High level expression and characterization of recombinant human hippocampus phenol sulfotransferase: A novel phenol-sulfating form of phenol sulfotransferase

Shin Rong Hwang, M. Palkóvits, Vivian Y H Hook

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2 Citations (Scopus)

Abstract

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 β-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 V(max(app)), values of 3 μM 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°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.

Original languageEnglish
Pages (from-to)125-134
Number of pages10
JournalProtein Expression and Purification
Volume11
Issue number1
DOIs
Publication statusPublished - Oct 1997

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Arylsulfotransferase
Phenol
Hippocampus
Sulfotransferases
Substrates
Chromatography
Application programs
Thiogalactosides
Escherichia coli Proteins
Ion Exchange Chromatography
Sequence Homology
Neuropeptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "High level expression and characterization of recombinant human hippocampus phenol sulfotransferase: A novel phenol-sulfating form of phenol sulfotransferase",
abstract = "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 β-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 V(max(app)), values of 3 μM 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°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.",
author = "Hwang, {Shin Rong} and M. Palk{\'o}vits and Hook, {Vivian Y H}",
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T1 - High level expression and characterization of recombinant human hippocampus phenol sulfotransferase

T2 - A novel phenol-sulfating form of phenol sulfotransferase

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AU - Palkóvits, M.

AU - Hook, Vivian Y H

PY - 1997/10

Y1 - 1997/10

N2 - 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 β-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 V(max(app)), values of 3 μM 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°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.

AB - 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 β-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 V(max(app)), values of 3 μM 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°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.

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