Purification and characterization of akr1b10 from human liver: role in carbonyl reduction of xenobiotics.
Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM-1 min-1 for dolasetron and 1.3, 0.43, and 0.47 mM-1 min-1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km=112 mM-1 min-1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.
Martin
Hans-Jörg
HJ
Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, D-24105 Kiel, Brunswikerstr. 10, Germany.
Breyer-Pfaff
Ursula
U
Wsol
Vladimir
V
Venz
Simone
S
Block
Simone
S
Maser
Edmund
E
D000449:Aldehyde Reductase / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D000074408:Aldo-Keto Reductases; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008658:Inactivation, Metabolic; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D015262:Xenobiotics / Q000737:chemistry / Q000493:pharmacokinetics*
D011994:Recombinant Proteins; D015262:Xenobiotics; C120471:AKR1B10 protein, human; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase