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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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Reconstitution of purified protein with DLPC and circular dichroism (CD). A.Purified protein was sonicated with three different concentrations of DLPC (0.5, 1.0, and 2 µM), and reconstitution was done as mentioned in ‘Materials and Methods’. EROD activity was measured from reconstituted protein. ** values are significant at p<0.01 compared to 0 uM of DLPC. # values are significant at p<0.05 compared to 0.5 uM of DLPC.+values are significant at p<0.05 compared to 1.0 uM of DLPC B. Circular dichroism (CD) from reconstituted protein was performed. α helicity was determined from curves by the method of Greenfield and Fasman (1969). Representative curves from three observations are presented. N indicates purified protein (1 ug), and A, B, and C indicate those curves sonicated with 0.5, 1.0, and 2.0 uM of DLPC with the same amount of purified protein. C. An amplified view of a representative curve (not the 4 B) at 200–225 nm portions as the hump at 208 nm indicates the α-helicity. D. α-helicity was calculated from curves in different conditions. N, A, B, and C indicate the purified protein, and DLPC inserted purified protein at concentrations of 0.5, 1.0, and 2.0 uM, respectively.
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pone-0057919-g004: Reconstitution of purified protein with DLPC and circular dichroism (CD). A.Purified protein was sonicated with three different concentrations of DLPC (0.5, 1.0, and 2 µM), and reconstitution was done as mentioned in ‘Materials and Methods’. EROD activity was measured from reconstituted protein. ** values are significant at p<0.01 compared to 0 uM of DLPC. # values are significant at p<0.05 compared to 0.5 uM of DLPC.+values are significant at p<0.05 compared to 1.0 uM of DLPC B. Circular dichroism (CD) from reconstituted protein was performed. α helicity was determined from curves by the method of Greenfield and Fasman (1969). Representative curves from three observations are presented. N indicates purified protein (1 ug), and A, B, and C indicate those curves sonicated with 0.5, 1.0, and 2.0 uM of DLPC with the same amount of purified protein. C. An amplified view of a representative curve (not the 4 B) at 200–225 nm portions as the hump at 208 nm indicates the α-helicity. D. α-helicity was calculated from curves in different conditions. N, A, B, and C indicate the purified protein, and DLPC inserted purified protein at concentrations of 0.5, 1.0, and 2.0 uM, respectively.

Mentions: The result of CD suggested that purified protein enriched with random coil in its conformation had been regaining α-helicity at a function of DLPC concentration; however, a synchronous loss of random coil clearly appeared in its conformation (Fig. 4B, 4D). The α-helicity of curves A, B, and C increased significantly compared to the curve of the only purified protein (curve N) (Table 2). The amplitude of α-helicity in the presence of DLPC was highly correlated with the enzymatic activity of the purified protein in the reconstitution system. Insertion of 1 uM and 2 uM of DLPC showed significantly higher (p<0.05 and p<0.01, respectively) EROD activity compared to 0.5 uM DLPC (Fig. 4A).


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

Ghosh MC, Ray AK - PLoS ONE (2013)

Reconstitution of purified protein with DLPC and circular dichroism (CD). A.Purified protein was sonicated with three different concentrations of DLPC (0.5, 1.0, and 2 µM), and reconstitution was done as mentioned in ‘Materials and Methods’. EROD activity was measured from reconstituted protein. ** values are significant at p<0.01 compared to 0 uM of DLPC. # values are significant at p<0.05 compared to 0.5 uM of DLPC.+values are significant at p<0.05 compared to 1.0 uM of DLPC B. Circular dichroism (CD) from reconstituted protein was performed. α helicity was determined from curves by the method of Greenfield and Fasman (1969). Representative curves from three observations are presented. N indicates purified protein (1 ug), and A, B, and C indicate those curves sonicated with 0.5, 1.0, and 2.0 uM of DLPC with the same amount of purified protein. C. An amplified view of a representative curve (not the 4 B) at 200–225 nm portions as the hump at 208 nm indicates the α-helicity. D. α-helicity was calculated from curves in different conditions. N, A, B, and C indicate the purified protein, and DLPC inserted purified protein at concentrations of 0.5, 1.0, and 2.0 uM, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057919-g004: Reconstitution of purified protein with DLPC and circular dichroism (CD). A.Purified protein was sonicated with three different concentrations of DLPC (0.5, 1.0, and 2 µM), and reconstitution was done as mentioned in ‘Materials and Methods’. EROD activity was measured from reconstituted protein. ** values are significant at p<0.01 compared to 0 uM of DLPC. # values are significant at p<0.05 compared to 0.5 uM of DLPC.+values are significant at p<0.05 compared to 1.0 uM of DLPC B. Circular dichroism (CD) from reconstituted protein was performed. α helicity was determined from curves by the method of Greenfield and Fasman (1969). Representative curves from three observations are presented. N indicates purified protein (1 ug), and A, B, and C indicate those curves sonicated with 0.5, 1.0, and 2.0 uM of DLPC with the same amount of purified protein. C. An amplified view of a representative curve (not the 4 B) at 200–225 nm portions as the hump at 208 nm indicates the α-helicity. D. α-helicity was calculated from curves in different conditions. N, A, B, and C indicate the purified protein, and DLPC inserted purified protein at concentrations of 0.5, 1.0, and 2.0 uM, respectively.
Mentions: The result of CD suggested that purified protein enriched with random coil in its conformation had been regaining α-helicity at a function of DLPC concentration; however, a synchronous loss of random coil clearly appeared in its conformation (Fig. 4B, 4D). The α-helicity of curves A, B, and C increased significantly compared to the curve of the only purified protein (curve N) (Table 2). The amplitude of α-helicity in the presence of DLPC was highly correlated with the enzymatic activity of the purified protein in the reconstitution system. Insertion of 1 uM and 2 uM of DLPC showed significantly higher (p<0.05 and p<0.01, respectively) EROD activity compared to 0.5 uM DLPC (Fig. 4A).

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