Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis

There are spectral and biochemical data suggesting that a tyrosine group(s) is involved in the cyclooxygenase reaction catalyzed by prostaglandin endoperoxide (PGH) synthase. Treatment with tetranitromethane, a reagent which nitrates tyrosine residues, abolishes cyclooxygenase activity, but this inactivation can be largely prevented by competitive cyclooxygenase inhibitors such as ibuprofen and indomethacin. To identify sites of nitration, native PGH synthase and indomethacin-pretreated PGH synthase were incubated with tetranitromethane, and the sequences of peptides containing nitrotyrosine were determined. Three unique tyrosines (Tyr-355, Tyr-385, and Tyr-417) were nitrated in the native enzyme but not in the indomethacin-treated PGH synthase. Using site-directed mutagenesis of sheep PGH synthase, each of these tyrosines, as well as two other tyrosine residues selected as controls (Tyr-254 and Tyr-262), were replaced with phenylalanine; cos-1 cells were transfected with constructs containing cDNAs coding for the native PGH synthase and each of the five phenylalanine mutants, and microsomes from these cells were assayed for cyclooxygenase and hydroperoxidase activities. The Phe-385 mutant of PGH synthase lacked cyclooxygenase activity but retained peroxidase activity; all other mutants expressed both enzyme activities. Our results establish that Tyr-385 is essential for the cyclooxygenase activity of PGH synthase and that nitration of this residue can be prevented by indomethacin. We conclude that Tyr-385 is at or near the cyclooxygenase active site of PGH synthase and could be the tyrosine residue proposed to be involved in the first step of the cyclooxygenase reaction, abstraction of the 13-proS hydrogen from arachidonate.     

Coupling of the peroxidase and cyclooxygenase reactions of prostaglandin H synthase

Interrelations between peroxidase and cyclooxygenase reactions catalyzed by prostaglandin endoperoxide synthase (prostaglandin H synthase) were analyzed in terms of the mutual influence of these reactions. The original branched-chain mechanism predicts competition between these two reactions for enzyme, so that peroxidase cosubstrate should inhibit the cyclooxygenase reaction and the cyclooxygenase substrate is expected to inhibit the peroxidase reaction. In stark contrast, the peroxidase reducing substrate is well known to strongly stimulate the cyclooxygenase reaction. In the present work the opposite effect, the influence of the cyclooxygenase substrate on the peroxidase reaction was studied. Experiments were conducted on the effect of arachidonic acid on the consumption of p-coumaric acid by prostaglandin H synthase and 5-phenyl-4-pentenyl-1-hydroperoxide. Neither the steady-state rates nor the total extent of p-coumaric acid consumption was affected by the addition of arachidonic acid. This suggests that the cyclooxygenase substrate does not influence observable velocities of the peroxidase reaction, namely oxidation and regeneration of the resting enzyme. The data support coupling of the cyclooxygenase and peroxidase reactions. A combination of the branched-chain and tightly coupled mechanisms is proposed, which includes a tyrosyl radical active enzyme intermediate regenerated through the peroxidase cycle. Numerical integration of the proposed reaction scheme agrees with the observed relations between peroxidase and cyclooxygenase reactions in the steady state.     

Immunoaffinity purification and cDNA cloning of human platelet prostaglandin endoperoxide synthase (cyclooxygenase).

The cDNA for prostaglandin endoperoxide synthase (cyclooxygenase) was cloned from human platelets by the polymerase chain reaction amplification method, and the primary structure of the enzyme was deduced from the nucleotide sequence. The enzyme was composed of 599 amino acids including 23-amino acid signal sequence, and the calculated molecular weight of the mature protein was 65,995. The enzyme was immunoaffinity-purified from human platelets. The N-terminal amino acid sequence determined by Edman degradation was Ala-Asp-Pro-Gly-Ala-Pro-Thr-Pro-, and the result confirmed the primary structure of the enzyme, which was deduced from the cDNA sequence.     

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase)

An oligonucleotide probe was used to isolate a clone encoding prostaglandin endoperoxide synthetase (cyclooxygenase, EC 1.14.99.1) from a sheep seminal vesicle cDNA library. The protein predicted from nucleic acid sequence contains 599 amino acids including a 23-amino acid signal sequence. Thus, the mature cyclooxygenase deduced from the cDNA compares favorably in molecular size to the 70-kDa protein determined by gel electrophoresis. A putative transmembrane region and potential carbohydrate addition sites for N-linked sugars can be inferred from the amino acid sequence. Significantly, sequence similarities exist between cyclooxygenase, myeloperoxidase, and several other heme-containing proteins. The putative glycosylation sites, transmembrane domain, and sequence similarities with functionally related enzymes have been incorporated into a model for the topology of cyclooxygenase in the endoplasmic reticulum.     

Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.     

Immunocytochemical localization of prostaglandin endoperoxide synthase in the bovine intestine

The localization of prostaglandin (PG) endoperoxide synthase in bovine intestine was examined immunocytochemically with polyclonal antibody raised against PG endoperoxide synthase purified from bovine seminal glands. The most intense positive staining reaction for the enzyme was present in mast cells. Mast cells were found to be widely distributed in the intestinal wall, and were particularly numerous in the lamina propria. Most of the mast cells in the lamina propria of the intestinal villi were elongated and oriented with their long axis parallel to the plane of the absorptive epithelium. In whole mount preparations of jejunal villi, mast cells were seen to form a two-dimensional network in the lamina propria. In addition to mast cells, smooth muscle cells of the inner circular muscle layer and muscularis mucosae, nerve cells and fibers, endothelial cells of arterioles, and serosal epithelial cells also showed faint to moderate staining for the enzyme. These results suggested that mast cells are the major source of PGs in the bovine intestinal wall. The characteristic arrangement of mast cells in the intestinal villi may be related to their functions in this portion of the bovine intestine.     

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.     

Not prostacyclin synthase but prostaglandin endoperoxide synthase increases with human placental development

Prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantitated with specific immunoradiometric assays in microsomes from human placentae (n = 20) obtained from 7 up to 17 weeks of gestation. Over that period, wherein trophoblastic invasion of the uterine spiral arteries occurs, the placentae showed a significant increase in concentrations of PGH synthase (r = 0.73, p less than 0.001; n = 20), but not in those of PGI synthase. While the variation between individual placentae was much larger for PGI synthase than for PGH synthase concentrations, there was no evidence for a large excess of PGI synthase over that of PGH synthase in any of these early placentae. The data indicate, first, that the developing placenta contains PGI synthase, but in amounts which are relatively small and do not appear to increase with advancing gestation. Second, they seem to indicate that the capacity for bioconversion of arachidonic acid into prostaglandin endoperoxides increases markedly with placental development.     

Not prostacyclin synthase but prostaglandin endoperoxide synthase increases with human placental development

Prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH sythase) and prostacyclin synthase (PGI synthase were quantitated with specific immunoradimetric assays in microsomes from human placentae (n=20) obtained from 7 up to 17 weeks of gestation. Over that period, wherein trophoblastic invasion of the uterine spiral arteries occurs, the placetae showed a significant increase in concentrations of PGH synthase (r=0.73, p<0.001; n=20), but not in those of PGI synthase. While the variation between individual placentae was much larger for PGI synthase than for PGH synthase concentrations, there was no evidence for a large excess of PGI synthase over that of PGH synthase in any of these early placentae. The data indicate, first, that the developing placenta contains PGI synthase, but in amount which are relatively small and do not appear to increase with advancing gestation. Second, they seem to indicate that the capacity for bioconversion of arachidonic acid into prostaglandin endoperoxides increases markedly with placental development.     

Target size analysis of prostaglandin endoperoxide synthase. Radiation inactivation of both cyclooxygenase and peroxidase correlated with the monomer of 72 kDa.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.     

Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxide H synthase-1

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18-22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co(3+) protoporphyrin IX-reconstituted ovine PGHS-1 (Co(3+)-oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-gamma-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. For LA, the carboxyl half binds in a more extended configuration than AA, which results in positioning C-11 next to Tyr-385. Val-349 and Ser-530, recently identified as important determinants for efficient oxygenation of DHLA by PGHS-1, play similar roles in the oxygenation of EPA and LA. Approximately 750- and 175-fold reductions in the oxygenation efficiency of EPA and LA were observed with V349A oPGHS-1, compared with a 2-fold change for AA. Val-349 contacts C-2 and C-3 of EPA and C-4 of LA orienting the carboxyl halves of these substrates so that the omega-ends are aligned properly for hydrogen abstraction. An S530T substitution decreases the V(max)/K(m) of EPA and LA by 375- and 140-fold. Ser-530 makes six contacts with EPA and four with LA involving C-8 through C-16; these interactions influence the alignment of the substrate for hydrogen abstraction. Interestingly, replacement of Phe-205 increases the volume of the cyclooxygenase site allowing EPA to be oxygenated more efficiently than with native oPGHS-1.     

Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase was studied throughout the rat estrous cycle. Animals were necropsied at 1300 h on each day of the 4-day cycle, and an additional group was necropsied at 2300 h on proestrus. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. At all stages of the cycle, intense immunostaining was observed in newly formed corpora lutea. Luteal cells were immunoreactive, but the connective tissue centrum was unstained. Interstitial tissue contained heavily labeled cells, whereas the germinal epithelium exhibited faint staining. During estrus, metestrus, and diestrus, thecal cells from preantral and antral follicles contained PGH synthase immunoreactivity, but granulosa cells were unstained. Faint staining of mural granulosa cells was observed first in 78% of preovulatory follicles (less than 400-microns diameter) in ovaries collected on the afternoon of proestrus. After the luteinizing hormone surge, 95% of the preovulatory follicles exhibited PGH synthase staining. The percentage of immunoreactive granulosa cells in these preovulatory follicles increased 4-fold in ovaries collected at 2300 h on proestrus. The presence of ovarian PGH synthase throughout the rat estrous cycle and the changes in cellular localization may reflect the potential role of PGs in follicular and luteal function.     

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.     

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.     

Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence

The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by digestion of the enzyme with carboxypeptidase Y and by automated Edman degradation of the intact enzyme polypeptide and peptide fragments obtained by limited proteolysis of the enzyme with Achromobacter proteinase I. Mature sheep prostaglandin endoperoxide synthase is shown to be composed of 576 amino acids with an Mr of 66,175. The precursor peptide is predicted to contain a 24-residue signal peptide. The serine residue susceptible to acetylation by aspirin is found to be located near the C-terminus of the enzyme polypeptide.     

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 ml-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and L-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5-120 microM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E2 and prostaglandin F2 alpha to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100 degrees C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays inhuman myometrium during the last trimester of pregnancy (n=23) and in non-pregnant controls (n=8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p < 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p < 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells.Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p < 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI₂ production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.     

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays in human myometrium during the last trimester of pregnancy (n = 23) and in non-pregnant controls (n = 8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p less than 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p less than 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells. Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p less than 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI2 production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.