Functional characterization of two human sulphotransferase cDNAs that encode monoamine- and phenol-sulphating forms of phenol sulphotransferase: substrate kinetics, thermal-stability and inhibitor-sensitivity studies.
The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated 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. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for 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 cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.
Veronese
M E
ME
Department of Clinical Pharmacology, Flinders University of South Australia, Adelaide.
Burgess
W
W
Zhu
X
X
McManus
M E
ME
D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D018076:DNA, Complementary; D009596:Nitrophenols; D010724:Phosphoadenosine Phosphosulfate; C015802:2,6-dichloro-4-nitrophenol; C084221:St1C1 N-hydroxyarylamine sulfotransferase; D015238:Sulfotransferases; D015239:Arylsulfotransferase; D004298:Dopamine; C024836:4-nitrophenol