Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

Sheets, Joel J.; Estabrook, Ronald W.

doi:10.1021/bi00344a043

Cited by 25 publications

(6 citation statements)

References 26 publications

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“…The experiments also suggest that the concerted mechanism is not a result of saturating 4 Despite the fact that 6/?,16a-diOHP is a metabolite of progesterone formed by purified cytochrome P-450g, it is clear from the results in Table IV that other cytochrome P-450 isozymes are responsible for this reaction in microsomes. It is interesting to note that 6/?,16a-dihydroxyandrostenedione is a product of the sequential oxidation of androstenedione in rat liver microsomes (Sheets & Estabrook, 1985). figure 8: Effect of added reductase on progesterone metabolism in microsomes from adult male rats.…”

Section: Resultsmentioning

confidence: 99%

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Swinney¹,

Ryan²,

Thomas³

et al. 1988

Biochemistry

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Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.

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Section: Resultsmentioning

confidence: 99%

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Swinney¹,

Ryan²,

Thomas³

et al. 1988

Biochemistry

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“…Further, the present study for the first time considers quantitatively the possible accumulation of intermediates in the membrane compartment. Assuming that steroid substrates and intermediates interact with the hydrophobic substrate binding site of the cytochrome P-450 from the membrane phase and not from the aqueous phase [7,12,23,[30][31][32], the actual concentration of a certain intermediate in the membrane compartment should be relevant for the 'handling' of that intermediate by the cytochrome P-450. In the case of the experiment described herein, the congruity of 3H/14C ratios in the membrane-associated 1 7a-hydroxyprogesterone pool and the androgen pool is indeed a clear indication that only the membrane-associated 1 7a-hydroxyprogesterone pool is accessible to the C-1 7,20-lyase function of the cytochrome P-450(C-17) and therefore determines androgen formation (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…As already indicated in previous studies emphasizing the role of a specific microenvironment for enzyme-substrate interactions [21][22][23], our study likewise demonstrates the significance of determining local substrate concentrations as close to the catalytic site as possible. In this context, we are aware of the possibility that there may be specific membrane domains (possibly in close proximity to the enzyme molecules [30,33]) which accumulate specific ligands to a higher degree than do other domains. However, this possible limitation apparently does not generally affect our model of a true intermediacy of the 17a-hydroxyprogesterone in the membrane compartment only.…”

Section: Discussionmentioning

confidence: 99%

Specific accumulation of 17α-hydroxyprogesterone in microsomal membranes during the process of cytochrome P-450(C-17)-catalysed androgen biosynthesis. A dynamic study of intermediate formation and turnover

Kühn‐Velten

Lessmann

Förster

et al. 1988

Biochemical Journal

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A complete dynamic analysis of cytochrome P-450(C-17)-catalysed androgen biosynthesis from a single dose of progesterone and 17 alpha-hydroxyprogesterone in a double-label double-substrate experiment was performed in order to elucidate the controversial intermediacy of 17 alpha-hydroxyprogesterone. Label distribution within the steroid fractions as well as in the membrane and buffer compartments yields direct evidence that the endogenously formed 17 alpha-hydroxyprogesterone (which is in an 'intermediate state') accumulates to a higher degree in microsomal membranes than does the exogenously added 17 alpha-hydroxyprogesterone (which is in a 'substrate state') under certain conditions. It is also demonstrated that endogenously formed 17 alpha-hydroxyprogesterone may partly leave the membrane compartment (in terms of a 'leakage' or 'overflow' phenomenon) and is then able to equilibrate with the pool of exogenously added 17 alpha-hydroxyprogesterone. Since only the label distribution in the membrane-associated (but not always in the aqueous) 17 alpha-hydroxyprogesterone pool corresponds to the label distribution in the androgen fraction, it is concluded that only the membrane-associated 17 alpha-hydroxyprogesterone pool is directly accessible to cytochrome P-450(C-17)-catalysed conversion into androgens.

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“…Although analysis of partition processes in multicompartment systems significantly improves quantification of local ligand concentrations in the microenvironment of a respective enzyme or receptor, there may be (possibly transient) differences in homogeneity within a membrane phase causing development of microdomains which specifically accumulate or exclude small hydrophobic ligands and modulate properties of organized enzyme systems in membrane-dependent metabolic sequences [13, [55][56][57][58][59][60][61][62].…”

Section: Methodological Aspectsmentioning

confidence: 99%

“…In particular, the putative steroid-binding site of rat testicular P45OXVII has been assigned to a hydrophobic cleft, with direct access to the membrane reservoir of steroid substrates [3]. Direct transmembrane steroid transfer during subsequent hydroxylation reactions on a single steroid molecule by liver monooxygenase systems points also to membrane-oriented binding sites [62]. The same conclusion has been reached on the basis of rate constants of steroid partition, association and dissociation in the adrenal P450XXI (the CYP2iAi gene product [26]) system [I 93.…”

Section: Methodological Aspectsmentioning

confidence: 99%

Thermodynamics and modulation of progesterone microcompartmentation and hydrophobic interaction with cytochrome P450XVII based on quantification of local ligand concentrations in a complex multi‐component system

Kühn‐Velten

1991

European Journal of Biochemistry

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An approach towards the determination of hydrophobic ligand distribution in endoplasmic reticulum membrane suspensions, and of hydrophobic ligand interaction with membrane-anchored proteins, based on calculations of local ligand pools, is presented. Rat testicular microsomes containing cytochrome P45OXVII (P45OXVII) were used as the model system and considered as consisting of three compartments, i.e. membrane lipid phase, aqueous phase and the ligand-binding protein, P45OXVII. Combinations of spectrophotometry, ultracentrifugation and equilibrium dialysis were used to quantify progesterone concentrations in each of the three compartments, as well as partition coefficients, Kp. Since the substrate-access channel of P45OXVII is likely to face the membrane-lipid phase, corrected spectral dissociation constants, K Y ' , were calculated on the basis of free, i.e. not enzyme-bound, progesterone concentrations in the membrane compartment. Modulation of individual components and construction of more complex systems demonstrated the validity of this concept for analysis of multicompartment systems. Although ligand distribution was considerably affected by both ligand and membrane concentrations, K p and Ki0" values were found to be independent of both parameters; Kp values amounted to 1920 and 3120, and Kiorr values amounted to 260 pM and 96 pM at 4°C and 25 "C, respectively. Thermodynamic parameters AH, AS and AG were calculated from Van't Hoff plots for progesterone partition into the membrane compartment, and for progesterone binding to P45OXVII. Both of these processes were entropy dominated, and free energy changes amounted to about -18 kJ/mol for Kp and -20 kJ/mol for Kiorr. Modification of P45OXVII by gonadotropininduced down-regulation, and by addition of a competitive inhibitor (estradiol) had no effect on progesterone partition. Consideration of Kp = 310 for estradiol allowed the determination of a corrected Ki = 3.09 mM. Modification of the membrane-lipid phase by detergents affected progesterone-P450XVII interaction solely by modulation of Kp; modification of the aqueous phase by addition of bovine serum albumin as a fourth compartment acted solely via additional steroid attraction. This model system therefore stresses the relevance of the local environment of membrane-bound enzymes or receptors for quantification of their interaction with substrates or ligands.

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Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

Cited by 25 publications

References 26 publications

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Specific accumulation of 17α-hydroxyprogesterone in microsomal membranes during the process of cytochrome P-450(C-17)-catalysed androgen biosynthesis. A dynamic study of intermediate formation and turnover

Thermodynamics and modulation of progesterone microcompartmentation and hydrophobic interaction with cytochrome P450XVII based on quantification of local ligand concentrations in a complex multi‐component system

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