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Acetylation of Ser-530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme. To determine the catalytic function of the hydroxyl group of Ser-530, we used site-directed mutagenesis to replace Ser-530 with an alanine. Cos-1 cells transfected with expression vectors containing the native (Ser-530) or mutant (Ala-530) cDNAs for sheep PGG/H synthase expressed comparable cyclooxygenase and hydroperoxidase activities. Km values for arachidonate (8 microM) and ID50 values for reversible inhibition by the cyclooxygenase inhibitors, flurbiprofen (5 microM), flufenamate (20 microM), and aspirin (20 mM), were also the same for both native and mutant PGG/H synthases; however, only the native enzyme was irreversibly inactivated by aspirin. Thus, the "active site" Ser-530 of PGG/H synthase is not essential for catalysis or substrate binding. Apparently, acetylation of native PGG/H synthase by aspirin introduces a bulky sidechain at position 530 which interferes with arachidonate binding. In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced. A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins. Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis. Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the heme-binding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase. The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/H synthase sequence is the axial heme ligand of this enzyme. more...

This article has no abstract. Keywords: acetylglucosamine 6-sulfatase [6S, Glc6S, G6S, GL6S]

We have examined the molecular mechanisms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated prostaglandin synthesis in Mardin Darvey canine kidney cells (MDCK). TCDD stimulates prostaglandin synthesis in these cells, at least in part, by elevating prostaglandin endoperoxide H2 synthase-2 (PGHS-2) levels. TCDD-stimulated transcription of the PGHS-2 gene was maximal (6-fold) within 2 h and resulted in a 100-fold increase in PGHS-2 mRNA and a 25-fold increase in PGHS-2 protein levels by 4 h. Transient transfection experiments using luciferase-reporter plasmids demonstrated that control element(s) responsible for TCDD activation of the murine PGHS-2 promoter in MDCK cells are located in the first 965 nucleotides upstream from the PGHS-2 transcriptional initiation site. A canonical xenobiotic response element, similar to those that control transcription of other well-known TCDD-sensitive genes, is present at position -157, but does not appear to be sufficient for halogenated aromatic hydrocarbon (HAH) activation of the PGHS-2 promoter. TCDD failed to stimulate transcription from the PGHS-2 promoter when reporter plasmids were transfected into Hepa 1c1c7 cells, a line which contains the functional aryl hydrocarbon receptor. It seems likely that inappropriate expression of PGHS-2 may contribute to the toxic effects of TCDD and other HAHs. In particular, PGHS-2 expression may affect those toxic reactions that involve inappropriate cellular growth, such as dermal hyperplasia and tumor formation. It is also likely that elevated synthesis of prostaglandins, which are potent regulators of immune function, could play a role in the immunotoxicity associated with HAH exposure. more...

The gene for the murine prostaglandin endoperoxide (PGH) synthase (8, 11, 14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) has been cloned. The gene was isolated from a mouse NIH 3T3 cell genomic library and is contained in four overlapping lambda FIXII bacteriophage clones. The gene spans approximately 22 kb and consists of 11 exons. Primer extension and RNAse protection assays indicate that transcription of the gene begins at an initiation site 63 nucleotides 5' to the ATG translation initiation codon. Neither TATA or CAAT boxes are present immediately upstream of the transcriptional start site, but SP1 binding sites are present at positions -47 to -42 and -30 to -25, relative to the transcription initiation site. Examination of the 5'-end and 2400 bp of the 5'-flanking sequence of the gene revealed sequences with homology to several transcriptional regulatory sequences. Three putative AP-1 binding sites were found, two within the first exon and intron and another at position -2097 to -2090. The AP-1 site at position -2097 is adjacent to a sequence with similarity to a negative glucocorticoid regulatory element (nGRE) (position -2123 to -2009). The presence of AP sites by themselves, or in conjunction with an nGRE sequence, suggests a possible interplay between jun/fos regulatory proteins and the glucocorticoid receptor for positive and negative regulation of the PGH synthase gene. An unexpected finding was the presence at position -403 to -385 of a putative dioxin responsive element, a sequence found to be responsible for the induction of transcription of the cytochrome P450IA1 gene (CYPIA1) and other genes involved in detoxification/activation of polycyclic aromatic hydrocarbons. more...

Factors such as serum (1), EGF (2, 3), PGDF (4, 5), hormones (6, 7), IL-1 (8, 9), and phorbol esters (8, 10) promote prostaglandin synthesis by two mechanisms. Within minutes these compounds activate specific phospholipases leading to the release of arachidonate and production of prostaglandin endoperoxide H2 (PGH2). This first committed step in the prostaglandin biosynthetic pathway is catalyzed by the enzyme prostaglandin endoperoxide H (PGH) synthase. Within a few hours, these same factors also increase prostaglandin synthesis by stimulating de novo synthesis of PGH synthase. New enzyme production increases the capacity of a cell to produce prostaglandins both by increasing the absolute PGH synthase levels, and by replacing inactive enzyme. PGH synthase is catalytically inactivated during production of PGH2 (11–13); therefore new enzymeis required for prolonged synthesis, and sustained production of prostaglandins is believed to be necessary for the elaboration of many biological events such as inflammation, ovulation and luteolysis, and for maintaining vascular homeostasis (reviewed in 14). The exact mechanisms controlling PGH synthase synthesis are not known, but many of the factors that increase de novo synthesis of the enzyme also increase PGH synthase mRNA levels (1, 4, 5, 15, 16). Thus, one likely mechanism for regulating PGH synthase enzyme expression may be by regulating transcription of the PGH synthase gene. We and others who have examined regulation of PGH synthase gene expression in cultured mouse 3T3 fibroblasts, have shown that mRNA levels of one PGH synthase gene can be elevated in response to serum (1), PDGF and phorbol esters (5). To begin to investigate how PGH synthase mRNA levels are regulated our laboratory has isolated genomic clones and determined the structure of one mouse PGH synthase gene. We have also determined the transcription start site and more than 2 kb of sequence from the 5’-flanking region of this gene. We have identified several enhancer sequences that may regulate PGH synthase transcription and here propose a model for the transcriptional regulation of PGH synthase gene expression. more...