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with AcOH (40 mL) and HCl (10 mL) for 2 h. After
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Enzyme section
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Sorbinil was a gift from Pfizer. Tolrestat was synthe-
sized according to a published procedure.38
Calf lenses for the purification of ALR2 were obtained
locally from freshly slaughtered animals. The capsule
was incized and the frozen lenses were suspended in
sodium potassium phosphate buffer pH 7 containing 5
mM DTT (1 g tissue/3.5 mL) and stirred in an ice-cold
bath for 1 h. The suspension was then centrifuged at
22,000g at 4 ꢀC for 40 min and the supernatant was
subjected to ion exchange chromatography on DE52.
Enzyme activity was measured by monitoring the
change in absorbance at 340 nm which accompanies the
oxidation of b-NADPH catalyzed by ALR2. The assay
was performed at 37 ꢀC as previously described,39 using
4.7 mM d,l-glyceraldehyde as substrate in 0.25 M
sodium phosphate buffer pH 6.8 containing 0.38 M
ammonium sulphate and 0.11 mM NADPH. The sensi-
tivity of ALR2 to inhibition by different ARIs was tes-
ted in the above assay conditions by including the
inhibitors dissolved in DMSO at the desired concentra-
tions in the reaction mixture. The DMSO in the assay
mixture was kept at constant concentration of 1%. A
reference blank containing all the above reagents
except the substrate was used to correct for the non-
enzymatic oxidation of NADPH. IC50 values (the con-
centration of the inhibitor required to produce 50%
inhibition of the enzyme catalyzed reaction) were
determined from least squares analyses of the linear
portion of the log dose–inhibition curves. Each curve
was generated using at least three concentrations of
inhibitor causing an inhibition between 20 and 80%
with two replicates at each concentration. The 95%
confidence limits (95% CL) were calculated from T
values for n-2, where n is the total number of determi-
nations.40
28. El-Kabbani, O.; Wilson, D. K.; Petrash, J. M.; Quiocho,
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Chem. 1999, 42, 2569.
References and Notes
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Chem. 1985, 28, 841.
2. Kador, P. F. Med. Res. Rev. 1988, 8, 325.
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Barlocco, D. Exp. Opin. Ther. Pat. 1997, 7, 843.
35. Murata, M.; Fujitani, B.; Minuta, H. Eur. J. Med. Chem.
1999, 34, 1061.