Modification in vitro by a cross-linking reagent of rabbit liver microsomal proteins induced by beta-naphthoflavone [proceedings].

Chemical cross-linking reagents are valuable tools for studying the structural relationships between proteins in complexes and organelles. Copper phenanthroline is an oxidizing agent that has been used to cross-link membrane proteins via adjacent free thiol groups. In the courseof such topographical studies of rabbit liver microsomal preparations, we noted that incubation of microsomal preparations from p-naphthoflavone-treated rabbits with copper phenanthroline decreased the intensity of two bands seen in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the microsomal fraction, and generated three new bands representing polypeptides of higher molecular weight. Unexpectedly, the changes could not be reversed by reducing agents, and thus can be distinguished from other changes brought about by copper phenanthroline. The two bands whose intensities decrease following treatment of the microsomal fraction in uitro with copper phenanthroline are those same bands that are increased in intensity by treatment in vivo of the rabbits with 8-naphthoflavone, an established inducer of certain cytochrome P-450 species (Haugen & Coon, 1976). Microsomal fractions were prepared from the livers of male New Zealand rabbits of weight 2.0-2.5 kg that had been deprived of food for 1 day by the method of van der Hoeven & Coon (1974). The following groups of three animals were used: (i) untreated, (ii) injected intraperitoneally with 70mg of p-naphthoflavone/kg 41 h before death, and (iii) exposed to 0.1 % sodium phenobarbitone in drinking water for 5 days before death. The microsomal fraction was washed twice in pyrophosphate buffer [0.1 Msodium pyrophosphate (pH 7,4)/1 .O~M-EDTA] and stored frozen at -20°C after resuspension in Coon’s buffer A [0.01 M-Tris/acetate (pH 7.4)/0.1 ~ M E D T A / ~ O % (v/v) glycerol]. Fig. 1 represents diagrammatically the resolution of microsomal proteins from these groups of animals on sodium dodecyl sulphate/polyacrylamide gels. Included in Fig. 2 are densitometric scans of portions of such gels. New bands (A, B and C) appear after the incubation of the microsomal fraction from 8-naphthoflavone-treated rabbits with 0.22m~-copper phenanthroline (Fig. 1, tracks 4, 5 and 6). CuClz alone produces only minor increases in bands A, B and C (track 2), whereas o-phenanthroline alone causes no increases at all (track 3). The new bands are seen clearly after reaction with copper phenanthroline for 15min at 4°C; reaction at 21 or 37°C brings about further increases in their intensities. Only very minor increases in bands A and B are seen if the microsomal fraction from untreated rabbits are incubated in the same manner with 0.22m~-copper phenanthroline (tracks 7 and 8); small increases in bands A, B and C are seen after corresponding treatment of the microsomal fraction from rabbits treated with sodium phenobarbitone (tracks 9 and 10). Further analysis of the sodium dodecyl sulphate/polyacrylamide gels is shown in Fig. 2. Fig. 2(a) represents superimposed densitometric traces of gels of microsomal fraction from both untreated and 8-naphthoflavone-treated rabbits. The two bands D and E are clearly of much higher intensity in the sample from p-naphthoflavone-induced rabbits. This effect is more clearly seen in Fig. 2(b), which represents the difference between the two traces shown in Fig. 2(a) (induced-untreated). Fig. 2(c) shows a similarly derived ‘difference-trace’ between scans of microsomal fraction from p-naphthoflavone-treated rabbits reacted with copper phenanthroline and unreacted microsomal fraction from 8-naphthoflavone-treated rabbits, and thus demonstrates the effects of copper phenanthroline treatment on microsomal fraction from 8naphthoflavone-induced rabbits. The appearance of the three bands A, B and C is accompanied by decreases in intensity of bands D and E. Band E, the major species induced by 8-naphthoflavone, is presumably the cytochrome P-450 species LM4 (see Haugen &