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Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta 4 and the delta 5 steroidogenic pathways can lead to testosterone production.
The kinetics of oxidation of eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse ferrocytochromes c, modified at lysine 7, 13, 25, 27, 60, 72, 86, or 87, and of one trinitrophenyl horse ferrocytochrome c, modified at lysine 13, by the 3- and 3+ inorganic complexes hexacyanoferrate(III) (Fe(CN)6(3-) ) and tris(1,10-phenanthroline)cobalt(III) (Co(phen)3(3+) ) have been characterized. The influence of the modified residues on the bimolecular rate constants for these reactions define the protein molecular surface involved. The site of electron exchange for both oxidants appears to be the solvent accessible edge of the heme prosthetic group or a closely related structure on the "front" surface of the molecule. The reaction with Fe(CN)6(3-) is most strongly influenced by modification of lysine 72, a residue to the left of the exposed heme edge. (CDNP lysine 72 cytochrome c yields a 3.6-fold decrease in the bimolecular rate constant, as compared to that for the native protein.) However, it is the region around lysine 27, to the right of the heme edge, that is most influential in the reaction with Co(phen)3(3+). (CDNP-lysine 27 cytochrome c exhibits a 7.3-fold increase in the rate constant, as compared to that for the native protein.) The kinetics of reaction of the CDNP-lysine 13, 60, 72, and 87 modified cytochromes c with Fe(CN)5(4-aminopyridine)2- as oxidant and Fe(CN)5(4-aminopyridine)3- and Fe(CN)5-(imidazole)3- as reductants have also been determined and further illustrate the influence of electrostatics on the kinetics of such protein-small molecule electron exchanges.
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)
In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.
Cytochrome P-450 (P-450) 2A6 was purified by chromatography of human liver microsomes. The final preparation was electrophoretically homogeneous and contained 16 nmol of P-450/mg of protein. The amino-terminal amino acid sequence of the protein (first 13 residues) matched that of the reported cDNA exactly. The UV-visible spectrum indicated that the isolated hemoprotein was in the low-spin form. The protein was recognized by rabbit antibodies raised against rat P-450 2A1, and a rabbit antiserum against the P-450 2A6 preparation was also prepared. With these antibodies, it was estimated that P-450 2A6 accounted for a maximum of 1% of the total P-450 present in the human liver microsomes; the level varied greater than 100-fold among the 20 samples examined. Purified P-450 2A6 catalyzed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation at rates similar to those measured in the human liver sample used to prepare P-450 2A6, and these two microsomal activities were strongly inhibited by the antibodies. The purified P-450 2A6 enzyme also catalyzed low levels of 4,4'-methylene-bis(2-chloroaniline) (MOCA) N-oxidation and activation of aflatoxin B1, 6-aminochrysene, 2-amino-3-methylimidazo[4,5-f]quinoline, and 2-amino-3,5-dimethylimidazo [4,5-f]quinoline to genotoxic products; the antibody inhibited the activity of purified P-450 2A6 towards aflatoxin B1 and 6-aminochrysene but did not inhibit these reactions in human liver microsomes (MOCA N-oxidation was inhibited approximately 20%). Human P-450 2A6 did not catalyze testosterone 7 alpha-hydroxylation, a characteristic activity of the related rat P-450 2A1 protein. These results emphasize the need to characterize individual P-450 enzymes in order to understand their functions in the context of more complex systems.
Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.