Interaction of 7-n-alkoxycoumarins with cytochrome P-4502 and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients

Abstract

A study was made of the binding of 7-ethoxy-, 7-n-propoxy-and 7-n-pentoxy-coumarin to cytochrome P-4502 reconstituted into large unilamellar liposomes composed of a mixture of egg L-a-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants QPP-increased linearly with increasing proteoliposomal concentration. When both cytochrome P-4502 and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants KmPP for 0-dealkylation of 7-methoxy-, 7-ethoxy-and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-nbutoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.

Quantitative structure-activity relationships (QSARs) for inhibitors and substrates of CYP2B enzymes: importance of compound lipophilicity in explanation of potency differences

“…O-dealkylation rates of 7-alkoxycoumarins mediated by CYP2B4 in the rabbit: HOMO energies and log P data. Reference [18].…”

“…In addition, CYP2B-related activities for a series of 7-alkoxycoumarins [18] and of barbiturates [19] were retrieved from previously reported studies. Compound lipophilicities in the form of log P values were obtained from tabulations of experimental data [20,21] or calculated via the ClogP (BioByte, Pomona, CA) and Pallas (CompuDrug, Budapest, Hungary) software systems.…”

Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether

“…Until now, the ether O-dealkylation activities have been widely found in mammalian cytochrome P450 systems. Rates of cytochrome P450-catalyzed O-dealkylation reactions usually decrease with bulkier alkyl chains of alkyl and benzyl ethers [29][30][31], as similarly observed in strain DEE5151 [22]. Besides, there are reports that several members of the genera Rhodococcus and Corynebacterium possess cytochrome P450 systems catalyzing the O-dealkylation reactions of 2-alkoxyphenols and 7-ethoxycoumarin [26][27][28].…”

“…Besides, there are reports that several members of the genera Rhodococcus and Corynebacterium possess cytochrome P450 systems catalyzing the O-dealkylation reactions of 2-alkoxyphenols and 7-ethoxycoumarin [26][27][28]. Rates of cytochrome P450-catalyzed O-dealkylation reactions usually decrease with bulkier alkyl chains of alkyl and benzyl ethers [29][30][31], as similarly observed in strain DEE5151 [22]. In this study it is yet uncertain whether a cytochrome P450 enzyme system in strain DEE5151 is involved in the oxidation of diethyl ether, because it is difficult to interpret the inhibition studies resulting in no inhibitory effect, if the permeability barrier, e.g., mycolate layer, of the cell surface might exclude some inhibitors (e.g., carbon monoxide) from the cytoplasm.…”

Charge-Mediated Localization of Conjugated Polythiophenes in Zwitterionic Model Cell Membranes

“…These values can be determined from the fluorescence intensity depending on whether there is quenching or enhancement, as described in the Experimental section. [43][44][45] The increased emission intensity and blue-shift observed for P3TMAHT is common for CPEs when combined with phospholipids, 18,24 oppositely-charged, 19,51 or neutral surfactants. 51,52 Such changes have been attributed to the surfactant-induced break-up of the CPE clusters to form new surfactant-CPE aggregates, 52 where the conformation of the CPE changes from an aggregated to more extended state.…”