6-aminoquinolyl-n-hydroxysuccinimidyl-carbamate and Leukemia-P388

Multidrug resistant protein 1 (MDR1) in a doxorubicin-resistant mouse leukemia cell line (P388/DOX) was identified using its amino acid composition combined with protein database searching (ExPASy and EMBL PROPSEARCH) via the World Wide Web. The proteins were separated by one-dimensional SDS-polyacrylamide gel electrophoresis, blotted onto a polyvinylidene fluoride membrane, and stained with Coomassie brilliant blue. A 160-kDa protein band was acid-hydrolyzed in the vapor phase (6 N HC1) 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 the sensitive AQC-amino acid analysis method, improving our previously described method. The improved method involved using a Cosmosil 5C8-MS column instead of a Pegasil C8; 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 using clean tools. This minimized contamination of amino acids at the low femtomolar level. A database search was carried out with bovine serum albumin as a calibration protein. MDR1 in P388/DOX was ranked first by both databases with high reliability (score 14 for ExPASy, distance 1.34 for EMBL).

Topics: Amino Acids; Aminoquinolines; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calibration; Carbamates; Chromatography, High Pressure Liquid; Databases, Factual; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Internet; Leukemia P388; Membranes, Artificial; Mice; Molecular Weight; Polyvinyls; Reproducibility of Results; Sensitivity and Specificity; Sequence Homology, Amino Acid; Serum Albumin, Bovine; Specimen Handling; Tumor Cells, Cultured