IMPROVED SEPARATION AND IMMUNODETECTION OF RAT CYTOCHROME-P450 4A FORMS IN LIVER AND KIDNEY

In the study of tissues that contain several forms of one cytochrome P 450 subfamily, it is useful to develop immunoblotting techniques so th at the various individual members of the family can be distinguished. This paper describes improvements in the immunoblotting technique to d istinguish members of the rat cytochrome P450 4A subfamily, 4A1, 4A2, and 4A3, as they are present in Sprague-Dawley rat liver microsomes. T his procedure was used to investigate differences in the cytochrome P4 50 4A forms observed under various conditions such as: untreated versu s peroxisome proliferator treated rats, Sprague-Dawley versus Fischer 344 male versus female rats, and liver versus kidney microsomes. In li ver microsomes of male Sprague-Dawley rats, forms 4A1, 4A2, and 4A3 we re induced by the peroxisome proliferators, clofibrate, di-(2-ethylhex yl) phthalate, dehydroepiandrosterone, aspirin, and ibuprofen. Express ion of the 4A forms shows strain specificity, A comparison of the cyto chrome P450 4A forms in male Sprague-Dawley and Fischer 344 rats treat ed with peroxisome proliferators demonstrated that three distinct prot ein bands are visible on immunoblots of liver microsomes of Sprague-Da wley rats, whereas only two distinct protein bands are detectable in l iver microsomes of Fischer 344 rats. The two protein bands in liver mi crosomes of male Fischer 344 rats migrate in positions corresponding t o the 4A2 and 4A3 bands in male Sprague-Dawley rats. There did not app ear to be a protein band corresponding to the 4A1 band of Sprague-Dawl ey rats, Expression of the 4A forms also shows gender specificity. In liver microsomes of female Sprague-Dawley rats, expression of the P450 4A2 form was not observed after treatment with a peroxisome prolifera tor. Expression of the 4A forms also shows tissue specificity. In kidn ey, 4A2 is the major protein band in male Sprague-Dawley rats with min or amounts of the 4A3 protein, whereas two prominent protein bands (4A 2 and 4A3) are seen in male Fischer 344 rats.