ISOFORM-I (MDR3) IS THE MAJOR FORM OF P-GLYCOPROTEIN EXPRESSED IN MOUSE-BRAIN CAPILLARIES - EVIDENCE FOR CROSS-REACTIVITY OF ANTIBODY-C219 WITH AN UNRELATED PROTEIN

P-glycoprotein (P-gp) is expressed in various non-cancerous tissues su ch as the endothelial cells of the blood-brain barrier. We used severa l monoclonal antibodies (mAbs) and isoform-specific polyclonal antibod ies to establish which P-gp isoforms are expressed in isolated mouse b rain capillaries. P-gp class I isoform was detected in capillaries wit h a Western immunoblotting procedure using a specific antiserum. No im munoreactivity was observed with either class II- or class III-specifi c antisera, Immunoreactivity was observed with mAb C219. However, this antibody detected two distinct immunoreactive proteins (155 and 190 k Da) in the isolated brain capillaries. These two proteins comigrated a s a broad band when the samples were submitted to heat prior to gel el ectrophoresis. The glycoprotein nature of these two antigens was evalu ated by their sensitivity to N-glycanase treatment. Following this tre atment, the size of the proteins was reduced from 190 and 155 kDa to 1 80 and 120 kDa, respectively. Triton X-114 phase-partitioning studies showed that the 190 kDa immunoreactive protein was poorly solubilized by Triton X-114, while the 155 kDa protein was partitioned in the dete rgent-rich phase. In labelling experiments, only the 155 kDa protein w as photolabelled with [I-125]iodoarylazidoprazosin. These results show that a 190 kDa protein detected by antibody C219 is an antigen unrela ted to the three P-gp isoforms presently known. Cross-reactivity of C2 19 with an unrelated protein emphasizes the fact that more than one an tibody should be used in the assessment of P-gp expression in cell lin es and tissues.