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 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).
Shindo
N
N
Division of Biochemical Analysis, Juntendo University School of Medicine, Tokyo, Japan. shindon@med.juntendo.ac.jp
Fujimura
T
T
Nojima-Kazuno
S
S
Mineki
R
R
Furusawa
S
S
Sasaki
K
K
Murayama
K
K
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
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