FIGURE 5.

Cross-linking of P450s in simple and binary BPC reconstituted systems with BS³ followed by immunoprecipitation with anti-CYP1A2 and immune blotting. The following reconstituted systems were prepared using BPC: 1) vesicles containing only CYP1A2, 2) vesicles containing only CYP2B4, 3) a mixture of CYP1A2 vesicles with CYP2B4 vesicles, and 4) a reconstituted system containing both CYP1A2 and CYP2B4 in the same vesicles. As described under “Experimental Procedures,” the systems were cross-linked (X-L) with BS³, immunoprecipitated (IP) with anti-CYP1A2, subjected to SDS-PAGE, and blotted with anti-CYP1A2 (A) or anti-CYP2B4 antibody (B). The reconstituted systems in lanes 1–4 were cross-linked with BS³, whereas those in lanes 7–10 were immunoprecipitated but not cross-linked. Lanes 1 and 7, CYP1A2-containing vesicles; lanes 2 and 8, CYP2B4-containing vesicles; lanes 3 and 9, a mixture of the CYP1A2 vesicles with the CYP2B4 vesicles; lanes 4 and 10, CYP1A2 and CYP2B4 in the same vesicles. Lane 5, a control using the immunoprecipitating CYP1A2 antibody without any reconstituted system; lane 6, molecular weight standards with the mass of the band designated (kDa). The figure also shows the approximate band locations for monomeric, dimeric, trimeric, and tetrameric complexes of P450 enzymes.