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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Abstract
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 P450XVII (P450XVII) 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, P450XVII. 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 P450XVII is likely to face the membrane-lipid phase, corrected spectral dissociation constants, Ks(corr), 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, Kp and Ks(corr) values were found to be independent of both parameters; Kp values amounted to 1920 and 3120, and Ks(corr) values amounted to 260 microM and 96 microM at 4 degrees C and 25 degrees C, respectively. Thermodynamic parameters delta H, delta S and delta G were calculated from Van't Hoff plots for progesterone partition into the membrane compartment, and for progesterone binding to P450XVII. Both of these processes were entropy dominated, and free energy changes amounted to about -18 kJ/mol for Kp and -20 kJ/mol for Ks(corr). Modification of P450XVII by gonadotropin-induced 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 K1 = 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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