Human liver thermostable phenol sulfotransferase its PST: CDNA cloning, bacterial expression and characterization

Dahn L Clemens, J. Cole, P. Kudlacek, C. Halgard, R. Andersen

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Sulfate conjugation is important in regulating the biologic activity of many compounds. Cytosolic TS PST catalyzes this reaction using such substrates as p-nitrophenol (pIMP) and triiodothyronine. To study TS PST in the absence of other potentially contaminating activities, we isolated and characterized a cDNA clone of an allelic variant of TS PST from a human liver cDNA library. To characterize this variant, the clone was expressed in transiently transfected COS-1 cells and as a fusion protein with the bacterial gene mal E. Both systems produced biologically active enzymes with Vmax values for COS-1 expressed and fusion protein activities of 0.01 and 611.7 nmol/min/mg protein, respectively. Apparent Km values for pNP were 0.67 μM and 0.25 νM, respectively; and apparent K.m values for 3'-phosphoadenosine 5' phosphosulfate were0.29/yM and O.tSfjM, respectively. The addition of the mal E coding region at the amino terminus of TS PST altered the enzyme activity by rendering it less thermostable, more sensitive to the inhibitor 2,6-dichloro-4-nitrophenol (DCNP) and more resistant to inhibition by NaCI. Conclusions: This TS PST allelic variant was similar to known TS PST variants. Definitive characterization of a fusion orotein is reauired to determine its similarity to the native protein.

Original languageEnglish (US)
Pages (from-to)A284
JournalFASEB Journal
Volume10
Issue number3
StatePublished - Dec 1 1996

Fingerprint

Arylsulfotransferase
sulfotransferases
Cloning
Liver
phenol
Organism Cloning
molecular cloning
p-nitrophenol
liver
Fusion reactions
Clone Cells
Phosphoadenosine Phosphosulfate
Proteins
Bacterial Proteins
proteins
COS Cells
Triiodothyronine
Enzymes
clones
Gene Library

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Human liver thermostable phenol sulfotransferase its PST : CDNA cloning, bacterial expression and characterization. / Clemens, Dahn L; Cole, J.; Kudlacek, P.; Halgard, C.; Andersen, R.

In: FASEB Journal, Vol. 10, No. 3, 01.12.1996, p. A284.

Research output: Contribution to journalArticle

Clemens, Dahn L ; Cole, J. ; Kudlacek, P. ; Halgard, C. ; Andersen, R. / Human liver thermostable phenol sulfotransferase its PST : CDNA cloning, bacterial expression and characterization. In: FASEB Journal. 1996 ; Vol. 10, No. 3. pp. A284.
@article{6b7ea454ddab427387845ceac385cfe5,
title = "Human liver thermostable phenol sulfotransferase its PST: CDNA cloning, bacterial expression and characterization",
abstract = "Sulfate conjugation is important in regulating the biologic activity of many compounds. Cytosolic TS PST catalyzes this reaction using such substrates as p-nitrophenol (pIMP) and triiodothyronine. To study TS PST in the absence of other potentially contaminating activities, we isolated and characterized a cDNA clone of an allelic variant of TS PST from a human liver cDNA library. To characterize this variant, the clone was expressed in transiently transfected COS-1 cells and as a fusion protein with the bacterial gene mal E. Both systems produced biologically active enzymes with Vmax values for COS-1 expressed and fusion protein activities of 0.01 and 611.7 nmol/min/mg protein, respectively. Apparent Km values for pNP were 0.67 μM and 0.25 νM, respectively; and apparent K.m values for 3'-phosphoadenosine 5' phosphosulfate were0.29/yM and O.tSfjM, respectively. The addition of the mal E coding region at the amino terminus of TS PST altered the enzyme activity by rendering it less thermostable, more sensitive to the inhibitor 2,6-dichloro-4-nitrophenol (DCNP) and more resistant to inhibition by NaCI. Conclusions: This TS PST allelic variant was similar to known TS PST variants. Definitive characterization of a fusion orotein is reauired to determine its similarity to the native protein.",
author = "Clemens, {Dahn L} and J. Cole and P. Kudlacek and C. Halgard and R. Andersen",
year = "1996",
month = "12",
day = "1",
language = "English (US)",
volume = "10",
pages = "A284",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "3",

}

TY - JOUR

T1 - Human liver thermostable phenol sulfotransferase its PST

T2 - CDNA cloning, bacterial expression and characterization

AU - Clemens, Dahn L

AU - Cole, J.

AU - Kudlacek, P.

AU - Halgard, C.

AU - Andersen, R.

PY - 1996/12/1

Y1 - 1996/12/1

N2 - Sulfate conjugation is important in regulating the biologic activity of many compounds. Cytosolic TS PST catalyzes this reaction using such substrates as p-nitrophenol (pIMP) and triiodothyronine. To study TS PST in the absence of other potentially contaminating activities, we isolated and characterized a cDNA clone of an allelic variant of TS PST from a human liver cDNA library. To characterize this variant, the clone was expressed in transiently transfected COS-1 cells and as a fusion protein with the bacterial gene mal E. Both systems produced biologically active enzymes with Vmax values for COS-1 expressed and fusion protein activities of 0.01 and 611.7 nmol/min/mg protein, respectively. Apparent Km values for pNP were 0.67 μM and 0.25 νM, respectively; and apparent K.m values for 3'-phosphoadenosine 5' phosphosulfate were0.29/yM and O.tSfjM, respectively. The addition of the mal E coding region at the amino terminus of TS PST altered the enzyme activity by rendering it less thermostable, more sensitive to the inhibitor 2,6-dichloro-4-nitrophenol (DCNP) and more resistant to inhibition by NaCI. Conclusions: This TS PST allelic variant was similar to known TS PST variants. Definitive characterization of a fusion orotein is reauired to determine its similarity to the native protein.

AB - Sulfate conjugation is important in regulating the biologic activity of many compounds. Cytosolic TS PST catalyzes this reaction using such substrates as p-nitrophenol (pIMP) and triiodothyronine. To study TS PST in the absence of other potentially contaminating activities, we isolated and characterized a cDNA clone of an allelic variant of TS PST from a human liver cDNA library. To characterize this variant, the clone was expressed in transiently transfected COS-1 cells and as a fusion protein with the bacterial gene mal E. Both systems produced biologically active enzymes with Vmax values for COS-1 expressed and fusion protein activities of 0.01 and 611.7 nmol/min/mg protein, respectively. Apparent Km values for pNP were 0.67 μM and 0.25 νM, respectively; and apparent K.m values for 3'-phosphoadenosine 5' phosphosulfate were0.29/yM and O.tSfjM, respectively. The addition of the mal E coding region at the amino terminus of TS PST altered the enzyme activity by rendering it less thermostable, more sensitive to the inhibitor 2,6-dichloro-4-nitrophenol (DCNP) and more resistant to inhibition by NaCI. Conclusions: This TS PST allelic variant was similar to known TS PST variants. Definitive characterization of a fusion orotein is reauired to determine its similarity to the native protein.

UR - http://www.scopus.com/inward/record.url?scp=33749116073&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33749116073&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:33749116073

VL - 10

SP - A284

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 3

ER -