Abstract

The effect of phosphatidylcholine, an essential component of the reconstituted liver microsomal enzyme system which hydroxylates drugs, on the sedimentation and other properties of cholate-solubilized cytochrome P-450 was studied. Upon centrifugation of the cytochrome P-450 fraction for 1 hr at 105,000 x g at 5°, 85-90% of the cytochrome P-450 remained in the supernatant fraction. The apparent sedimentation coefficient of the cytochrome P-450 was 13 S, whether this fraction was centrifuged alone or in the presence of the reductase fraction and phosphatidylcholine. When the concentration of bound cholate was reduced by dialysis to approximately 1 µg/mg of protein, corresponding to about 1 mole/mole of cytochrome P-450, almost full catalytic activity was retained and an apparent sedimentation coefficient of 18 S was observed. This value was not altered by the addition of the reductase fraction and the lipid. The molecular weight of the dialyzed cytochrome P-450 was judged to be about 350,000 by sedimentation velocity measurements in the ultracentrifuge, sucrose density gradient centrifugation, and gel exclusion chromatography, but this figure must be considered provisional in view of the heterogeneity of the preparation. The effect of sedimentation on the distribution of enzyme activity was also determined. After centrifugation of the complete reaction mixture containing the cytochrome P-450 fraction, the reductase fraction, phosphatidylcholine, TPNH, and benzphetamine for 1 hr at 105,000 x g, over 90% of the original hydroxylation activity remained in the supernatant layer. The resuspended pellet was inactive when supplemented with TPNH and substrate. When the reconstituted enzyme system was subjected to gel exclusion chromatography, the cytochrome P-450 and TPNH—cytochrome P-450 reductase were well separated from each other and from the phospholipid, thereby indicating that these components behave as separate entities rather than as a tightly associated complex. The solubilized cytochrome P-450 fraction was examined in the electron microscope and found to have the appearance of individual protein molecules rather than of highly complex structures. Similarly, a mixture of the cytochrome P-450 fraction, the reductase fraction, and phospholipid was devoid of structures which could be attributed to aggregation. These findings indicate that, although a dissociable complex containing cytochrome P-450, reductase, and phosphatidylcholine may function in catalysis, the phospholipid exerts its effect on drug hydroxylation without causing the formation of aggregates or membrane-like structures.