A SPECIFIC INTERACTION BETWEEN NADPH CYTOCHROME REDUCTASE AND PHOSPHATIDYLSERINE AND PHOSPHATIDYLINOSITOL

In the present study the interaction of NADPH-cytochrome reductase wit h phospholipids was investigated using P-31-NMR, thin-layer chromatogr aphy combined with chemical analysis, fluorescence spectroscopy and ki netic studies with purified rat liver cytochrome P450 IIB1. P-31-NMR a nalysis demonstrates that the composition of the phospholipids that re main associated to NADPH-cytochrome reductase upon its purification is significantly different from the phospholipid composition of the micr osomal membrane. Thin-layer chromatography followed by chemical analys is of the phospholipid composition demonstrates that the isolated NADP H-cytochrome reductase was enriched in L-alpha-1,2-diacyl-sn-glycero-3 -phosphoserine (acyl2GroPSer) and L-alpha-1,2-diacyl-sn-glycero-3-phos phoinositol (acyl2GroPIns) compared to the microsomal membrane. The ob served preference of NADPH-cytochrome reductase for acyl2GroPSer and a cyl2GroPIns appeared not to be a result of the procedure for solubilis ation and/or purification of the protein. The specific interaction of NADPH - cytochrome reductase with acyl2GroPSer and acyl2GroPIns was fu rther investigated by comparison of the effect of acyl2GroPSer and acy l2GroPIns with that of acyl2GroPCho and acyl2GroPEtn on the glycero-3- phosphocholine-(DphPamGroPCho)-dependent quenching of the tryptophan f luorescence of purified NADPH-cytochrome reductase. The results demons trate that the addition of acyl2GroPSer or acyl2GroPIns affects the Dp hPamGroPCho-dependent quenching of the tryptophan fluorescence in a ma nner significantly different from the addition of acyl2GroPCho or acyl 2GroPEtn. 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 con formation of NADPH-cytochrome reductase induced by the latter two lipi ds. Finally, the possible consequences of this special interaction of acyl2GroPSer and acyl2GroPIns with NADPH-cytochrome reductase on the k inetic characteristics of the cytochrome P450 system were studied usin g cytochrome-P450-IIB1-dependent 0-dealkylation of pentoxyresonifin as the model reaction. These studies demonstrate that a 1:1 mixture of a cyl2GroPCho and acyl2GroPSer results in a significantly higher apparen t maximum rate (V) of 0-dealkylation than a 1:1 mixture of acyl2GroPCh o and acyl2GroPEtn or acyl2GroPCho alone. This increase in the apparen t V can be ascribed to an acyl2GroPSer-dependent improvement of the in teraction of NADPH-cytochrome reductase with cytochrome P450. This imp rovement of the interaction of the proteins cannot, however, be exclus ively ascribed to the negative charge of acyl2GroPSer, since the other negatively charged.phospholipid investigated, namely acyl2GroPIns, re sulted in a significant decrease in the apparent V. In both cases the substrate apparent K(m) was not affected. This opposite effect of acyl 2GroPSer and acyl2GroPIns on die kinetics of the cytochrome P450 IIB1 system might provide a means for phospholipid-mediated regulation of t his cytochrome P450 enzyme.