Stereoselective carbonyl reductases from rat skin and leukocyte microsomes converting 12-ketoeicosatetraenoic acid to 12(S)-HETE

AbstractCell-free preparations from rat polymorphonuclear leukocytes and skin were found to catalyze the reduction of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) to 12-hydroxyeicosatetraenoic acid (12-HETE). The reductase activity was associated with the microsomal fraction and showed a marked preference for NADH over NADPH as reducing cofactor. Characterization of the reaction product by chiral phase HPLC of the methyl ester derivative indicated that 12-KETE reduction generated almost exclusively 12(S)-HETE. The results demonstrate that rat skin and leukocyte microsomes possess an NADH-dependent 12-KETE reductase activity that forms 12(S)-HETE as a major product. The identification of Stereoselective 12-KETE reductases provides a basis for further defining the role these enzymes may play in the regulation of 12-KETE levels and in the protection against degradation of 12-KETE to the pro-inflammatory 12(R)-HETE by selectively generating 12-HETE of the S configuration

Fatty acid reductase : an enzyme complex catalyzing the synthesis of aldehydes for bacterial bioluminescence

The biosynthesis of long chain aliphatic aldehydes required for light emission by bacterial luciferase has been studied in Photobacterium phosphoreum. A fatty acid reductase has been identified and shown to catalyze the conversion of fatty acids to their corresponding aldehydes in a reaction that requires NADPH and cleavage of ATP to AMP. The fatty acid reductase activity was measured using a sensitive luminescence coupled assay with luciferase or from the reduction of {('3)H}tetradecanoic acid to {('3)H}aldehyde using thin layer chromatography to resolve the product of the reaction. The measurement of the enzyme activity in extracts during bacterial growth indicated that the fatty acid reductase activity is induced at a late stage of growth during development of bioluminescence.The fatty acid reductase has also been shown to catalyze the ATP-dependent activation of the fatty acid to an acyl-protein (acyl-protein synthetase activity) and the reduction of tetradecanoyl-CoA to the aldehyde (acyl-CoA reductase activity). Acyl-protein synthetase and acyl-CoA reductase were resolved into functional components and shown to restore fatty acid reductase activity upon mixing. The acyl-CoA reductase has been purified to apparent homogeneity, giving a single polypeptide of molecular weight of 58,000 as analyzed by SDS-polyacrylamide gel electrophoresis. The changes in physical and kinetic properties of these enzymes after their resolution demonstrate that they are associated into an enzyme complex which catalyzes the reduction of fatty acids in bioluminescent bacteria

Cyclo-oxygenase-2 contributes to constitutive prostanoid production in rat kidney and brain

Cyclo-oxygenases (COXs) catalyse the synthesis of PGH(2) (prostaglandin H(2)), which serves as the common substrate for the production of PGE(2), PGD(2), PGF(2α), prostacyclin (or PGI(2)) and TXs (thromboxanes). While COX-1 is the major isoform responsible for prostanoid synthesis in healthy tissues, little information is available on the contribution of constitutive COX-2 to the various prostanoid synthetic pathways under non-inflammatory conditions. To evaluate further the role of COX-2 in prostanoid biosynthesis, rats were acutely treated with the selective COX-1 inhibitor SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole] or the selective COX-2 inhibitors MF tricyclic [3-(3,4-difluorophenyl)-4-(4-(methylsulphonyl)phenyl)-2-(5H)-furanone] and DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2-(5H)-furanone]. Selected tissues were then processed for a complete analysis of their prostanoid content by liquid chromatography MS. Whereas the treatment with SC-560 caused a 60–70% inhibition in the total prostanoid content of most tissues examined, a significant decrease (35–50%) in total prostanoid content following selective COX-2 inhibition was solely detected for kidney and brain tissues. Analysis of the individual prostanoids reveals significant inhibition of 6-oxo-PGF(1α), PGE(2), PGD(2), PGF(2α) and TXB(2) in the kidney and inhibition of all these prostanoids with the exception of PGD(2) in the forebrain. These results demonstrate that constitutively expressed COX-2 contributes to the production of prostanoids in kidney and brain for each of the PGE(2), PGI(2) and TXB(2) pathways under non-inflammatory conditions. Approaches to modulate inflammation through specific inhibition of terminal synthases, such as mPGES-1 (microsomal PGE(2) synthase-1), thus have the potential to differ from COX-2 inhibitors and non-selective non-steroidal anti-inflammatory drugs with regard to effects on constitutive prostanoid synthesis and on renal function