Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.
Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.
Imaoka
S
S
Laboratory of Chemistry, Osaka City University Medical School, Japan.
Imai
Y
Y
Shimada
T
T
Funae
Y
Y
D000818:Animals; D001555:Benzene Derivatives / Q000494:pharmacology; D001571:Benzoflavones / Q000494:pharmacology; D002384:Catalysis; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008012:Lidocaine / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009543:Nifedipine / Q000378:metabolism; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D013739:Testosterone / Q000378:metabolism
D001555:Benzene Derivatives; D001571:Benzoflavones; D002793:Cholic Acids; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; C015153:1,2-dilauroylphosphatidylcholine; D013739:Testosterone; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D008012:Lidocaine; D009251:NADPH-Ferrihemoprotein Reductase; C017251:1,2-oleoylphosphatidylcholine; D019826:Cholic Acid; D009543:Nifedipine; C007164:cumene hydroperoxide