Interactions of phospholipase D and cytochrome P450 protein stability.
Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.
Zangar
Richard C
RC
Pacific Northwest National Laboratory, Richland, WA 99352, USA. richard.zangar@pnl.gov
Fan
Yang-Yi
YY
Chapkin
Robert S
RS
D000704:Analysis of Variance; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004795:Enzyme Stability; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D009529:Nicardipine / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000378:metabolism
D002121:Calcium Channel Blockers; D010618:Phenanthrolines; D010743:Phospholipids; D003577:Cytochrome P-450 Enzyme System; D009529:Nicardipine; D010738:Type C Phospholipases; D010739:Phospholipase D; D004121:Dimethyl Sulfoxide