INHIBITION OF HUMAN GLUTATHIONE S-TRANSFERASES BY DOPAMINE, ALPHA-METHYLDOPA AND THEIR 5-S-GLUTATHIONYL CONJUGATES

The reversible and irreversible inhibition of human glutathione S-tran sferases (GST) by dopamine, alpha-methyldopa and their 5-S-glutathiony l conjugates (termed 5-GSDA and 5-GSMDOPA, respectively) was studied u sing purified isoenzymes. The reversible inhibition, using CDNB as sub strate and expressed as I-50, ranged from 0.18-0.24 (GST M1a-1a), 0.19 -0.24 (GST M1b-1b) to 0.5-0.54 mM (GST A1-1) for 5-GSDA and 5-GSMDOPA, respectively. About 20% inhibition was observed for GST A2-2 and P1-1 , using 0.5 mM of both 5-GSDA and 5-GSMDOPA. No significant reversible inhibition was observed with dopamine and alpha-methyldopa. Tyrosinas e was used to generate ortho-quinones from dopamine and alpha-methyldo pa which may bind covalently to GST and thereupon irreversibly inhibit GST. In this respect, GST P1-1 was by far the most sensitive enzyme. The inhibition (expressed as a % of control) after incubating 0.5 mu M GST in the presence of 100 units/ml tyrosinase with 5 mu M of the cat echolamines for 10 min at 25 degrees C, was 99% and 67% for dopamine a nd alpha-methyldopa, respectively. Moderate irreversible inhibition of GST A1-1 by both dopamine and alpha-methyldopa (33% and 25%, respecti vely), and of GST M1b-1b by dopamine (45%) was also observed. GST P1-1 is also the only isoenzyme susceptible to irreversible inhibition by 5-GSDA (33% inhibition), while no significant inhibition was observed with 5-GSMDOPA. A minor part of the inhibition by dopamine (23%), and the complete inhibition by 5-GSDA was restored by reduction with dithi otreitol. This suggests that GST P1-1 is inhibited by disulfide format ion in the case of 5-GSDA, while this oxidative pathway also substanti ally contributes to the inactivation by dopamine. This was supported b y the HPLC-profile of the GST P1-1 subunit which was strongly affected by dopamine, while for 5-GSDA after reduction with dithiotreitol the original elution profile of the subunit returned.