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Glycerol Increases Content and Activity of Human Cytochrome P‐4502E1 in a Transduced HepG2 Cell Line by Protein Stabilization
Author(s) -
Yang MingXue,
Cederbaum Arthur I.
Publication year - 1997
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.1997.tb03770.x
Subject(s) - glycerol , cytochrome , cycloheximide , biochemistry , methionine , cyp2e1 , chemistry , cytochrome c , microsome , cytochrome b , microbiology and biotechnology , protein biosynthesis , biology , enzyme , amino acid , mitochondrion , gene , mitochondrial dna
Glycerol is widely used to stabilize cytochrome P‐450 and prevent its transformation to cytochrome P‐420. The effect of glycerol on the content and activity of human cytochrome P‐4502E1 (CYP2E1) in a HepG2 cell line that stably and constitutively expresses this P‐450 was evaluated by immunoassays and oxidation of P ‐nitrophenol. Addition of 100 to 200 mM glycerol to the culture medium resulted in a 2½‐ to 3‐fold increase in the content and activity of CYP2E1 in microsomes isolated from the cells. Increases could be observed within 4 to 8 hr after addition of glycerol to the culture medium. Glycerol had no effect on the content of cytochrome b 5 or activities of NADPH‐cytochrome P‐450 reductase or NADH‐cytochrome b 5 reductase. Upon the addition of cycloheximide to stop protein synthesis, CYP2E1 content and activity decreased with apparent half‐lives of 6 and 4 hr, respectively. Glycerol prevented or decreased this loss of CYP2E1 content and activity. Labeling CYP2E1 with [ 35 S]methionine, followed by pulse‐chase experiments with cold methionine and immunoprecipitation of CYP2E1 indicated a half‐life for CYP2E1 of ∼3 hr. Glycerol increased the half‐life to ∼11 hr. Stabilization of CYP2E1 protein by glycerol was not additive or synergistic with the increase of CYP2E1 by ethanol or 4‐methylpyrazole, suggesting that all three agents elevate CYP2E1 by a similar type of mechanism in this model. These results indicate that glycerol can interact with human CYP2E1 to stabilize it against proteolytic degradation, increasing the half‐life of the enzyme and thereby elevating the content and activity of CYP2E1.