Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Myasoedova, K. N.; Arutyunyan, Alexander M.; Magretova, Natalia N.

doi:10.1007/s10540-006-9005-0

Cited by 6 publications

(6 citation statements)

References 31 publications

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“…16–21 Attempts to prevent this aggregation using detergents, salts, and lipids have been shown to affect ET. 22–24 Due to these difficulties, it has not been possible to determine, how oligomer formation alters metabolism, and it is very possible that aggregation affects ET. A related factor is whether CPR binding to P450s, an obligatory step in the P450 catalytic cycle, alters ET.…”

Section: Introductionmentioning

confidence: 99%

Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

Bostick¹,

Flora²,

Gannett³

et al. 2015

Nanotechnology

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Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site.

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Section: Introductionmentioning

confidence: 99%

Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

Bostick¹,

Flora²,

Gannett³

et al. 2015

Nanotechnology

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“…The individual electron transfer complexes represent tight associations of proteins that co-purify even under strong detergent or salt conditions [4]. This is not always the case with protein assemblies.…”

Section: Introductionmentioning

confidence: 99%

The mitochondrial inner membrane protein Mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled‐coil‐helix coiled‐coil‐helix domain‐containing protein 3 and 6 and DnaJC11

Xie

Marusich²,

Souda³

et al. 2007

FEBS Letters

189

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A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.

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“…In contrast, CYP1A2 was found to require much higher concentrations of Emulgen 913 to produce monomeric forms, and at the higher detergent concentrations, a loss of monooxygenase activity was seen 71 . However, non-ionic detergents were later shown to modulate P450 metabolism at concentrations that would produce no changes in aggregation state of the P450 72 and thus confound interpretation of data from prior studies utilizing detergents.…”

Section: P450-p450 Interaction Effects On Metabolismmentioning

confidence: 99%

“…To fully interpret results obtained from immobilized proteins, it is crucial to have a deep understanding of the protein layer thickness, the density of adsorbed protein, orientation of the protein, secondary and tertiary structure of the adsorbed protein, protein stability, and most importantly the effect of adsorption on the biological function of the protein 171 Generally, information about protein secondary and tertiary structure can be obtained by Circular Dichroism (CD) spectroscopy 72 . CD measurements are generally conducted in solution, thus labs have employed the use of nanoparticles to observe how protein adsorption affects structure 173,174 .…”

Section: Characterization Of Immobilized P450smentioning

confidence: 99%

“…P450s are known to aggregate, forming homo-and hetero-oligomers in solution, and a range of effects from these interactions can occur, such as alteration in catalytic rate or degree of binding to the binding partner CPR [64][65][66]68,194,196 . Attempts to prevent this aggregation using detergents, salts, and lipids have been shown to affect ET 57,72,235 . Due to these difficulties, it has not been possible to determine, how oligomer formation alters metabolism, and it is very possible that aggregation affects ET.…”

Section: Introductionmentioning

confidence: 99%

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Use of protein immobilization to measure cytochrome P450 conduction and metabolism kinetics

Bostick¹

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USE OF PROTEIN IMMOBILIZATION TO MEASURE CYTOCHROME P450 METABOLISM KINETICS AND ITS EFFECTORS by Christopher D. Bostick Cytochrome P450s (P450s) are a large family (>11,000) of heme thiolated proteins that are responsible for ~ 75% of the metabolism of pharmaceuticals on the market. Understanding P450 mediated metabolism is crucial for accurate in vitro predictions of drug metabolism. P450 protein-protein interactions have been shown to alter enzyme catalytic activity. Furthermore, these interactions are isoform specific, and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show these effects are also dependent on the protein binding partner cytochrome P450 reductase (CPR), and the order of protein addition to purified reconstituted enzyme systems. In the current work, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drugmetabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homo/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real-time utilizing surface plasmon resonance (SPR). Lastly, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems. Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4 in rank order. KD values obtained via SPR show strong binding, in the nanomolar range, of the above pairs, with CYP2D6 yielding the lowest KD, followed by CYP2C9, CPR, and CYP3A4. Metabolic incubations show immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 in solution, but inhibited when CYP2C9 was immobilized, both at saturating and sub-saturating CPR concentrations. Order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced magnitude of inhibition for CYP3A4, but not CYP2D6. These results indicate isoform specific P450 interactions and effects on P450 mediatedmetabolism. These findings are important in evaluating how in vitro results are obtained for measuring P450 kinetics, and provide a better mechanistic understanding of P450-P450 interactions to allow for better prediction of in vivo metabolism from in vitro data. We also demonstrate that gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized P450s without aggregation. Given that transfer of the 1 st electron to the P450 heme group acts as the gating step for the catalytic cycle, understanding electron transfer in P450s could shed light on metabolism kinetics. Conductance measurements of nanopillars with immobilized CYP2C9 using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 ...

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Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Cited by 6 publications

References 31 publications

Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

The mitochondrial inner membrane protein Mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled‐coil‐helix coiled‐coil‐helix domain‐containing protein 3 and 6 and DnaJC11

Use of protein immobilization to measure cytochrome P450 conduction and metabolism kinetics

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