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Human microsomal pr...
Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.
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- Thoren, S (författare)
- Karolinska Institutet
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Weinander, R (författare)
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Saha, S (författare)
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- Jegerschold, C (författare)
- Karolinska Institutet
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Pettersson, P L (författare)
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- Samuelsson, B (författare)
- Karolinska Institutet
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- Hebert, Hans (författare)
- Karolinska Institutet,Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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- Hamberg, M (författare)
- Karolinska Institutet
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- Morgenstern, R (författare)
- Karolinska Institutet
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- Jakobsson, P J (författare)
- Karolinska Institutet
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(creator_code:org_t)
- 2003
- 2003
- Engelska.
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Ingår i: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 278:25, s. 209-22199
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http://dx.doi.org/10... (free)
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
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Thoren, S
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Weinander, R
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Saha, S
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Jegerschold, C
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Pettersson, P L
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Samuelsson, B
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visa fler...
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Hebert, Hans
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Hamberg, M
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Morgenstern, R
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Jakobsson, P J
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visa färre...
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Journal of Biolo ...
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Lunds universitet
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Karolinska Institutet