Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.
(2003) In Journal of Biological Chemistry 278(25). p.209-22199- Abstract
- Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 µmol min–1 mg–1) and high kcat/Km (310 mM–1 s–1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 µmol min–1 mg–1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also... (More)
- Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 µmol min–1 mg–1) and high kcat/Km (310 mM–1 s–1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 µmol min–1 mg–1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 µmol min–1 mg–1), 5-hydroperoxyeicosatetraenoic acid (0.043 µmol min–1 mg–1), and 15-hydroperoxy-PGE2 (0.04 µmol min–1 mg–1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 µmol min–1 mg–1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1. (Less)
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http://lup.lub.lu.se/record/128535
- author
- Thoren, S; Weinander, R; Saha, S; Jegerschold, C; Pettersson, P L; Samuelsson, B; Hebert, Hans LU ; Hamberg, M; Morgenstern, R and Jakobsson, P J
- organization
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 278
- issue
- 25
- pages
- 209 - 22199
- publisher
- ASBMB
- external identifiers
-
- scopus:0038266571
- ISSN
- 1083-351X
- DOI
- 10.1074/jbc.M303227200
- language
- English
- LU publication?
- yes
- id
- 4cd67a42-28d4-4b23-83e4-fdac3aff422e (old id 128535)
- date added to LUP
- 2007-07-09 16:36:52
- date last changed
- 2019-07-09 01:53:12
@article{4cd67a42-28d4-4b23-83e4-fdac3aff422e,
abstract = {Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 µmol min–1 mg–1) and high kcat/Km (310 mM–1 s–1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 µmol min–1 mg–1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 µmol min–1 mg–1), 5-hydroperoxyeicosatetraenoic acid (0.043 µmol min–1 mg–1), and 15-hydroperoxy-PGE2 (0.04 µmol min–1 mg–1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 µmol min–1 mg–1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.},
author = {Thoren, S and Weinander, R and Saha, S and Jegerschold, C and Pettersson, P L and Samuelsson, B and Hebert, Hans and Hamberg, M and Morgenstern, R and Jakobsson, P J},
issn = {1083-351X},
language = {eng},
number = {25},
pages = {209--22199},
publisher = {ASBMB},
series = {Journal of Biological Chemistry},
title = {Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.},
url = {http://dx.doi.org/10.1074/jbc.M303227200},
volume = {278},
year = {2003},
}