Glucocorticoid effects on gastric peroxidase activity

The peroxidase activity in rat gastric mucosa is inhibited after administration of glucocorticoids. The synthetic steroid dexamethasone is more potent than the naturally occurring steroids, such as cortisone or corticosterone. Almost complete inhibition of the enzyme occurs after 24 h with a single dose of 100 μg dexamethasone/120 g body weight. Other mitochondrial enzyme activities, like monoamine oxidase, succinic dehydrogenase and Mg²⁺-ATPase, remain unaltered under the same experimental condition. Submaxillary peroxidase and thyroid peroxidase activity are not inhibited by dexamethasone. Gastric peroxidase activity is increased 200–250% on the 6th day after adrenalectomy. This effect is blocked by the administration of dexamethasone. In fact, the enzyme becomes more sensitive to dexamethasone after adrenalectomy, since it is inhibited by more than 90% at the dose of 25 μg/120 g body weight. The inhibition by dexamethasone in normal animals is reversible. The enzyme is also inhibited after the administration of a single dose of ACTH. The apparent K_m of the enzyme for H₂O₂ is not altered after dexamethasone treatment or after adrenalectomy. The increase in enzyme activity following adrenalectomy is not blocked by actinomycin D or by α-amanitin, but is prevented by puromycin or cycloheximide. After administration of dexamethasone, the iodide concentration process in the gastric mucosa is not affected, but the organification of iodide is significantly diminished.     

Characteristics of estrogen-induced peroxidase in mouse uterine luminal fluid.

Peroxidase activity in the uterine luminal fluid of mice treated with diethylstilbestrol was measured by the guaiacol assay and also by the formation of 3H2O from [2-3H]estradiol. In the radiometric assay, the generation of 3H2O and 3H-labeled water-soluble products was dependent on H2O2 (25 to 100 microM), with higher concentrations being inhibitory. Tyrosine or 2,4-dichlorophenol strongly enhanced the reaction catalyzed either by the luminal fluid peroxidase or the enzyme in the CaCl2 extract of the uterus, but decreased the formation of 3H2O from [2-3H]estradiol by lactoperoxidase in the presence of H2O2 (80 microM). NADPH, ascorbate, and cytochrome c inhibited both luminal fluid and uterine tissue peroxidase activity to the same extent, while superoxide dismutase showed a marginal activating effect. Lactoferrin, a major protein component of uterine luminal fluid, was shown not to contribute to its peroxidative activity, and such an effect by prostaglandin synthase was also ruled out. However, it was not possible to exclude eosinophil peroxidase, brought to the uterus after estrogen stimulation, as being the source of peroxidase activity in uterine luminal fluid.     

Quantitative modulation of thyroid iodide peroxidase by thyroid stimulating hormone

The activity of rat thyroid iodide peroxidase fell to 8% of the normal value 48 hours after hypophysectomy. Rats given injections of thyroid stimulating hormone manifested an enzyme activity indistinguishable from that of the sham-operated animals. Cycloheximide prevented the thyroid stimulating hormone-induced restoration of the enzyme activity. The incorporation of ¹⁴C-leucine into the thyroid gland decreased gradually and reached two thirds of the sham-operated group by 48 hours after hypophysectomy. Thyroid stimulating hormone administration prevented this decrease, as observed for iodide peroxidase activity. Thyroidal RNA contents decreased also in hypophysectomized rats, thyroid stimulating hormone treatment prevented the reduction of RNA contents and no significant change was observed in thyroidal DNA contents. These data are consistent with the idea that protein biosynthesis is involved in thyroid stimulating hormone regulation of thyroidal iodide peroxidase and that the life span of the peroxidase is less than 48 hours.     

Presence of uterine peroxidase activity in the rat early pregnancy

Peroxidase has been associated with estrogen action in the uterus. This enzyme plays an important role in the control of hydrogen peroxide levels and in catechol estrogen production. Since the uterus, during early pregnancy, is subjected to estrogen and progesterone regulation, we analyzed the changes of peroxidase activity in relation to receptivity and uterine early response to the embryo. Soluble and microsomal peroxidase activity were determined in the rat uterus during the estrus phase and early pregnancy (days 3 through 6). Soluble peroxidase activity increased significantly (p < 0.01) from day 3 (1.50 +/- 0.24) to day 4 (3.5 +/- 0.3) and 5 (5 +/- 0.5 U/mg protein, mean +/- S.D., n = 6) of pregnancy. During day 6, a significant decrease was noted in both the implantation site and the nonimplantation uterine tissue. Microsomal calcium-extractable peroxidase showed a similar pattern, with lower specific activity than, the soluble peroxidase. During estrus, the uterine tissue showed the highest activity of calcium-extracted peroxidase (8.7 +/- 1.35 U/mg protein), statistically greater when compared with days 3, 4, 5 and 6 of pregnancy. In conclusion, high peroxidase activity was associated with uterine receptivity. The decrease of activity on day 6 might be due to a progesterone-estrogen interaction, and consequently, hydrogen peroxide can be utilized for hydroxile production by means of the Fenton reaction. Lipoperoxidation may be necessary for changes in membrane fluidity for embryo attachment to endometrial epithelium.     

Peptidases from pig reproductive tract: purification and properties of aminopeptidases from uterine secretions, allantoic fluid, and amniotic fluid

In ovariectomized sows, aminopeptidase is secreted into the uterine lumen under the influence of progesterone. The enzyme also accumulates in allantoic and amniotic fluids of pregnant animals. We have purified the predominant form of this enzyme from uterine flushings, allantoic fluid, and amniotic fluid by the following steps: ammonium sulfate precipitation, Sepharose 6B chromatography, ion-exhange chromatography on diethylaminoethyl cellulose, and affinity chromatography usingl-leucylglycine immobilized on agarose. The overall procedure gave approximately 974-, 110-, and 230-fold purifications of the allantoic, uterine, and amniotic enzymes, respectively. The enzymes from all three sources are glycoproteins with pI's around 4 and molecular weights of about 480,000. They may be dissociated into six apparently identical subunits of molecular weight 80,000 as judged by sodium dodecyl sulfate gel electrophoresis. With l-leucyl-β-naphthylamide as substrate the pH optimum and apparent K_m value for each enzyme were 7.1 and 14 μm, respectively. However, the uterine and allantoic aminopeptidases exhibited V values of 0.35 μmol of substrate hydrolyzed/min/mg of protein, whereas the V for the amniotic enzyme was at least sixfold greater. The amniotic enzyme also differed from the other two in pH and temperature stability. The activity of all three enzymes was stimulated by Co²⁺ and inhibited by Cu²⁺, Fe³⁺, and chelating agents, while iodoacetate and mercaptoethanol had no effect on catalysis. The effect of Co²⁺ on the allantoic enzyme was investigated in further detail. The stimulation of peptidase activity by Co²⁺ was shown to be a complex process but consistent with Co²⁺ replacing another metal at the active site and at some other additional site on the enzyme. The function of the aminopeptidases in the pregnant uterus is unknown.     

Glucocorticoid effects on gastric peroxidase activity

The peroxidase activity in rat gastric mucosa is inhibited after administration of glucocorticoids. The synthetic steroid dexamethasone is more potent than the naturally occurring steroids, such as cortisone or corticosterone. Almost complete inhibition of the enzyme occurs after 24 h with a single dose of 100 micrograms dexamethasone/120 g body weight. Other mitochondrial enzyme activities, like monoamine oxidase, succinic dehydrogenase and Mg2+-ATPase, remain unaltered under the same experimental condition. Submaxillary peroxidase and thyroid peroxidase activity are not inhibited by dexamethasone. Gastric peroxidase activity is increased 200-250% on the 6th day after adrenalectomy. This effect is blocked by the administration of dexamethasone. In fact, the enzyme becomes more sensitive to dexamethasone after adrenalectomy, since it is inhibited by more than 90% at the dose of 25 micrograms/120 g body weight. The inhibition by dexamethasone in normal animals is reversible. The enzyme is also inhibited after the administration of a single dose of ACTH. The apparent Km of the enzyme for H2O2 is not altered after dexamethasone treatment or after adrenalectomy. The increase in enzyme activity following adrenalectomy is not blocked by actinomycin D or by alpha-amanitin, but is prevented by puromycin or cycloheximide. After administration of dexamethasone, the iodide concentration process in the gastric mucosa is not affected, but the organification of iodide is significantly diminished.     

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10⁵ M^-1 as determined by competition with [³H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.     

Inhibition of intestinal peroxidase activity by nonsteroidal antiinflammatory drugs

The peroxidase activity of the mitochondrial fraction of rat intestine is inhibited in vitro by non-steroidal antiinflammatory drugs (NSAIDs), such as indomethacin (IMN) and acetylsalicylic acid (ASA), the former being more potent than the latter. The peroxidase was solubilised by cetab-NH₄Cl extraction and purified to apparent homogeneity by Sephadex G-150 gel filtration and affinity chromatography on Con-A Sepharose. The purified enzyme activity was 80% inhibited by 150 μM IMN and 50% by 2.67 mM ASA. IMN could also inhibit lactoperoxidase activity to the same extent but not the horseradish peroxidase activity. The inhibition of peroxidase-catalysed iodide oxidation by IMN and ASA was optimal at pH 5.5 and 4.5, respectively. Kinetic studies revealed that the inhibition by IMN was competitive with respect to iodide or guaiacol, while the inhibition by ASA was noncompetitive and reversible in nature. Studies of some structural analogues showed that indole-3-acetic acid was as effective as IMN, while salicylic acid was more potent than ASA. Spectral studies showed a small bathochromic shift of the Soret band of the enzyme by IMN, suggesting its possible interaction at or near the heme moiety. The competitive nature of IMN may be explained as due to its oxidation by the peroxidase to a product absorbing at 412 nm, the formation of which is inhibited by iodide. We suggest that IMN inhibits intestinal peroxidase activity by acting as a competitive substrate for the enzyme. As intestinal peroxidase is mainly contributed by the invading eosinophils, NSAIDs may affect the host defence mechanism by inhibiting the activity of the enzyme.     

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [³H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.     

Immunological relationship between peroxidases in eosinophils, uterus and other tissues of the rat.

A rabbit antibody against purified pig intestinal peroxidase was shown by means of Western blotting and immunodetection to bind to peroxidases in various rat tissues, including eosinophils, uterus, uterine fluid and mammary tumours, and also to bind to bovine lactoperoxidase. The peroxidase in all rat tissues had an Mr of 53 000, except for uterine fluid, in which the cross-reacting band had an Mr of 80 000. The results indicate that while some of the peroxidase present in uterine tissue could be derived from eosinophils, the enzyme secreted into the lumen of the uterus is likely to have a different origin. They also suggest that mammary tumour peroxidase could originate from infiltration by eosinophils.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF_2α the treatment with progesterone (4 mg.day⁻¹, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol- 17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE₂, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE₂ catabolism into 15-keto- PGE₂ as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Hormonal regulation of collagen degradation in the uterus: Inhibition of collagenase expression by progesterone and cyclic AMP

Dibutyryl cyclic AMP markedly increases the ability of progesterone to prevent the expression of collagenase activity in cultures of post-partum rat uterus. Dibutyryl cyclic AMP itself and, to a lesser extent, native cyclic AMP, are capable of producing a partial decrease in enzyme activity, but complete abolition is not observed at high cyclic nucleotide concentrations (5 mM) in the culture medium. Theophylline, when added to cultures, mimics the effect of dibutyryl cyclic AMP. Other cyclic nucleotides were without effect on levels of collagenase activity in the uterine cultures.When non-inhibitory concentrations of either dibutyryl cyclic AMP (1 · 10^?4 M) or theophylline (1 · 10^?4 M) are added to cultures together with a non-inhibitory concentration of either progesterone (5 · 10^?6 M) or the potent progesterone analogue Provera (1 · 10^?8 M) the ability of the tissue to produce collagenase is decreased by 40–70%. Collagenase activity is consistently diminished more than additively by combinations of steroid and cyclic nucleotide. Theophylline mimics the effect of dibutyryl cyclic AMP on steroid activity in culture. In the presence of dibutyryl cyclic AMP, diminution of collagenase activity can be observed at concentrations of steroid more than two orders of magnitude lower than the normal minimally inhibitory dose. Reduction of collagenase activity is reflected in all experiments by a concomitant decrease in the normal proteolytic degradation of collagen in the tissue ex-plants. The possibility that progesterone acts in the uterus to raise cyclic AMP levels is suggested by the fact that uterine tissue, when cultured in the presence of progesterone, contains reduced levels of cyclic nucleotide phosphodiesterase.These data suggest that, in some way a cyclic AMP-mediated system is critically involved in the control of collagenase activity by progesterone in the rat uterus.     

Progesterone-induced estrogen receptor-regulatory factor is not 17β-hydroxysteroid dehydrogenase

These studies were done to determine if the progesterone-induced estrogen receptor-regulatory factor (ReRF) in hamster uterus is 17β-hydroxysteroid dehydrogenase (17β-HSD), i.e. that rapid loss of nuclear estrogen receptor (Re) might be due to enhanced estradiol oxidation to estrone catalyzed by 17β-HSD. Treatment of proestrous hamsters with progesterone (~25 mg/kg BW) for either 2 h or 4 h had no effect on 17β-HSD activity measured as the rate of conversion of [6,7-³H]estradiol to [³H]estrone by whole uterine homogenstes at 35°C. During this same time interval, progesterone treatment increased the rate of inactivation of the occupied form of nuclear Re as determined during a 30 m1n incubation of uterine nuclear extract in vitro at 36°C. Since we previously demonstrated that such in vitro Re-inactivating activity represents ReRF, the present studies show that ReRF is not 17β-HSD or a modifier of that enzyme.     

Depression of estrogen-induced uterine peroxidase in alloxan-diabetic rats

The influence of alloxan diabetes on reproductive function and the estradiol-stimulated increase in uterine peroxidase was investigated. Alloxan monohydrate in a dose of 75 mg/kg body weight effectively produced permanent diabetes. In adult rats, 20 days of diabetes resulted in cessation of the estrous cycle and a significant reduction in the gain of body weight, the weights of anterior pituitary gland, ovary, uterus, the level of serum progesterone and the activity of the estradiol-stimulated uterine peroxidase (P less than 0.05). After 10 days of insulin treatment, the ovarian weight, the estrous cycle and the level of ovarian hormones were restored to normal whereas the uterine weight and the estradiol-stimulated uterine peroxidase activity were only partially recovered. Persistent depression of the uterine response in the insulin-treated diabetic rats to both endogenous and exogenous ovarian hormone stimulation suggests that the uterus was directly affected by diabetes. The direct effect of diabetes upon the uterus was further demonstrated in the ovariectomized immature rat in which diabetes depressed the stimulatory action of estradiol on both uterine weight and uterine peroxidase activity.     

Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy

Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy.     

An essential role of active site arginine residue in iodide binding and histidine residue in electron transfer for iodide oxidation by horseradish peroxidase

The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min^-1 M^-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection constant (K_p) for iodide and guaiacol are 500 and 10 M respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased K_d value to 571 mM from the native enzyme (K_d = 150 mM). Although arginine-modified enzyme reacts with H₂O₂ to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding.The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 0.66 min^-1 M^-1. Both the nitrogens (, ) of histidine imidazole were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme. However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H₂O₂ suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl group during oxidation.     

Nonsteroidal anti-inflammatory drugs inhibit gastric peroxidase activity

The peroxidase activity of the mitochondrial fraction of rat gastric mucosa was inhibited with various nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro. Indomethacin was found to be more effective than phenylbutazone (PB) or acetylsalicylic acid (ASA). Mouse gastric peroxidase was also very sensitive to indomethacin inhibition. Indomethacin has no significant effect on submaxillary gland peroxidase activity of either of the species studied. Purified rat gastric peroxidase activity was inhibited 75% with 0.15 mM indomethacin showing half-maximal inhibition at 0.04 mM. The inhibition could be withdrawn by increasing the concentration of iodide but not by H2O2. NSAIDs inhibit gastric peroxidase activity more effectively at acid pH (pH 5.2) than at neutral pH. Spectral studies showed a bathochromic shift of the Soret band of the enzyme with indomethacin indicating its interaction at or near the heme part of the enzyme.     

In vitro myometrial inhibition by the partitioned aqueous fraction of Anthocleista djalonensis leaves

This study investigated the effect on the uterus of the aqueous fraction of the partitioned methanol crude extract of the leaves of Anthocleista djalonensis (AD) and the possible mechanism of AD activity. AD inhibited the concentration-response curves induced by oxytocin and CaCl2 on the rat uterus in vitro and significantly reduced the EC50 in a concentration-dependent manner (p?< 0.05). A similar effect was observed with salbutamol and verapamil on the concentration-response curves obtained for oxytocin and CaCl2. The inhibitory effect of AD was not attenuated in the presence of propranolol. AD, salbutamol, and verapamil also produced a concentration-dependent relaxation on K+-induced sustained uterine contraction. In Ca2+-free medium, AD and salbutamol similarly inhibited oxytocin-induced contraction, but verapamil failed to produce this effect. The present results suggest that AD, being a mixture of phytochemicals, probably exerts inhibitory activity on in vitro uterine contractions of the nonpregnant, diethylstilboestrol-treated rat by multiple mechanisms that do not involve interaction with β-adrenergic receptors and do not solely depend on inhibition of calcium influx.     

The in vivo and in vitro influence of methyl jasmonate on oxidative processes in Arabidopsis thaliana leaves

In Arabidopsis thaliana leaves a strong increase of H₂O₂ content was induced by application of methyl jasmonate (JAMe) through the root system, but the induction only slightly depended on JAMe concentration. The activity of superoxide dismutase and ascorbic acid peroxidase increased at lower JAMe concentrations and decreased at higher ones. Catalase activity decreased proportionally to JAMe concentration (in comparison with control plants). The sum of ascorbic acid and dehydroascorbate content at 10⁻⁶ M JAMe was similar to the control, but at higher concentrations it increased, especially due to a higher ascorbate accumulation. Methyl jasmonate applied directly to the extract of leaves (in vitro experiment) also induced a strong increase in H₂O₂ level, even at a low concentration (10⁻⁸ M). Since lower JAMe concentrations induced weak superoxide dismutase and did not change catalase and peroxidase activity, it is suggested that in this case a high level of hydrogen peroxide was not the result of the activity of the mentioned enzymes. JAMe-induction of H₂O₂ increase at the highest JAMe concentration resulted from SOD activity. Our in vivo and in vitro experiments suggest that jasmonate can influence oxidative stress not only through gene expression but also by its direct effect on enzyme activity.     

Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin

Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.