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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.

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Related in: MedlinePlus

SDS-PAGE electrophoresis and chromatogram of eluted protein. A.SDS-PAGE (8%) was performed of polyethylene glycol (PEG) fractionated protein from solubilized microsome. Lane1, marker; lane 2, microsome; lane 3, solubilized microsome with CHAPS and sodium-cholate; lanes 4–6; solubilized microsomal protein with 10, 20, and 30% PEG, respectively. B. Chromatogram of eluted protein from DE-52 column. Protein was eluted from the column by linear gradient of KCl, and 10-ml fractions were collected. Four conspicuous peaks in terms of protein content measured at 280 nm were observed in the eluate as; Hb, A, B, C, and D (upper graph). Fractions were pooled, and heme content was tested at 416 nm (lower graph). C. The pooled fractions of D peak were added to the equilibrated hydroxyapatite column. The protein was eluted from the column by step gradient of phosphate buffer, and 5-ml fractions were collected. Fractions were pooled. There were two peaks, H1 and H2, in terms of protein content (upper graph). The lower graph represents the heme content of the pooled fractions.
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pone-0057919-g002: SDS-PAGE electrophoresis and chromatogram of eluted protein. A.SDS-PAGE (8%) was performed of polyethylene glycol (PEG) fractionated protein from solubilized microsome. Lane1, marker; lane 2, microsome; lane 3, solubilized microsome with CHAPS and sodium-cholate; lanes 4–6; solubilized microsomal protein with 10, 20, and 30% PEG, respectively. B. Chromatogram of eluted protein from DE-52 column. Protein was eluted from the column by linear gradient of KCl, and 10-ml fractions were collected. Four conspicuous peaks in terms of protein content measured at 280 nm were observed in the eluate as; Hb, A, B, C, and D (upper graph). Fractions were pooled, and heme content was tested at 416 nm (lower graph). C. The pooled fractions of D peak were added to the equilibrated hydroxyapatite column. The protein was eluted from the column by step gradient of phosphate buffer, and 5-ml fractions were collected. Fractions were pooled. There were two peaks, H1 and H2, in terms of protein content (upper graph). The lower graph represents the heme content of the pooled fractions.

Mentions: Using CF-treated fish livers, we purified the cytochrome P4501A protein enriched in EROD activity. SDS-PAGE analysis of solubilized microsome after PEG-cut showed that 20% was the most suitable for availability of P450 protein (Fig. 2A). Therefore, protein obtained at 20% PEG was collected and loaded onto a DE-52 column. Four pooled fractions (Fraction-A, Fraction-B, Fraction-C, and Fraction-D) eluted from the DE-52 column were collected and used to measure specific activity (Fig. 2B). Fraction-D demonstrated the maximum ratio of cytochrome P450 protein when tested for heme and protein, respectively (Fig. 2B, lower graph). In the reconstitution study, Fraction-D showed maximum reactivity to 7-ethoxyresorufin in comparison to any other fraction (Table 1).


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

Ghosh MC, Ray AK - PLoS ONE (2013)

SDS-PAGE electrophoresis and chromatogram of eluted protein. A.SDS-PAGE (8%) was performed of polyethylene glycol (PEG) fractionated protein from solubilized microsome. Lane1, marker; lane 2, microsome; lane 3, solubilized microsome with CHAPS and sodium-cholate; lanes 4–6; solubilized microsomal protein with 10, 20, and 30% PEG, respectively. B. Chromatogram of eluted protein from DE-52 column. Protein was eluted from the column by linear gradient of KCl, and 10-ml fractions were collected. Four conspicuous peaks in terms of protein content measured at 280 nm were observed in the eluate as; Hb, A, B, C, and D (upper graph). Fractions were pooled, and heme content was tested at 416 nm (lower graph). C. The pooled fractions of D peak were added to the equilibrated hydroxyapatite column. The protein was eluted from the column by step gradient of phosphate buffer, and 5-ml fractions were collected. Fractions were pooled. There were two peaks, H1 and H2, in terms of protein content (upper graph). The lower graph represents the heme content of the pooled fractions.
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Related In: Results  -  Collection

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

pone-0057919-g002: SDS-PAGE electrophoresis and chromatogram of eluted protein. A.SDS-PAGE (8%) was performed of polyethylene glycol (PEG) fractionated protein from solubilized microsome. Lane1, marker; lane 2, microsome; lane 3, solubilized microsome with CHAPS and sodium-cholate; lanes 4–6; solubilized microsomal protein with 10, 20, and 30% PEG, respectively. B. Chromatogram of eluted protein from DE-52 column. Protein was eluted from the column by linear gradient of KCl, and 10-ml fractions were collected. Four conspicuous peaks in terms of protein content measured at 280 nm were observed in the eluate as; Hb, A, B, C, and D (upper graph). Fractions were pooled, and heme content was tested at 416 nm (lower graph). C. The pooled fractions of D peak were added to the equilibrated hydroxyapatite column. The protein was eluted from the column by step gradient of phosphate buffer, and 5-ml fractions were collected. Fractions were pooled. There were two peaks, H1 and H2, in terms of protein content (upper graph). The lower graph represents the heme content of the pooled fractions.
Mentions: Using CF-treated fish livers, we purified the cytochrome P4501A protein enriched in EROD activity. SDS-PAGE analysis of solubilized microsome after PEG-cut showed that 20% was the most suitable for availability of P450 protein (Fig. 2A). Therefore, protein obtained at 20% PEG was collected and loaded onto a DE-52 column. Four pooled fractions (Fraction-A, Fraction-B, Fraction-C, and Fraction-D) eluted from the DE-52 column were collected and used to measure specific activity (Fig. 2B). Fraction-D demonstrated the maximum ratio of cytochrome P450 protein when tested for heme and protein, respectively (Fig. 2B, lower graph). In the reconstitution study, Fraction-D showed maximum reactivity to 7-ethoxyresorufin in comparison to any other fraction (Table 1).

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
Related in: MedlinePlus