Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column.
MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Yasuhisa
Y
Kimura
Seiji
S
Shibasaki
Kei
K
Morisato
Norio
N
Ishizuka
Hiroyoshi
H
Minakuchi
Kazuki
K
Nakanishi
Michinori
M
Matsuo
Teruo
T
Amachi
Mitsuyoshi
M
Ueda
Kazumitsu
K
Ueda
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects; D000244:Adenosine Diphosphate / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000737:chemistry; D012680:Sensitivity and Specificity; D014025:Titanium / Q000737:chemistry*; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009711:Nucleotides; C009495:titanium dioxide; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D014700:Verapamil; D014025:Titanium; D008274:Magnesium; D001608:Beryllium; D005459:Fluorides