Our studies with PGHS-1 indicate that a Tyr radical is formed by the peroxidase activity of PGHS-1 and that this radical is involved in the self-inactivation that occurs during arachidonic acid metabolism. Our data suggest that the Tyr radical is not the reactive intermediate that initiates arachidonic acid oxygenation. To obtain further support for this conclusion we are examining the formation of Tyr radical in human PGHS-2. This enzyme was selected since expression and purification of the native and mutants proteins in the necessary large amounts can be accomplished. Nitric oxide(NO) is reported to enhance prostaglandin formation by direct enhancement of PGHS activity. We have investigated this problem and found that NO is a substrate for the peroxidase but does not enhance the cyclooxygenase activity. Also, NO does not stimulate the expression of PGHS or alter LPS dependent PGHS-2 expression. NO could act by altering the activity or expression of cPLA2 which we are currently studying. We have also cloned rat PGHS-1 and -2 and studied their expression by rat tracheal cells (EGV-6). We observed an aberrantly sliced PGHS-1 mRNA in addition to the complete mRNA. Furthermore, we observed in these cells that TPA up-regulated PGHS-1 which in most cells is not upregulated by ligands but constitutively regulated. This may be related to the transformed nature of the EGV-6 cells. We have shown that EGF regulates the formation of 13-HODE in Syrian hamster embryo (SHE) cells. Considerable effort was devoted to cloning this apparent 15-lipoxygenase in an attempt to develop the tools necessary to investigate its regulation by EGF (see cell proliferation project). Despite considerable effort and several approaches, we were unable to clone this 15-lipoxygenase. This suggests that the enzyme is not related to any characterized lipoxygenase. We did successfully clone, sequence and express in, E. coli, a hamster 5-lipoxygenase. Our efforts to understand the regulation by EGF of 13-HODE formation will shift to human breast cell cultures which also metabolize linoleic acid to 13-HODE and is regulated by EGF. The necessary tools are available for the human enzyme.
