Effects of Ionic Strength on the Functional Interactions between CYP2B4 and CYP1A2

Kelley, Rusty; Reed, James R.; Backes, Wayne L.

doi:10.1021/bi0477900

Cited by 53 publications

(95 citation statements)

References 59 publications

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“…In contrast, maximal EROD activity of the CYP1A2 system increased to a maximum at 200 mM HEPES and declined with further increases in buffer concentration. Declining activities with increasing buffer concentrations were expected and have been attributed to the disruption of electrostatic interactions between reductase and P450, consistent with previous studies (6)(7)(8)(9)(10)(11)(12)(13)(14)16,17,24,39). When comparing EROD in the mixed reconstituted system to the sum of the rates of the binary systems (which can be seen by comparing the third and fourth bars from each group), a significant synergistic stimulation of EROD was observed at lower ionic strength; higher concentrations of HEPES relieved this synergism.…”

Section: Resultssupporting

confidence: 91%

“…Such a functional interaction between these P450 enzymes has not previously been reported. The behavior of these proteins in mixed reconstituted systems is analogous to that previously reported for CYP1A2 and CYP2B4 (21,22), a response that was shown to be sensitive to changes in ionic strength (24). If the functional complexes between these P450s during 7-ER turnover are governed by electrostatic charge, then disruption by high ionic strength would be expected to attenuate EROD synergism in the mixed reconstituted systems.…”

supporting

confidence: 74%

“…The interaction has also been reported in microsomal preparations (23). Interestingly, the inhibition of PROD reported in these mixed reconstituted systems was relieved at high ionic strength, consistent with disruption of a charge-paired CYP2B4-CYP1A2 complex (24).…”

supporting

confidence: 64%

“…The observation of interactions in mixed reconstituted systems containing CYP1A2 (i.e. CYP1A2/CYP2B4 and CYP1A2/CYP2E1) taken together with the lack of detectable interactions with the CYP2B4/CYP2E1 system strongly suggests that some feature of CYP1A2 may facilitate the formation of functional interactions with other P450 enzymes.CYP1A proteins have previously been shown to interact with CYP3A subfamily P450s (13,21,24,40). Alston et al (40) provided evidence for a physical interaction between CYP3A2 and CYP1A1 by treating liver microsomes with a bifunctional crosslinking agent to covalently link closely associated proteins.…”

mentioning

confidence: 99%

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Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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Mixed reconstituted systems containing CYP2B4, CYP1A2 and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin-Odealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin-Odeethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.The cytochrome P450 (P450) superfamily of proteins is functionally diverse, and well distributed in nature. In addition to their roles in the metabolism of numerous endogenous compounds, P450s are important contributors to drug metabolism, primarily by catalyzing the insertion of an oxygen atom into a substrate molecule. NADPH cytochrome P450 reductase (reductase) is the major electron transfer partner, and is required for the majority of NADPHdependent oxidation reactions. Multiple forms of cytochrome P450 exist in the endoplasmic reticulum in a large excess over reductase (1), with the ratio of P450 to reductase dependent † These studies were supported by a US Public Health Service Research Grant from the National Institute of Enviromental Health Sciences ES004344 (WLB), and a predoctoral fellowship from the Stanley Scott Cancer Center (RWK). * Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, La 70112, Voice 504-568-6557, FAX 504-568-6888, emailwbacke@lsuhsc.edu SUPPORTING INFORMATION AVAILABLE The supporting information includes ...

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Section: Resultssupporting

confidence: 91%

supporting

confidence: 74%

supporting

confidence: 64%

mentioning

confidence: 99%

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Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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“…Recombinant rabbit NADPH cytochrome P450 reductase (plasmid: pSC-CPR, provided by Dr. Lucy Waskell (Univ. Michigan); constructed from plasmid pCWori-rabbit reductase and plasmid pOR263-rat reductase, utilizing a T7 promoter) was expressed in E. coli C41, solubilized and purified as described previously (18)(19)(20). Both preparations of NADPH-cytochrome P450 reductase showed similar enzyme activities.…”

Section: Enzymesmentioning

confidence: 99%

Inhibition of CYP2B4 by 2-ethynylnaphthalene: Evidence for the co-binding of substrate and inhibitor within the active site

Cheng¹,

Harris²,

Reed³

et al. 2007

Archives of Biochemistry and Biophysics

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Abstract2-Ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components, (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.Cytochrome P450 refers to a superfamily of enzymes that catalyze the oxidation of a wide variety of exogenous and endogenous chemicals. The enzyme system most commonly supports oxygen insertion into a substrate molecule, generating a hydroxylated product; however, the initial monooxygenation can lead to a wide variety of reactions such as dealkylation, oxidative deamination, sulfoxidation, and epoxidation (1).The broad substrate selectivity of the P450 enzymes is due not only to the multiplicity of P450 enzymes, but also due to the characteristics of the active site. The active site for several of the P450 enzymes has been shown to be relatively large and capable of binding and metabolizing substrates of diverse chemical size and structure. A consequence of the large active site is its ability to accommodate multiple substrate/effector molecules. This effect is most commonly associated with CYP3A4 (2;3), where the presence of multiple compounds within the active site has been shown to alter the kinetics to exhibit cooperativity (4;5), and both substrate and product inhibition (2;6). The binding of multiple substrate/inhibitor molecules has also been documented for CYP2C9 (4), CYP ERYF (7;8), and P450 cam (9). The presence of active sites on other P450 enzymes that are sufficiently large to bind multiple ligands is clearly possible and likely, based on the relative size of the ligands as compared to the active sites of these nonspecific enzymes. 2-Ethynylnaphthalene (2EN) is a selective mechanism-based inhibitor of CYP2B4. CYP2B4 catalyzes the conversion of 2EN to the highly reactive intermediate, 2-naphthylacetic acid, which covalently modifies the apoprotein and results in its inactivation (10;11). In addition to its ability to inhibit CYP2B4-mediated reactions, 2EN could also act as a reversible inhibitor of both CYP1A1 and CYP1A2 (12). Although earlier studies reported that 2EN could act as a mechanism-based inhibitor of CYP1A proteins (...

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Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein

Huber

Backes

2008

Analytical Biochemistry

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Free heme binds to heme oxygenase as a prosthetic group and substrate in the conversion of heme to biliverdin, carbon monoxide, and free iron. Current methods for quantifying heme oxygenase-1 (HO-1) involve reconstitution of the enzyme with heme, followed by a hydroxyapatite column to remove the excess heme. As a result of the hydroxyapatite chromatography, there are significant losses of purified protein. We have developed a method which allows accurate quantitation of HO-1 using a heme titration and elimination of the final hydroxyapatite column, increasing the amount of purified protein.Heme oxygenase-1 (HO-1) is an inducible enzyme responsible for the oxidative cleavage of heme to biliverdin, carbon monoxide and free iron (1). Heme is a heterocyclic porphyrin with an iron center which serves as cofactor or prosthetic group for a number of proteins classified as hemoproteins. The binding of heme to HO-1 is unique because heme serves as both cofactor and substrate, and is degraded upon the addition of reducing equivalents. Recombinant HO-1 proteins from a variety of species have been expressed and purified, with human and rat most commonly studied (2,3). The deletion of 23 C-terminal amino acids, which serve as the membrane spanning domain (4), allowed for a swift purification of truncated human HO-1 (sHO-1) in large quantities. Standard purification procedures of sHO-1 produce a purified protein that must be reconstituted with heme to calculate the specific HO-1 concentration (3). Excess heme is removed from the HO-1-heme complex via passage through a final hydroxyapatite (HA) column. The HO-1-heme spectrum includes a Soret band at 405 nm, which is commonly utilized to quantify the complex with an extinction coefficient of 140 mM −1 cm −1 (5). The HO-1-heme complex produces a distinct absorption spectrum in comparison to the apoprotein. This report describes an improved method to quantify HO-1 using a heme titration, eliminating the final HA column, and effectively increasing the total purified protein.For these studies, truncated, human HO-1 was purified by a previously described method, with minor modifications (3). Following the expression of sHO1 in E. coli strain DH5α, the media appeared green in color, due to the accumulation of biliverdin (6). The bacterial cell pellet remained green throughout the purification procedure prior to the ammonium sulfate precipitation. The increase in ionic strength forced the disassociation of the biliverdin from the sHO-1, forming a colorless protein product. Following standard column chromatography, the Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology& The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112, Tel. 504-568-6557; FAX 504-568-6888; E-mail: wbacke@lsuhsc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the man...

show abstract

Effects of Ionic Strength on the Functional Interactions between CYP2B4 and CYP1A2

Cited by 53 publications

References 59 publications

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Inhibition of CYP2B4 by 2-ethynylnaphthalene: Evidence for the co-binding of substrate and inhibitor within the active site

Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein

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