A SPECIFIC INTERACTION BETWEEN NADPH CYTOCHROME REDUCTASE AND PHOSPHATIDYLSERINE AND PHOSPHATIDYLINOSITOL

In the present study the interaction of NADPH-cytochrome reductase with phospholipids was investigated using P-31-NMR, thin-layer chromatography combined with chemical analysis, fluorescence spectroscopy and kinetic studies with purified rat liver cytochrome P450 IIB1. P-31-NMR analysis demonstrates that the composition of the phospholipids that remain associated to NADPH-cytochrome reductase upon its purification is significantly different from the phospholipid composition of the microsomal membrane. Thin-layer chromatography followed by chemical analysis of the phospholipid composition demonstrates that the isolated NADPH-cytochrome reductase was enriched in L-alpha-1,2-diacyl-sn-glycero-3-phosphoserine (acyl2GroPSer) and L-alpha-1,2-diacyl-sn-glycero-3-phosphoinositol (acyl2GroPIns) compared to the microsomal membrane. The observed preference of NADPH-cytochrome reductase for acyl2GroPSer and acyl2GroPIns appeared not to be a result of the procedure for solubilisation and/or purification of the protein. The specific interaction of NADPH - cytochrome reductase with acyl2GroPSer and acyl2GroPIns was further investigated by comparison of the effect of acyl2GroPSer and acyl2GroPIns with that of acyl2GroPCho and acyl2GroPEtn on the 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine-(DphPamGroPCho)-dependent quenching of the tryptophan fluorescence of purified NADPH-cytochrome reductase. The results demonstrate that the addition of acyl2GroPSer or acyl2GroPIns affects the DphPamGroPCho-dependent quenching of the tryptophan fluorescence in a manner significantly different from the addition of acyl2GroPCho or acyl2GroPEtn. The relatively larger DphPamGroPCho-induced quenching of the tryptophan fluorescence of NADPH-cytochrome reductase in the presence of acyl2GroPSer and acyl2GroPIns must result from a change in die conformation of NADPH-cytochrome reductase induced by the latter two lipids. Finally, the possible consequences of this special interaction of acyl2GroPSer and acyl2GroPIns with NADPH-cytochrome reductase on the kinetic characteristics of the cytochrome P450 system were studied using cytochrome-P450-IIB1-dependent 0-dealkylation of pentoxyresonifin as the model reaction. These studies demonstrate that a 1:1 mixture of acyl2GroPCho and acyl2GroPSer results in a significantly higher apparent maximum rate (V) of 0-dealkylation than a 1:1 mixture of acyl2GroPCho and acyl2GroPEtn or acyl2GroPCho alone. This increase in the apparent V can be ascribed to an acyl2GroPSer-dependent improvement of the interaction of NADPH-cytochrome reductase with cytochrome P450. This improvement of the interaction of the proteins cannot, however, be exclusively ascribed to the negative charge of acyl2GroPSer, since the other negatively charged.phospholipid investigated, namely acyl2GroPIns, resulted in a significant decrease in the apparent V. In both cases the substrate apparent K(m) was not affected. This opposite effect of acyl2GroPSer and acyl2GroPIns on die kinetics of the cytochrome P450 IIB1 system might provide a means for phospholipid-mediated regulation of this cytochrome P450 enzyme.