagaries of a bovine adrenal microsomal cytochrome P-450 introduced and expressed in transformed monkey kidney cells.
abstract: The present studies illustrate the ability to carry out kinetic measurements of steroid hydroxylation using a cytochrome P-450 expressed in a tissue culture cell system. For these experiments a single species of cDNA, incorporated into a suitable expression vector, has been introduced via transfection. A number of interesting preliminary observations have been made on the function of the cytochrome P-450 associated with adrenocortical microsomes which catalyses the 17-hydroxylation of progesterone and pregnenolone. In confirmation of earlier reports the adrenal 17-OHase possesses both 17-hydroxylase as well as C17,20-lyase activities. However, the latter is only functional with 17-OH pregnenolone and not with 17-OH progesterone as substrate. This result differs from the numerous reports that a lyase activity for both substrates is associated with this P-450. The reason for this difference between a delta 4 and a delta 5 steroid remains unresolved although initial experiments indicate that the 5-alpha reduced progesterone is a suitable substrate for both the 17-OHase as well as lyase reactions. This result suggests an inhibitory effect of the delta 4 double bond preventing the carbon-carbon cleavage of the C-17,20 bond in 17-OH progesterone. Clearly more experiments will be required to resolve this question. Measurements of substrate affinity for the cytochrome P-450 expressed in COS cells appears to be influenced by a permeability barrier to the steroid effecting the transport of the steroid across the cell membrane into the cells. This conclusion is suggested by the presence of a time lag before the onset of metabolism as well as by the discrepancy in the concentration of substrate required to give half-maximal rates of metabolism, cf. the results obtained where the initial concentration of progesterone present in the reaction medium is altered versus those experiments measuring the kinetics of substrate depletion. The presence of such a barrier to the free movement of steroid across the membrane is interesting to contemplate when considering the build up of 17-OH pregnenolone required for the lyase reaction. Most unexpected where the results obtained when a comparable expression vector containing the cDNA for cytochrome b5 was cotransfected with pCD17 alpha 2. Cytochrome b5 has been postulated to be an electron transfer component participating in the cyclic function of some cytochromes P-450 (Hildebrandt and Estabrook, 1971).(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: R W
authors.initials: RW
authors.lastname: Estabrook
authors.forename: J I
authors.initials: JI
authors.lastname: Mason
authors.forename: C
authors.initials: C
authors.lastname: Martin-Wixtrom
authors.forename: M
authors.initials: M
authors.lastname: Zuber
authors.forename: M R
authors.initials: MR
authors.lastname: Waterman
mesh_terms: D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D015786:Cytochromes b5; D004247:DNA / Q000235:genetics; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D014162:Transfection
chemical_list: D003573:Cytochrome b Group; D011374:Progesterone; D011284:Pregnenolone; D004247:DNA; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System