Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes

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Abstract
1. In liver microsomal membranes from adult rabbits treated with beta-naphthoflavone, reaction with Cu2+ salts plus 1,10-phenanthroline leads to the cross-linking of the two specifically beta-naphthoflavone-inducible cytochrome P-450 species, form 4 and form 6, to form homo- and hetero-dimer species. 2. The cross-linking is not reversed by treatment with 2-mercaptoethanol, so that it can be observed conveniently and specifically on conventional reducing sodium dodecyl sulphate/polyacrylamide gels. 3. The reaction occurs rapidly, and significant cross-linking is observed after 30s at all temperatures from −10 to 40 degrees C. 4. The cross-linking can be brought about by Cu2+ alone at concentrations greater than 0.5 mM, but not by 1,10-phenanthroline alone; at low Cu2+ concentrations, 1,10-phenanthroline enhances the cross-linking reaction, but high concentrations of 1,10-phenanthroline are inhibitory; the optimal molar ratio of Cu2+ to 1,10-phenanthroline is 4:1.5. The effect of Cu2+ is not mimicked by Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+ or Ag+; Cu+ is probably also ineffective. 6. The cross-linking reaction is inhibited by the prior addition of high concentrations of EDTA or thiol compounds, by sodium dodecyl sulphate at greater than or equal to 0.1% and by sodium deoxycholate and non-ionic detergents at greater than or equal to 1%; the reaction cannot be reversed by incubation with EDTA or with thiol compounds after reaction with cupric phenanthroline; the cross-linking reaction is not inhibited by prior treatment of microsomal membranes with N-ethylmaleimide. 7. The chemical nature of the cross-linking reaction is unknown, but it is most unlikely that it involves the formation of intermolecular disulphide bonds. 8. The great specificity of the reaction makes it a promising tool for the study of molecular interactions between cytochrome P-450 species in intact microsomal membranes.