Identification of multidrug resistant protein 1 of mouse leukemia P388 cells on a PVDF membrane using 6-aminoquinolyl-carbamyl (AQC)-amino acid analysis and World Wide Web (WWW)-accessible tools

Multidrug resistant protein 1 (MDR1) in a doxorubicin-resistant mouse leuke mia cell Line (P388/DOX) was identified using its amino acid composition co mbined with protein database searching (ExPASy and EMBL PROPSEARCH) via the World Wide Web. The proteins were separated by one-dimensional SDS-polyacr ylamide gel electrophoresis, blotted onto a polyvinylidene fluoride membran e, and stained with Coomassie brilliant blue, A 160-kDa protein band was ac id-hydrolyzed in the vapor phase (6 N HCl) and converted to 6-aminoquinolyl -carbamyl (AQC)-amino acids without extraction of the amino acids from the membrane. The amino acid composition of the protein was determined using th e sensitive AQC-amino acid analysis method, improving our previously descri bed method. The improved method involved using a Cosmosil 5C(8)-MS column i nstead of a Pegasil C-8; replacement of the mobile phase A, constituent, 75 mM ammonium phosphate (pH 7.5), with 30 mM sodium phosphate buffer (pH 7.2 ); and slight modification of the separation program (9). All manipulations for protein hydrolysis and AQC derivatization were carried out in a hood u sing clean tools, This minimized contamination of amino acids at the low fe mtomolar level. A database search was carried out with bovine serum albumin as a calibration protein. MDR1 in P388/DOX was ranked first by both databa ses with high reliability (score 14 for ExPASy, distance 1.34 for EMBL). (C ) 1998 Academic Press.