Metabolism of estradiol by true and pseudoperoxidases

The activation of 14C-labeled estradiol by "true" and "pseudo" peroxidases to form conjugates and other products was compared in four model systems using H2O2, glutathione, Mn2+ or irradiated riboflavin. Albumin was used as acceptor except in the glutathione system. The binding of estradiol to glutathione in the presence of the true peroxidases, lacto- or uterine peroxidase (no H2O2 added), was also examined and the conditions shown to differ from those required with the pseudoperoxidases, microperoxidase or trypsin-digested cytochrome c. The conjugates were purified by chromatography after elution from Amberlite XAD-2 and the relative amounts of these products assessed by autoradiography. The ratio of steroid to glutathione in the main water-soluble metabolite formed with lactoperoxidase was found to be approx 1:1 in a double label experiment with [14C]estradiol and [3H]glutathione. It was also shown, using estradiol labeled with 3H in different positions of the steroid molecule, that lactoperoxidase acts non-specifically in catalyzing the formation of glutathionyl conjugates as indicated by the release of 3H2O. The possible role of peroxidase and glutathione in the metabolism of estrogens and in the formation of artifactual products is discussed.     

Lactoperoxidase-catalyzed oxidation of [4-14C]estradiol

The conversion of [4-14C]estradiol to water-soluble products by lactoperoxidase (EC 1.11.1.7) in the presence of added or generated H2O2 was studied using albumin or tyrosine as acceptor. The enzyme was able to catalyze the oxidation and binding of estradiol to albumin even in the absence of 2,4-dichlorophenol at very low concentrations of hydrogen peroxide. Other systems in which H2O2 was replaced by oxygen and Mn2+, light-sensitized riboflavin or glutathione was also shown to be active in the conversion of estradiol to water-soluble products and the effect of inhibitors on these reactions was investigated. Possible mechanisms for the peroxidase-catalyzed formation of these estradio metabolites are discussed.     

Endogenous acetylcholine release and labelled acetylcholine formation from [3H]choline in the myenteric plexus of the guinea-pig ileum.

The spontaneous release of acetylcholine (ACh) from the guinea-pig myenteric plexus - longitudinal muscle preparation superfused at a constant rate in the presence of physostigmine was 10 nmol-g-1-h-1. This release was decreased to one-third by tetradotoxin or by MnCl2 and increased 2.5 times by 0.1 Hz and 20 times by 16 Hz stimulation. The formation of [3H]ACh from [3H]choline increased from 3 to 33 nmol-g(-1)-h(-1) when the concentration of [3H]choline was increased from 1 muM to 50 muM. The rate of [3H]ACh formation was not affected by tetrodotoxin, MnCl2, or physostigmine in the absence of stimulation. It was increased by 50% by 0.1 Hz and by 100% by 16 Hz stimulation during the first 9 min of exposure to [3H]choline but not subsequently. The myenteric plexus - longitudinal muscle preparation contains 200 nmol/g choline. Results suggest that the apparent small [3H]ACh formation from low concentrations of [3H]choline is due to the dilution of [3H]choline by endogenous choline. The major part of [3H]ACh formation appears to be due to the intracellular turnover of ACh while the evoked release of [3H]ACh appears to originate from a small pool.     

A comparison of methods measuring aromatase activity in human placenta and rat ovary

The single step chromatographic product isolation method using [4-14C]-androstenedione and the tritiated water method using either [1 beta, 2 beta-3H]- or [1 beta-3H]-androstenedione have been used to determine a suitable method to measure the aromatase activity in rat ovarian 1000 g supernatant and human placental microsomes. The single step product isolation method using [4-14C]A4 reveals the presence of four distinct [4-14C]-labelled products in the rat ovary of which only the synthesis of estradiol is markedly inhibited by CGS 16949A, a well established aromatase inhibitor. In the human placenta, the formation of both [4-14C]-estrone and [4-14C]-estradiol is strongly inhibited by CGS 6949A. Therefore, in the rat ovary spurious results are obtained if accumulative radiolabelled product formation is measured without characterisation of the products. The Vmax in the rat ovary using [1 beta, 2 beta-3H]-A4 as a substrate is 13.7 pmol/h/mg compared to 2.9 pmol/h/mg when [1 beta-3H]-A4 is used. In the human placenta, the Vmax is similar using either [1 beta, 2 betat-3H]-A4 or [1 beta-3H]-A4 (1.21 and 1.27 nmol/h/mg, respectively). Consistent results are obtained for the human placenta using either the single step chromatographic product isolation method or the tritiated water method. However, in the rat ovary the more suitable method of the two used to measure the aromatase activity is the tritiated water method employing [1 beta-3H]-A4 as a substrate. Aromatase activity in the rat ovary during estrus cycle was measured using the tritiated water method employing [1 beta-3H]-A4 as a substrate. A peak of aromatase activity at proestrus was seen which returned rapidly to its basal level at estrus. Plasma estradiol concentrations were in parallel with the aromatase activity.     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

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.     

Immunological probe of estrogen biosynthesis. Evidence for the 2 beta-hydroxylative pathway in aromatization of androgens

The terminal hydroxylation in placental estrogen biosynthesis from androgens is at the 2 beta position. The 2 beta-hydroxy-19-oxoandrogen derivative collapses nonenzymatically to estrogen and is therefore the proximate precursor of the female hormone. To establish the role of this pathway in biological aromatization, an immunological approach was employed in which an antibody was obtained which recognizes 2 beta-hydroxy-19-oxygenated androgens but not intermediates oxygenated at C-19 only. Binding of the 2 beta-hydroxy-19-oxo intermediate by the antibody stabilizes it so that its nonenzymatic transformation to estrogen is delayed and results in slower estrogen formation. When placental microsomes were incubated with [1,2-3H]androstenedione in the presence of the antibody antiserum, a 50% decrease in [3H]estradiol formation and 3H2O release was observed when compared with identical incubations containing normal rabbit serum alone. This inhibition is blocked when the antibody is inactivated by presaturation with 2 beta, 19-dihydroxyandrostenedione. Precipitation of immunoglobulins from the incubations followed by heating liberated the 2 beta-hydroxy-19-oxo intermediate (30%) from the antibody, and resulted in its nonenzymatic collapse to estrogen with concomitant release of 3H2O. Control normal rabbit serum or blocked antibody incubations did not show a similar increase in [3H]estradiol or 3H2O yields in the precipitate. Heat treatment (90 degrees C) of the antibody but not normal rabbit serum incubations resulted in a similar increase in [3H]estradiol and 3H2O yields. These results are consistent with the hypothesis that the final and rate-determining hydroxylation in aromatization of androgens is at the 2 beta position and that this pathway is the dominant, if not the sole, route of estrogen biosynthesis by placental aromatase. The antibody probe also permits the characterization of aromatization mechanisms in tissues other than the placenta.     

Estradiol metabolism in Ishikawa endometrial cancer cells

Estrogen-responsive human cells derived from a specimen of well differentiated endometrial adenocarcinoma (Ishikawa line) were incubated with [3H]estradiol (E2) at various concentrations and the medium was sampled at 3, 6 and 24 h to evaluate the kinetics of removal of the hormone and the formation of unconjugated or sulfated metabolites. The detectable products of metabolism were estrone and the conjugate estradiol-3-sulfate. The latter was identified by high pressure chromatography, before and after acetylation, oxidation, and hydrolysis. The disappearance of [3H]E2 from the medium was found to follow first order kinetics between 3 and 24 h, with half-lives increasing from 4.7 to 53 h as the initial concentrations of the hormone were raised from 10(-8) to 10(-6)M. At the lowest concentration, practically all of the [3H]E2 added to the cultures was converted to estradiol-3-sulfate in 24 h, whereas at 10(-6)M oxidation to estrone was quantitatively more important than sulfation. These results indicate the presence in Ishikawa cells of an estrogen sulfotransferase of low Michaelis constant for E2, and 17 beta-oxidoreductase activity that significantly contributes to the metabolism of E2 only at higher concentrations of substrate.     

Radioligand purification prior to routine receptor assays

The need to repurify the commercially available radioligands [3H]estradiol and [3H]testosterone before use in routine assays was investigated. Storage of these products for 2 months after delivery led to appreciable degradation of [3H]estradiol compared to [3H]testosterone. Unexpectedly, TLC and even HPLC procedures were ineffective in completely restoring the purity of [3H]-estradiol and the unremoved polar products induced important variations in our estrogen receptor assays. An increase in non-specific binding and a comcomitant decrease in total binding were observed resulting in an underestimation of specific binding sites and of the affinity constant. In some cases Scatchard analysis was not possible. We therefore strongly recommend the repurification of low-stability radioligands and propose an economic time-saving procedure for the purification of [3H]estradiol by solvent differential partition which requires no high-cost investment in apparatus.     

Estrogen 1,2-epoxides or estrogen quinones/semiquinones

Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o-quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2-hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o-quinones/semiquinones and not estrogen 1, 2-epoxide.     

Radiochemical evidence for estradiol-17-fatty acid esters in human blood

The C-17 fatty acid esters of estradiol, known as the lipoidal derivatives of estradiol, LE2, are metabolites of estradiol that were originally isolated from various tissues after in vitro incubations with estradiol. These steroidal esters are active estrogens with extremely prolonged potencies. The present study investigates the existence of LE2 in human blood using a radiochemical isotope dilution technique. The LE2 fraction from blood was isolated, saponified, and the hydrolyzed estradiol was then acetylated with [3H]acetic anhydride. It was found that 3H was incorporated into estradiol diacetate, demonstrating that LE2 is present in human blood. Thus these steroidal esters represent a new class of endogenous estrogens which have not been previously considered in the physiology of the female sex steroids.     

Estradiol binding by intact cells isolated from DMBA-induced mammary tumors of the rat

Study of hormone binding in intact cells enables one to examine binding under conditions which elicit a biological response. Cells from 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of the rat were enzymatically dispersed. More than 80% of these cells excluded trypan blue and were used to study binding of [3H] estradiol-17 beta. Specific binding was determined by subtracting the amount of [3H]estradiol bound in the absence and presence of 200-fold excess unlabeled estradiol. Specific binding at 37 degrees was maximal after 15 min. Steroid competition studies indicated that [3H]estradiol binding sites were relatively specific for estrogens, although there was a 9-18% inhibition of binding by androgens and progestins when present at 150-fold molar excess. Scatchard analyses of [3H]estradiol (0.15-5.0 nM) binding by whole cells suggest a single, high-affinity binding site (Kd = 7.5 x 10-10M) of low capacity (6.1 fmol/10(6) cells). More [3H]estradiol was translocated to the nucleus after 1 hr at 37 degrees than at 0 degrees. Preliminary studies indicated that incubations at 37 degrees result in appreciable metabolism of [3H]estradiol to other steroids and/or conjugates when examined by silica gel thin layer chromatography.     

A simple method for determining total glucose utilisation by isolated adipocytes using (5-3H)-glucose

Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-¹⁴C]-glucose utilisation was calculated by summing the ¹⁴C found in CO₂, triglycerides, and anions. [³H]-H₂O formation from [5-³H]-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the [5-³H]-glucose is simpler to use than [U-¹⁴C]-glucose and gives substantially more reproducible results than glucose oxidase.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Protein synthesis in rabbit reticulocytes: characteristics of CO-eIF-2 protein complex.

A high molecular weight reticulocyte protein factor, named Co-eIF-2, contains Co-eIF-2A, Co-eIF-2B, and Co-eIF-2C activities and stimulates Met-tRNAf binding to eIF-2 both in the presence and absence of Mg2+. Some characteristics of this stimulation in the absence of Mg2+ are: (1) Stimulation is most pronounced at low eIF-2 levels. (2) Stimulation is partially resistant to heat and NEM treatment, and thus appears to be due to the combined action of both heat and NEM-insensitive Co-eIF-2A, and heat and NEM-sensitive Co-eIF-2C activities. (3) [3H]GDP bound in eIF-2 . [3H]GDP complex is rapidly displaced by unlabelled GTP during ternary complex formation Co-eIF-2 stimulates Met-tRNAf binding to eIF-2 even when added after the [3H]GDP from eIF-2 . [3H]GDP has been completely displaced. This indicates that Co-eIF-2-stimulation is not due to GDP displacement from eIF-2 . GDP. We propose that eIF-2 molecules become inactive in the presence of Mg2+ and at high dilution, and Co-eIF-2 restores the inactive eIF-2 molecules into an active form.     

Androgen metabolism by hepatic and renal tissues of the fetal rhesus monkey

Liver and kidney from fetal monkeys (day 125 of gestation) were fractionated into low speed pellets, microsomal and cytosolic fractions. Liver cytosols converted as much testosterone (T) to 5 beta-androstane-3 alpha,17 beta-diol (5 beta-diol) at 0 degrees C as at 4 degrees-45 degrees C without exogenous cofactors. The principal product formed from 5 alpha-dihydrotestosterone (5 alpha-DHT) was 5 alpha-diol. A 1000-fold molar excess of radioinert 5 beta- or 5 alpha-DHT inhibited 5 beta-diol formation from [3H]T by cytosols and increased 5 beta-DHT formation. Similarly, using 5 alpha-DHT as substrate, 5 alpha-diol formation was inhibited. Microsomal and low speed pellets with added cofactors formed products which recrystallized with either etiocholanolone or androsterone from [3H]T or [3H]DHT, respectively. Little product was formed without cofactor. Whole liver homogenates produced 5 beta-reduced products from [3H]T in the presence of an NADPH generating system whereas kidney homogenates produced 5 alpha-reduced products. These data provide new information on the capacity of fetal monkey liver and kidney to metabolize androgens. The 3 alpha-reductases are cytosolic. The 5 alpha- and 5 beta-reductases are mostly in the low speed pellet but are sufficiently represented in cytosols to mediate diol formation. The 17-hydroxysteroid dehydrogenases are in the microsomal fraction. Our results suggest that 5 alpha-DHT is the active androgen in fetal liver since testosterone is metabolized to 5 beta-DHT and 5 beta-diol which are inactive androgens.     

Estrogen synthesis and metabolism in the hamster blastocyst, uterus and liver near the time of implantation

The steroidogenic potential of hamster tissues, just prior to implantation of the blastocyst in the uterus, was characterized by incubating blastocysts (14) and pieces of endometrium with [1, 2-3H]-androstenedione for 24 h. [3H]-2-Methoxyestradiol was synthesized, but intermediate estrogens were not found. To obtain a more quantitative assessment and comparison of steroidogenic activity, especially aromatase activity, in these tissues as well as in the uterine myometrium and liver and to increase the possibility of recovering estradiol, microsomes were isolated from 244 blastocysts and portions of the other tissues. Microsomes were incubated with [1 alpha, 2 alpha-3H]-testosterone plus [1 beta,2 beta-3H]-testosterone for 6 h. During this time [3H]-metabolites were synthesized by all tissues as indicated by HPLC. [3H]-Androstenedione was noted and values were higher than control levels (medium alone or microsomes from uterine flush fluid) in all samples but liver. [3H]-Estradiol was detected at an elevated level only in the blastocyst sample; however, addition of unlabeled estradiol during the subsequent incubation of endometrial, myometrial and liver microsomes increased the recovery of [3H]-estradiol. Identities of [3H]-2-methoxyestradiol from the first experiment and [3H]-androstenedione and [3H]-estradiol from the second experiment were confirmed by recrystallization. The formation of 3H2O from [beta-3H]-testosterone was used as an index of aromatase activity. After subtracting control medium values, blastocysts were 24-fold more active (dpm/microgram protein) than the endometrium and myometrium in synthesizing 3H2O. While there was no difference in synthetic potential between endometrium and myometrium, aromatase activity in these tissues was greater than that of the liver. Microsomes from uterine flush fluid displayed no capacity for synthesizing 3H2O indicating that the elevated blastocyst levels were not caused by contaminating endometrial cells. These results indicate that all of the tissues examined have the capacity to metabolize C19-steroids to a variety of hormones, including estrogens, and further, that estrogen metabolism occurs rapidly in these tissues. This capacity may be important for providing a suitable hormonal milieu at the time of implantation.     

Dose-dependent and sequence-sensitive effects of amyloid-beta peptide on neurosteroidogenesis in human neuroblastoma cells

Interactions between neurosteroidogenesis and proteins involved in age-related diseases are unknown. High concentrations of amyloid-beta (A beta) peptides induce plaques in Alzheimer's disease but several studies demonstrated that physiological or non-toxic doses are neuroprotective. We compared the effects of non-toxic and toxic concentrations of A beta 1-42 and A beta 25-35 on neurosteroidogenesis in human neuroblastoma SH-SY5Y cells. Viability assays revealed that nanomolar doses of A beta are devoid of cytotoxicity while 12 microM induced cell death. Pulse-chase, high-performance liquid chromatography and flow-scintillation analyses showed that non-toxic A beta 1-42 concentrations, acting selectively, decreased [3H]progesterone but increased [3H]estradiol production from the precursor [3H]pregnenolone. Non-toxic A beta 25-35 doses reduced [3H]progesterone formation but had no effect on [3H]estradiol biosynthesis. At 12 microM, both A beta 1-42 and A beta 25-35 inhibited [3H]progesterone formation but only A beta 1-42 reduced [3H]estradiol production. The results demonstrate a selective and amino-acid sequence-dependent action of A beta on neurosteroidogenesis. The fact that non-toxic A beta 1-42 doses stimulated neuroprotective-neurosteroid estradiol synthesis, which is inhibited by high A beta 1-42 doses, may explain A beta 1-42 ability to exert either protective or deleterious effects on nerve cells.     

H2 histaminic receptors in rat cerebral cortex. 1. Binding of [3H]histamine

Saturable binding of [3H]histamine in equilibrium with homogenates of rat cerebral cortex reveals Hill coefficients between 0.4 and 1.0, depending upon the conditions. Data from individual experiments are well described assuming one or two classes of sites. Only the sites of higher affinity (KP1 = 3.9 +/- 0.5 nM) are observed when binding is measured by isotopic dilution at a low concentration of the radioligand (less than 1.5 nM) in the presence of magnesium or by varying the concentration of the radioligand. The sites of lower affinity (KP2 = 221 +/- 26 nM) appear during isotopic dilution at higher concentrations of the radioligand or at lower concentrations either upon the addition of guanylyl imidodiphosphate (GMP-PNP) or upon the removal of magnesium. Estimates of the second- and first-order rate constants for association and dissociation of [3H]histamine agree well with KP1. Apparent capacities corresponding to KP1 and KP2 are of the order of 100 ([R1]t) and 1300 pmol/g of protein ([R2]t), respectively. Simple interconversion cannot account for the changes in binding that occur upon adding GMP-PNP or removing magnesium, since the increase in [R2]t exceeds the decrease in [R1]t. Moreover, the apparent amount of high-affinity complex exhibits a biphasic dependence on the concentration of [3H]histamine; an increase at low concentrations is offset by a decrease that occurs at higher concentrations. The latter appears to be positively cooperative and concomitant with formation of the low-affinity complex. These and other observations indicate that the binding of histamine is inconsistent with models commonly invoked to rationalize the binding of agonists to neurohumoral receptors. GMP-PNP and magnesium reciprocally alter capacity at the sites of higher affinity, however, and the reduction caused by GMP-PNP reflects a substantial increase in the rate constant for dissociation at the sites that appear to be lost. The sites labeled by [3H]histamine thus reveal the properties of neurohumoral receptors linked to a nucleotide-specific G/F protein.     

Somatostatin enhances binding of [3H]estradiol to a cytosolic protein in rat pancreas. Possible role of oligopeptide coligands in secretion

The cytosol fraction of rat pancrease can bind [3H] estradiol specifically and extensively. In contrast to the rat uterus, the binding protein in pancreas requires an accessory factor as a coligand in the steroid-binding reaction. Removal of this accessory factor by passage of the cytosol through CM Affi-Gel blue columns renders eluate fractions virtually incompetent with respect to binding of [3H]estradiol (10 nM). Certain synthetic oligopeptides such as N-benzoyl-L-argininyl-p-nitroanilide, as well as an endogenous accessory factor, can reactivate binding of [3H]estradiol. Thus, localization of the protein that binds [3H]estradiol following chromatography with CM Affi-Gel blue columns can be determined readily by assaying eluate fractions in the absence and presence of either accessory factor or N-benzoyl-L-argininyl-p-nitroanilide. Addition of somatostatin (tetradecapeptide referred to as SRIF14; somatotropin release inhibiting factor) to the activatable, but incompetent, eluate fractions, also enhanced binding of [3H]estradiol. The effect of SRIF14 was biphasic. The threshold concentration required for activation of [3H]estradiol binding was about 1 microM, and maximal stimulation occurred at 25 microM. At higher concentrations of SRIF14, binding declined and reached basal levels at about 75 microM. The concentrations of somatostatin required for activation of binding of [3H]estradiol in vivo may be lower than those indicated above since 1) preparations containing [3H]estradiol-binding protein also contained an SRIF14 peptidase. Following incubation of [125I-Tyr1]SRIF14 with these preparations there was loss of binding of radiolabeled peptide with SRIF14 antiserum. 2) The biphasic nature of SRIF14 activation may reflect feedback inhibition of [3H]estradiol binding by a degradation product of SRIF14. Since SRIF14 has been identified in the delta- (or D-) islet cells of the pancreas, and in concentrations that may be in the microM range, the possibility is raised that these cells serve a paracrine function with respect to acinar cell secretion.