Abstract
BackgroundHematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD2. Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated.MethodsH-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates.ResultsA specific activity of 24 μmol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 μg/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with Ki = 41 μg/ml towards GSH, and non-competitive inhibition towards CDNB with Ki = 7.7 μg/ml and Ki′ = 9.2 μg/ml.ConclusionThe data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation.
Highlights
Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione
Coomassie blue staining and Western blot for poly-His tag hematopoietic prostaglandin D2 synthase (H-PGDS) was expressed in E. coli and was purified by Nickel affinity chromatography
The H-PGDS was purified to homogeneity and a single band was obtained on SDSPAGE analyses
Summary
Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inflammatory process may be defined as a sequence of events that occurs in response to noxious stimuli, an infection or trauma [1]. It is the first response of the immune system to infection or irritation and is a protective attempt by the organism to remove injurious stimuli and initiate the healing process [2]. Prostaglandin D2 is an acidic lipid mediator which is responsible for the regulation of body temperature, hormone release, olfactory reaction, sleep, prevention of platelet aggregation and pain responses [5]. DP2 receptors mediate eosinophil chemotaxis and are involved in Th2 related inflammation [2]
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