FUNCTIONAL-CHARACTERIZATION OF 2 HUMAN SULFOTRANSFERASE CDNAS THAT ENCODE MONOAMINE-SULFATING AND PHENOL-SULFATING FORMS OF PHENOL SULFOTRANSFERASE - SUBSTRATE KINETICS, THERMAL-STABILITY AND INHIBITOR-SENSITIVITY STUDIES

The present paper describes the functional characterization of two hum an aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously is olated by us from liver and brain, respectively [Zhu, Veronese, Sansom , and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. C ommun. 195, 120-127]. These appear to encode the two major forms of ph enol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulp hating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNA s were functionally expressed in COS-7 cells and kinetically character ized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HA ST1 was shown to be enzymically active in sulphating p-nitrophenol wit h high affinity (K-m 0.6 mu M), whereas dopamine was the preferred sub strate for HAST3 (K-m 9.7 mu M). HAST1 could also sulphate dopamine, a s could HAST3 sulphate p-nitrophenol, but the K-m for these reactions were at least two orders of magnitude greater than for the preferred s ubstrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical wi th human liver cytosolic P-PST and M-PST activities respectively. Ther mal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent w ith P-PST being termed the TS PST and M-PST being termed the thermolab ile (TL) PST. Western immunoblot analyses of the expressed PST protein s using an antibody generated to a bacterially expressed rat liver ary l/phenol ST showed that HAST1 and HAST3 migrated as single proteins wi th different electrophoretic mobilities (32 versus 34 kDa). This is co nsistent with the differences in electrophoretic mobilities observed f or P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNA s provides important information on the structural as well as function al relationships of human PSTs, which sulphate a vast array of exogeno us and endogenous compounds.