Ligand partitioning into membranes: Its significance in determining KM and KS values for cytochrome P‐450 and other membrane bound receptors and enzymes

Parry, Gordon; Palmer, David N.; Williams, David

doi:10.1016/0014-5793(76)80348-1

Cited by 91 publications

(24 citation statements)

References 8 publications

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“…The PGIS lipophilic substrate, PGH # , derived from arachidonic acid by PGHS, would then have ready access to the PGIS active site without even leaving the membrane to contact the polar medium. This is consistent with the results from the studies of the relationship between the ER membrane and the substrate access channel for other P450s using hydrophobic drugs [27,28], and fluorescence energy transfer [29]. Alternative hypotheses can be envisaged, including the possibility of proteinprotein interaction.…”

Section: Discussionsupporting

confidence: 88%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I # synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A H (1 A H l 0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare sitespecific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the

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Section: Discussionsupporting

confidence: 88%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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“…For the shorter-alkylchain coumarin ethers, however, the fluorescence model is a valuable alternative model for the determination of their liposomal partition coefficients. Parry et al (1976) distinguished between aqueous-and lipidfaced binding sites of membrane-bound proteins. Backes et al (1982Backes et al ( , 1984 Backes et al, 1982).…”

Section: Discussionmentioning

confidence: 99%

“…A similar dependence on enzyme concentration has been observed for the apparent dissociation constants K.Sy" of the interaction of liver microsomal cytochrome P-450 with a number of amphipathic ligands (Waterman et al, 1973;Ebel et al, 1978;Backes et al, 1982Backes et al, , 1984 and of the steroid binding to testicular microsomal cytochrome P-450 (Barbieri et al, 1981;Kuhn-Velten et al, 1989) and adrenal cytochrome P-450 reconstituted in liposomal membranes (Kominami et al, 1986(Kominami et al, , 1988. Emphasizing the biphasic nature of membrane suspensions and assuming that accumulation of ligands and substrates into the membranes can be considered as a partition equilibrium, Parry et al (1976) developed a formalism for the description of ligand and substrate binding to membrane-bound proteins. In this model, the sizes of the aqueous and lipid phases are defined in terms of their volume fractions.…”

Section: Introductionmentioning

confidence: 99%

“…Komori et al (1984) purified a cytochrome P-450 isoenzyme, called cytochrome P-4502, with high affinity for 7-nalkoxycoumarins. The present paper reports on the determination of the membrane partition coefficients of several 7-nalkoxycoumarins based on application of existing models (Parry et al, 1976; to binding and kinetic studies performed with cytochrome P-4502 reconstituted into liposomal material. The validity of these methods was assessed by direct determination of liposomal partition coefficients based on quenching of the fluorescence of the ligands by the liposomal material.…”

Section: Introductionmentioning

confidence: 99%

“…where kc.t is the catalytic constant (Fersht, 1985), [EO] the total concentration of enzymic sites, Klm the effective Michaelis constant By analogy with the kinetic model, expressions for ligand binding to membrane-bound acceptors can be formulated (Parry et al, 1976;. For the equilibrium reaction:…”

Section: Introductionmentioning

confidence: 99%

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

Vermeir

Boens

Heirwegh

1992

Biochemical Journal

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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.

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Influence of membrane partitioning on inhibitors of membrane-bound enzymes

Homan

Hamelehle

2001

Journal of Pharmaceutical Sciences

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Ligand partitioning into membranes: Its significance in determining K_M and K_S values for cytochrome P‐450 and other membrane bound receptors and enzymes

Cited by 91 publications

References 8 publications

Substrate access channel topology in membrane-bound prostacyclin synthase

Substrate access channel topology in membrane-bound prostacyclin synthase

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

Influence of membrane partitioning on inhibitors of membrane-bound enzymes

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