On the role of phospholipids in the reconstituted cytochrome P-450 system. A model study using dilauroyl and distearoyl glycerophosphocholine.
The difference in pentoxyresorufin O-dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P-450b (IIB1) system. The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P-450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P-450 for the NADPH-cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho-dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme. We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P-450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P-450 molecules in these systems. Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P-450 molecule, but also on the efficiency of electron transfer from NADPH-cytochrome reductase to cytochrome P-450.
Rietjens
I M
IM
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Ancher
L J
LJ
Veeger
C
C
D000818:Animals; D002846:Chromatography, Affinity; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D046911:Macromolecular Substances; D010713:Phosphatidylcholines; C015153:1,2-dilauroylphosphatidylcholine; D003577:Cytochrome P-450 Enzyme System; C010942:1,2-distearoyllecithin; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital