Glycerol increases content and activity of human cytochrome P-4502E1 in a transduced HepG2 cell line by protein stabilization.
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 2 1/2- 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 b5 or activities of NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 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 [35S]methionine, followed by pulse-chase experiments with cold methionine and immunoprecipitation of CYP2E1 indicated a half-life for CYP2E1 of approximately 3 hr. Glycerol increased the half-life to approximately 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.
Yang
M X
MX
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Cederbaum
A I
AI
D000818:Animals; D002461:Cell Line, Transformed; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics*; D010450:Endopeptidases / Q000502:physiology; D004795:Enzyme Stability / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology*; D006801:Humans; D008114:Liver Neoplasms, Experimental; D014161:Transduction, Genetic / Q000187:drug effects / Q000235:genetics*
D019392:Cytochrome P-450 CYP2E1; D010450:Endopeptidases; D005990:Glycerol