Back to Search
Start Over
Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS
- Source :
- Archives of Biochemistry and Biophysics. 424:128-136
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), respectively. These enzyme activities were measured by a new method, the liquid chromatographic-electrospray ionization-mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M-H](-)), 353 ([M-H](-)), 351 ([M-H](-)), 333 ([M-H-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF(2alpha) and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.
- Subjects :
- Spectrometry, Mass, Electrospray Ionization
Restriction Mapping
Biophysics
Prostaglandin
Reductase
Biochemistry
Substrate Specificity
chemistry.chemical_compound
Enzyme Stability
Humans
Ethanolamide
Prostaglandin-F synthase
Selected ion monitoring
Enzyme Inhibitors
Molecular Biology
chemistry.chemical_classification
Chromatography
biology
Prostaglandin D2
Substrate (chemistry)
Cloprostenol
Amides
Lipids
Recombinant Proteins
Enzyme assay
Bimatoprost
Enzyme
chemistry
Prostaglandins F, Synthetic
Hydroxyprostaglandin Dehydrogenases
biology.protein
Chromatography, Liquid
Subjects
Details
- ISSN :
- 00039861
- Volume :
- 424
- Database :
- OpenAIRE
- Journal :
- Archives of Biochemistry and Biophysics
- Accession number :
- edsair.doi.dedup.....885a8dabf65d904cb6048f829c9e838a