title: 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.
abstract: 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).
authors.affiliation: Division of Biochemical Analysis, Juntendo University School of Medicine, Tokyo, Japan. shindon@med.juntendo.ac.jp
authors.forename: N
authors.initials: N
authors.lastname: Shindo
authors.forename: T
authors.initials: T
authors.lastname: Fujimura
authors.forename: S
authors.initials: S
authors.lastname: Nojima-Kazuno
authors.forename: R
authors.initials: R
authors.lastname: Mineki
authors.forename: S
authors.initials: S
authors.lastname: Furusawa
authors.forename: K
authors.initials: K
authors.lastname: Sasaki
authors.forename: K
authors.initials: K
authors.lastname: Murayama
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis*; D000634:Aminoquinolines; D000818:Animals; D002138:Calibration; D002219:Carbamates; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D020407:Internet; D007941:Leukemia P388; D008567:Membranes, Artificial; D051379:Mice; D008970:Molecular Weight; D011145:Polyvinyls; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine; D013048:Specimen Handling; D014407:Tumor Cells, Cultured; D005465:Fluorocarbon Polymers
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000596:Amino Acids; D000634:Aminoquinolines; D002219:Carbamates; D008567:Membranes, Artificial; D011145:Polyvinyls; D012710:Serum Albumin, Bovine; C078178:6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers