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. 2015 Feb;56(2):342–357. doi: 10.1194/jlr.M054718

Fig. 4.

Fig. 4.

Inhibition of ALDH1A and RA formation by WIN 18,446. The inhibition kinetics of WIN 18,446 were characterized and used to determine the fraction of atRA formation by ALDH1A2 in the human testis. The reversible inhibition kinetics of WIN 18,446 with recombinant ALDH1A1 (A) (IC50 = 102 nM) and ALDH1A3 (B) (IC50 = 187 nM) were determined together with the effect of WIN 18,446 on atRA formation in testicular S10 fractions (C) (IC50 = 88 nM). Due to the time-dependent inhibition of ALDH1A2 by WIN 18,446, the rate of inactivation was determined with increasing concentrations of inhibitor (D, inset) and plotted as a function of inhibitor concentration (D) to determine the KI and kinact. In order to measure the fm for ALDH1A2 in the testis, S10 protein was preincubated with inhibitor for 0.25, 10, and 30 min to inactivate any ALDH1A2 protein (E). The preincubation was diluted out to remove reversible inhibition, and the ALDH1A activity was determined. The 25% decrease in ALDH1A activity (E) can be attributed to the contribution by ALDH1A2. The predicted relative contribution of each ALDH1A enzyme (green, ALDH1A1; blue, ALDH1A2; red, ALDH1A3) to atRA formation as a function of at-retinal concentration is shown in (F).