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Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum
C H Yun1, T Ahn, F P Guengerich
1Department of Biochemistry, Pai-Chai University, Taejon, 302-735, Korea. chyun@woonam.paichai.ac.kr
Abstract:
Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized. Purified human liver microsomal cytochromes P450 (P450)-P450 1A2 and P450 2E1-were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline. The activity was confirmed using recombinant and mutated human P450s expressed in bacteria. In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1. Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties.
Insights
Cytochromes P450 (P450) exhibit phospholipase D (PLD) activity in the liver endoplasmic reticulum (ER). This P450-mediated PLD activity impacts cell signaling and membrane properties.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) remains uncharacterized.
- Cytochromes P450 (P450) are crucial enzymes in liver metabolism.
- The role of P450s in phospholipid hydrolysis is not well understood.
Purpose of the Study:
- To characterize Phospholipase D (PLD) activity within the mammalian liver endoplasmic reticulum (ER).
- To investigate the potential role of Cytochromes P450 (P450) as PLD enzymes in the liver.
- To elucidate the mechanisms by which P450-mediated PLD activity influences cellular functions.
Main Methods:
- Purification and activity assays of human liver microsomal P450 enzymes (P450 1A2, P450 2E1).
- Enzymatic hydrolysis studies using phosphatidylcholine as a substrate.
- Confirmation of activity using recombinant and mutated P450s expressed in bacteria.
- Immunoinhibition assays in human liver microsomes using specific anti-P450 antibodies.
Main Results:
- Purified human liver P450 1A2 and P450 2E1 demonstrated significant PLD activity, hydrolyzing phosphatidylcholine to generate phosphatidic acid (PA) and choline.
- Recombinant and mutated P450s confirmed this enzymatic activity.
- Immunoinhibition studies indicated that anti-P450 1A2 showed the strongest inhibition, followed by anti-P450 2C and anti-P450 2E1, suggesting their contribution to PLD activity.
Conclusions:
- Cytochromes P450 (P450) function as significant phospholipase D (PLD) enzymes in the human liver ER.
- P450-mediated PLD activity can modulate cellular signaling pathways.
- This activity also contributes to changes in membrane properties, impacting overall cellular function.