Limits...
Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

Ghosh MC, Ray AK - PLoS ONE (2013)

Bottom Line: Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites.In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver.We purified the carbofuran-induced cytochrome P4501A protein from catfish liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America. ghosh.manik7@gmail.com

ABSTRACT
Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Show MeSH
Biochemical characterization of purified protein. A.Capillary electrophoresis of purified fraction D was performed using amine capillary column 57 cm long as described in ‘Materials and methods’. The amount of protein added to the column was 1 µg. The result showed only a single peak at 2.14 min, indicating the presence of a protein of a single molecular weight. B. Coomassie and silver stain of H1 eluate was performed upon resolving the protein in 8% SDS-PAGE according to the procedure mentioned in ‘Materials and methods’. C. Dot-blot of purified fraction D using the polyclonal antibody against rabbit. 1, 2, 3, 4, 5, and 6 indicated BSA, 20, 10, 5, 2, and 1 µg of purified protein, respectively. D. Induction of cytochrome P4501A1 in carbofuran-treated hepatocytes. Cultured catfish hepatocytes were treated with different doses of carbofuran for 24 h. Lysate was made, and protein was run on 8% SDS-PAGE gel. Immunoblot was performed as described in ‘Materials and methods’ using the antibody against purified protein. Lane1, marker; lane 2, control; lanes 3–7, microsomal protein obtained from hepatocytes treated with various doses of CF: 0.1, 1.0, 10, 100, and 1000 nM, respectively. E. Immunoblot was performed by running all the fractions in 8% SDS-PAGE. The antibody raised against fraction D was used as a primary antibody. The rest of the procedure was followed as described in ‘Materials and methods’.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585281&req=5

pone-0057919-g003: Biochemical characterization of purified protein. A.Capillary electrophoresis of purified fraction D was performed using amine capillary column 57 cm long as described in ‘Materials and methods’. The amount of protein added to the column was 1 µg. The result showed only a single peak at 2.14 min, indicating the presence of a protein of a single molecular weight. B. Coomassie and silver stain of H1 eluate was performed upon resolving the protein in 8% SDS-PAGE according to the procedure mentioned in ‘Materials and methods’. C. Dot-blot of purified fraction D using the polyclonal antibody against rabbit. 1, 2, 3, 4, 5, and 6 indicated BSA, 20, 10, 5, 2, and 1 µg of purified protein, respectively. D. Induction of cytochrome P4501A1 in carbofuran-treated hepatocytes. Cultured catfish hepatocytes were treated with different doses of carbofuran for 24 h. Lysate was made, and protein was run on 8% SDS-PAGE gel. Immunoblot was performed as described in ‘Materials and methods’ using the antibody against purified protein. Lane1, marker; lane 2, control; lanes 3–7, microsomal protein obtained from hepatocytes treated with various doses of CF: 0.1, 1.0, 10, 100, and 1000 nM, respectively. E. Immunoblot was performed by running all the fractions in 8% SDS-PAGE. The antibody raised against fraction D was used as a primary antibody. The rest of the procedure was followed as described in ‘Materials and methods’.

Mentions: The purity of the H1 eluate was further checked by capillary electrophoresis with a loading amount of 1 µg. The profile showed a major and sharp peak at 2.16 min with optical density of 0.15 at 280 nm. No secondary, or so-called noise, peak was observed, meaning that the protein added in that column was a single molecule with a homogeneous mass (Fig. 3A). In the SDS-PAGE analysis, the H1 eluate revealed pure protein with a presumptive molecular mass of 58 KD determined by Gel-Doc software (Bio-Rad, Tokyo, Japan) (Fig. 3B). Data of both coomassie and silver staining supported the observation of capillary electrophoresis demonstrating a major single peptide at around 58 KD. Upon confirming the molecular identity of the H1 eluate, we decided to develop an antibody against the purified protein by using rabbit (Fig. 3B).


Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

Ghosh MC, Ray AK - PLoS ONE (2013)

Biochemical characterization of purified protein. A.Capillary electrophoresis of purified fraction D was performed using amine capillary column 57 cm long as described in ‘Materials and methods’. The amount of protein added to the column was 1 µg. The result showed only a single peak at 2.14 min, indicating the presence of a protein of a single molecular weight. B. Coomassie and silver stain of H1 eluate was performed upon resolving the protein in 8% SDS-PAGE according to the procedure mentioned in ‘Materials and methods’. C. Dot-blot of purified fraction D using the polyclonal antibody against rabbit. 1, 2, 3, 4, 5, and 6 indicated BSA, 20, 10, 5, 2, and 1 µg of purified protein, respectively. D. Induction of cytochrome P4501A1 in carbofuran-treated hepatocytes. Cultured catfish hepatocytes were treated with different doses of carbofuran for 24 h. Lysate was made, and protein was run on 8% SDS-PAGE gel. Immunoblot was performed as described in ‘Materials and methods’ using the antibody against purified protein. Lane1, marker; lane 2, control; lanes 3–7, microsomal protein obtained from hepatocytes treated with various doses of CF: 0.1, 1.0, 10, 100, and 1000 nM, respectively. E. Immunoblot was performed by running all the fractions in 8% SDS-PAGE. The antibody raised against fraction D was used as a primary antibody. The rest of the procedure was followed as described in ‘Materials and methods’.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585281&req=5

pone-0057919-g003: Biochemical characterization of purified protein. A.Capillary electrophoresis of purified fraction D was performed using amine capillary column 57 cm long as described in ‘Materials and methods’. The amount of protein added to the column was 1 µg. The result showed only a single peak at 2.14 min, indicating the presence of a protein of a single molecular weight. B. Coomassie and silver stain of H1 eluate was performed upon resolving the protein in 8% SDS-PAGE according to the procedure mentioned in ‘Materials and methods’. C. Dot-blot of purified fraction D using the polyclonal antibody against rabbit. 1, 2, 3, 4, 5, and 6 indicated BSA, 20, 10, 5, 2, and 1 µg of purified protein, respectively. D. Induction of cytochrome P4501A1 in carbofuran-treated hepatocytes. Cultured catfish hepatocytes were treated with different doses of carbofuran for 24 h. Lysate was made, and protein was run on 8% SDS-PAGE gel. Immunoblot was performed as described in ‘Materials and methods’ using the antibody against purified protein. Lane1, marker; lane 2, control; lanes 3–7, microsomal protein obtained from hepatocytes treated with various doses of CF: 0.1, 1.0, 10, 100, and 1000 nM, respectively. E. Immunoblot was performed by running all the fractions in 8% SDS-PAGE. The antibody raised against fraction D was used as a primary antibody. The rest of the procedure was followed as described in ‘Materials and methods’.
Mentions: The purity of the H1 eluate was further checked by capillary electrophoresis with a loading amount of 1 µg. The profile showed a major and sharp peak at 2.16 min with optical density of 0.15 at 280 nm. No secondary, or so-called noise, peak was observed, meaning that the protein added in that column was a single molecule with a homogeneous mass (Fig. 3A). In the SDS-PAGE analysis, the H1 eluate revealed pure protein with a presumptive molecular mass of 58 KD determined by Gel-Doc software (Bio-Rad, Tokyo, Japan) (Fig. 3B). Data of both coomassie and silver staining supported the observation of capillary electrophoresis demonstrating a major single peptide at around 58 KD. Upon confirming the molecular identity of the H1 eluate, we decided to develop an antibody against the purified protein by using rabbit (Fig. 3B).

Bottom Line: Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites.In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver.We purified the carbofuran-induced cytochrome P4501A protein from catfish liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America. ghosh.manik7@gmail.com

ABSTRACT
Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Show MeSH