High level expression and characterization of recombinant human hippocampus phenol sulfotransferase: a novel phenol-sulfating form of phenol sulfotransferase.
Phenol sulfotransferases (PSTs) represent a family of sulfotransferase enzymes that modify the biologic activities and excretion of phenolic compounds and monoamines. A novel human hippocampal PST (H-PST) cDNA with homology to phenol (P) and monoamine (M) forms of PST was previously isolated from brain. To compare the biochemical properties of H-PST with that of phenol (P-PST) and monoamine (M-PST) sulfotransferases, high level expression of recombinant H-PST was achieved in this study with the pET3c vector in BL21(DE3) Escherichia coli cells. Expression was demonstrated by isopropyl beta-D-thiogalactopyranoside induction of 34-kDa H-PST that represented 5-10% of total E. coli proteins. Purification by ion-exchange chromatography on DEAE-Sepharose yielded more than 2 mg of H-PST. Characterization showed that H-PST exists as a homodimer of 60-65 kDa by gel filtration chromatography. H-PST prefers p-nitrophenol as substrate and does not sulfate dopamine or neuropeptide substrates. Kinetic studies showed that H-PST possessed K(m(app)) and Vmax(app) values of 3 microM p-nitrophenol and 160 nmol/min/mg, respectively. H-PST was sensitive to inhibition by DCNP (2,6-dichloro-4-nitrophenol). H-PST is thermolabile since its activity was reduced upon preincubation at 37 degrees C. These results indicate that H-PST shows similarities and differences compared to P-PST and M-PST sulfotransferases. P-PST prefers p-nitrophenol as substrate, is sensitive to inhibition by DCNP, and is thermostable; in contrast, M-PST prefers monoamines as substrate, is not sensitive to DCNP, and is thermolabile. The distinct profile of biochemical properties of H-PST, and its primary sequence homology to P-PST and M-PST, suggests that H-PST represents a novel allelic variant of human phenol sulfotransferases. Importantly, this study demonstrates that high level expression of H-PST allows determination of distinguishing characteristics of variant forms of PSTs.
Hwang
S R
SR
Department of Medicine, University of California, San Diego 92093, USA.
Palkovits
M
M
Hook
V Y
VY
D000483:Alleles; D000595:Amino Acid Sequence; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001792:Blood Platelets / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006624:Hippocampus / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000494:pharmacology; D011994:Recombinant Proteins; D016415:Sequence Alignment
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009596:Nitrophenols; D011994:Recombinant Proteins; C015802:2,6-dichloro-4-nitrophenol; D015239:Arylsulfotransferase