BACTERIAL EXPRESSION AND KINETIC CHARACTERIZATION OF THE HUMAN MONOAMINE-SULFATING FORM OF PHENOL SULFOTRANSFERASE

The cDNA for the human monoamine-sulfating form of phenol sulfotransfe rase (hM-PST) was isolated from a T47D human breast carcinoma lambda g t10 cDNA library, and the active enzyme was expressed in Escherichia c oli, Expressed hM-PST was very similar to the brain, intestinal, and p latelet forms of the enzyme in its physical, immunological, and kineti c properties. The ability of hM-PST to sulfate a number of xenobiotics was examined and compared with the bacterially expressed human phenol -sulfating form of PST (hP-PST), The translation product of the T47D h M-PST cDNA was 92% identical to that of liver hP-PST. Monoamine neurot ransmittors, such as epinephrine and dopamine, were maximally conjugat ed at lower concentrations by expressed hM-PST (2 and 20 mu M, respect ively) than by hP-PST (1 and 1 mM, respectively). In contrast, simple phenols-such as p-nitrophenol, acetaminophen, and alpha-naphthol-were maximally conjugated at lower concentrations(4 mu M, 20 mu M, and 0.5 mu M, respectively) by hP-PST than by hM-PST (1 mM, 1.5 mM, and 50 mu M, respectively). Minoxidil was sulfated at similar rates and concentr ations (7 mM) by both forms of PST. None of the estrogens or related c ompounds, such as beta-estradiol, 17 alpha-ethinylestradiol, diethylst ilbestrol, equilenin, or genistein tested as substrates were sulfated by hM-PST; however, all of these compounds were substrates for hP-PST, As with hP-PST, the hydroxysteroids dehydroepiandrosterone and cortis ol were not sulfated by hM-PST. In addition, inhibition studies sugges t that the amino acid differences between hM-PST and hP-PST are of ade quate significance to prevent compounds with a sterol-like structure f rom binding the active site of hM-PST, These results indicate that the re are important differences in the substrate reactivities of the two PSTs and that expression of the individual human STs in bacteria is a valuable tool for the investigation of differences in drug and xenobio tic metabolism.