Dextran-coated Charcoal Adsorption Research Articles

Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?

Non-denatured human placental cytosol fractions displaced tracer binding in parallel with gonadotrophin-releasing hormone (GnRH) isoform and agonist peptides in GnRH-specific radioimmunoassays and radioreceptor assays. However, placental immuno- and receptor binding-GnRH-like activity was highly correlated with inactivation of GnRH tracers, suggesting that placental GnRH-like factors may be an artefact of ligand degradation during assay. The properties and inhibitor sensitivities of the major (125)I-labelled GnRH-degrading enzymes of term placental cytosol were studied using a dextran-coated charcoal (DCC) adsorption assay as a rapid screen for GnRH tracer inactivation. Three different activities were demonstrable: (i) a cathepsin D-like enzyme (M(r) 55 kDa), active against all radiolabelled GnRH isoforms and agonists tested, optimal at acid pH, and inhibited specifically by pepstatin; (ii) a metallo-thiol endopeptidase activity (M(r) 70 kDa) optimal at alkaline pH (7-9) which degraded GnRH isoforms to a greater extent than GnRH analogues, inhibited dose-dependently by low concentrations of thiol reagents (N-ethylmaleimide, thimerosal), chelating agents (o-phenanthroline, EDTA), and by tosyl-phenylalanyl-chloromethyl ketone but not by other serine protease inhibitors; and (iii) a bacitracin-sensitive enzyme optimal at physiological pH. These observations permitted the development of a robust radioreceptor assay which minimized GnRH tracer degradation. Under these assay conditions, the GnRH-like radioreceptor assay activity of human placental cytosol fractions was markedly reduced.

Analytical performance and clinical usefulness of two binding assays for growth hormone binding protein (GHBP) measurement: high performance liquid chromatography (HPLC)-gel filtration and dextran-coated charcoal adsorption

We compared two binding assays for growth hormone binding protein (GHBP) measurements, which differ in the method of bound and free GH separation: HPLC-gel filtration or dextran coated-charcoal adsorption (DCC). Two pools of sera (high and medium GHBP activity) were used for quality-control assessment. Moreover, 62 samples from 34 children and 28 adults with different nutritional status were studied. Total, between- and intra-iodination coefficients of variation (CVs) from the two methods were not different. Although percentage binding measured in the pool sera significantly differed, the concentrations assessed by Scatchard plot were comparable. Results obtained by the two methods in the 62 sera were significantly correlated ( r=0.77, P<0.001). With both methods GHBP activity correlated with chronological age and body mass index (BMI) and differed among groups with different nutritional status. Although HPLC and DCC separation methods for GHBP measurement differ in their practicability, our study demonstrates that performance and the clinical usefulness of the two methods are comparable.

Physicochemical Parameters Affecting the Charcoal Adsorption Assay for Quantitative Retinoid-Binding Measurement

The different parameters affecting the accuracy and reliability of the dextran-coated charcoal adsorption assay for characterization of retinoic acid receptors ligand binding activity were investigated. Using dextran-coated charcoal (DCC) at a final 10 mg/ml concentration, an efficient adsorption of free [3H]retinoic acid was observed with a yield in the range 99.2 to 99.8% for ligand concentrations varying from 10−9 to 10−4 M. Nonspecific adsorption of retinoic acid reached 50% to polystyrene and silanized glass and 70% to uncoated glass. Results obtained by the DCC method and by gel-filtration assay were correlated; however, the DCC assay appeared easier to perform and gave more reproducible results. When a careful measurement of free retinoid concentration was performed, the apparent equilibrium dissociation constant (KD) of retinoic acid was 3.1 ± 0.4 nM and the KD of CD367, a synthetic retinoid, was 1.8 ± 0.3 nM. Optimal pH for the binding of [3H]retinoic acid or [3H]CD367 was in the range 7.5 to 8.5. Under the conditions described for the adsorption assay, bound retinoid measurement was linearly related to the protein concentration between 0.05 and 0.25 mg/ml. At a lower protein concentration, addition of bovine serum albumin exerted a stabilizing effect on retinoid binding, allowing an accurate measurement of the number of specific binding sites. Using retinoic acid as ligand, bacterial extracts often resulted in a level of nonspecific binding in the range 10-25%. It could be lowered (4-10%) when resorting to [3H]CD367. The extent of nonspecific binding could be significantly reduced when organic solvents (dimethylformamide or dimethyl sulfoxide) were added at a 3-6% final concentration during the adsorption step with charcoal, whereas gelatin was unefficient. The DCC adsorption assay used under optimized conditions provides a rapid and useful method for an accurate and quantitative measurement of retinoic acid receptors.

Characterization of estrogen receptor in human gastric cancer

Estrogen receptors (ER) were examined in cytosol, nuclear potassium chloride (KCl) extractable fraction, and nuclear KCl unextractable fraction by the dextran-coated charcoal adsorption method in various gastric cancer tissue. The overall ER-positive rate in the cytosol and nuclear fraction was 19.2%. The maximum binding site (Bmax) was 36.0 to 175.0 fmol/mg of protein, and the dissociation constant (Kd) was 0.6 to 1.6 X 10(-9) in cytosol fraction. In the nuclear fraction, Bmax was 7.5 fmol/mg of DNA and Kd was 2.3 X 10(-9). Estrogen receptors were characterized in cytosol protein. In cytosol, the estrogen (E2)-ER complex was sedimented at approximately the 5S and 8S regions by 5% to 20% linear sucrose gradient centrifugation. A steroid specificity study of ER showed the presence of an binder in gastric cancer tissue. In conclusion, these results that gastric cancer tissue has E2 binding sites with the same biochemical characteristics as in breast cancer and endometrial cancer strongly suggest the hormonal dependency of gastric cancer.

Oestrogen receptor expression and the effects of oestrogen and tamoxifen on the growth of human ovarian carcinoma cell lines

To assess the role of oestrogen regulation in the growth of ovarian cancer, we examined the effects of an oestrogen, 17 beta-oestradiol, and an anti-oestrogen, tamoxifen, on oestrogen receptor (ER) -positive and -negative human ovarian carcinoma cell lines. As measured by a dextran-coated charcoal adsorption assay, cell lines PEO1, PEO4 and PEO6 possessed moderate concentrations of ER (96-132 fmol mg-1 protein), PEA1 and PEA2 had low values (12-23 fmol mg-1 protein) and PEO14, TO14, PEO23 and PEO16 were ER-negative. Addition of 17 beta-oestradiol (10 nM or 0.1 nM) to the ER +ve cell line, PEO4, increased the growth rate. This oestrogen stimulation could be blocked by 1 microM tamoxifen. In contrast, the growth rate of the ER -ve cell line PEO14 was unaffected by the addition of 17 beta-oestradiol or tamoxifen. Concentrations of tamoxifen in excess of 8 microM were required to produce complete cytostasis in all lines. This concentration of tamoxifen over 72 hours also inhibited 50% colony formation when cells were plated on plastic. These data indicate that some ovarian carcinoma cell lines contain ER and their growth can be sensitive to oestrogen and anti-oestrogen modulation.

Stabilization of soluble active rat liver glucagon receptor

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (<8 h) lost activity. Either inclusion of 1× Hanks' balanced salt solution in the 3 m m Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from ~10 to >80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 °C). Of the Hanks' solution components, either NaCl (137 m m) or CaCl 2 (1.26 m m) was effective in increasing specific binding to ~70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC 50 value of 10–20 n m (slope factor ~1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which cosolubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 °C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 °C, and its size.

Response to endocrine manipulation and oestrogen receptor concentration in large operable primary breast cancer

Forty-three patients with large (greater than or equal to 4 cm) but operable carcinoma of the breast have been treated by endocrine manipulation before definitive local surgery. This has allowed the study of the relationship between response to therapy and pretreatment oestrogen receptor (ER) concentration, as measured by a dextran-coated charcoal adsorption method. Premenopausal patients (17) were treated by surgical (4) or medical (13) oophorectomy. Post-menopausal patients (26) received either tamoxifen (10) or an aromatase inhibitor (16). Response was assessed from statistical analysis of the changes in tumour size. On completion of 12 weeks of endocrine therapy, there was significant regression of tumour size in 18 of the 43 patients. All 18 patients had tumours with ER concentrations of greater than or equal to 20 fmol mg-1 cytosol protein. Conversely all patients except one progressing on treatment had tumours with ER concentrations of less than 20 fmol mg-1 cytosol protein. This relationship applied for both premenopausal and post-menopausal patients. The overall response rate of patients with tumours of ER concentration greater than or equal to 20 fmol mg-1 cytosol protein was 60%.

Metastatic enhancement and growth inhibition by glucocorticoid and its receptors in mouse teratocarcinoma OTT6050 in vivo.

To elucidate the mechanism of glucocorticoid-induced metastases and growth inhibition of mouse teratocarcinoma OTT6050, syngeneic 129/Sv-CP+SlJ mice were subcutaneously (s.c.) injected with the tumor and treated with daily intraperitoneal (i.p.) injections of hydrocortisone in doses of 1.5 to 12.5 mg per mouse. The number of lymph-node and visceral metastases was greatly increased by the treatment with hydrocortisone unrelated to the doses and sexes. Tumor growth (the products of three principal diameters) of lymph-node metastases was dose-dependently inhibited by the treatment. Histologically, lymph-node metastatic tumors contained large masses of well-differentiated tissues compared with the s.c. transplanted tumor of the same size. The glucocorticoid receptors were measured using dextran-coated charcoal adsorption techniques with [3H] hydrocortisone. Glucocorticoid receptor contents were higher in lymph-node metastatic tumor (100 fmole/mg cytoplasmic protein, mean) than in s.c. transplanted tumors (74.5 fmole/mg, mean) respectively treated with 12.5 mg of hydrocortisone. The metastatic growth inhibition by hydrocortisone might be closely related to the receptor-mediated mechanisms and histological differentiation.

Sex steroid receptors in human meningiomas.

Results of experiments on the presence of steroid receptors in a series of 67 meningioma tissues, which were derived from 64 patients are presented. All tissues were studied using dextran-coated charcoal adsorption and Scatchard plot analysis. Progestin receptors were present in a high concentration in more than 80% of the specimens. Very low concentrations of an oestrogen binding agent were found in a limited number of samples. Thus, most meningiomas contain high levels of progestin receptors in the virtual absence of oestrogen receptors. Relations were not observed between progestin receptor-content and the age and sex of the patients, or the location and the histological type of the tumour. The role and action of progestin on meningiomas is still unknown. The presence of progestin receptors, however, suggests that meningioma may be regarded as a true target tissue for progestins.

Increased dihydrotestosterone receptor levels in high-stage renal adenocarcinoma

Primary renal adenocarcinoma tissue, metastatic deposits, and normal kidney parenchyma from 16 patients were assayed for sex hormone receptors by dextran-coated charcoal adsorption and sucrose gradient centrifugation techniques. Dihydrotestosterone receptors (DHTR) were found in all renal carcinomatous tissue (20/20) and in 93% (13/14) autologous normal kidneys analyzed. Testosterone receptors were found in 84% (16/19) of tumors and 93% (14/15) or normal kidneys analyzed. Estrogen receptors in small amounts (ER) were detected in only 5% (1/19) of tumors and in 7% (1/15) of normal kidneys. Progesterone receptors (PR) in low quantities were detected in 30% (6/20) of renal tumors and in 40% (6/15) of normal kidneys. DHTR levels in high-stage tumors (T3, T4) were significantly elevated over levels in autologous normal kidney, whereas in low-stage tumors localized to the kidney (T1 and T2 tumors) DHTR levels were not significantly different from autologous normal kidney. The mean levels of DHTR in high-stage kidney tumors were significantly elevated over levels in low-stage tumors (P less than 0.001). DHTR estimation in renal neoplasms may help in biologic staging of renal adenocarcinoma and could define a group of patients in whom anti-androgen therapy may be worth a trial.

A microassay for the determination of binding parameters of estrogen and androgen receptors employing affinity immobilization on Cibacron blue 3GA-Sepharose 6B

The problems of currently available ligand-binding assays for sex-steroid receptor proteins include the relatively large mass of tissue required, the interference by sex hormone-binding globulin (SHBG), and use in the androgen receptor (AR) assay of the unstable synthetic ligand methyltrienolone. To overcome these difficulties the stabilizing effect of the dye Cibacron blue 3GA on AR and estrogen receptor (ER) proteins, and its ability to bind to these proteins, was utilized in developing an assay system for each receptor that could be applied to small samples. Use of the affinity gel Cibacron blue 3GA-Sepharose 6B (Blue gel) for the immobilization of AR, ER, and the steroid ligands bound to these receptors in the standard two-tier column assay system enabled the use of a 1:100 (original tissue weight:volume) concentration, making possible full (5–7 point) Scatchard analysis on tissue specimens of a mass as low as 15–20 mg. Significant stabilization of AR and ER was observed and association constants for these receptors were of a similar order of magnitude to those obtained either by Sephadex LH-20 gel filtration or the dextran-coated charcoal adsorption technique. Inactivation by dilution was shown to be largely prevented based on results obtained with cytosol concentrations from 1:5 to 1:100 (original tissue weight:volume). Because Blue gel does not bind SHBG, the natural steroid 5α-androstan-17β-ol-3-one (DHT) may be employed as a ligand in the AR assay. An additional advantage of SHBG not being retained by the Blue gel was that simultaneous determination of DHT binding to this protein in the column eluates could be performed. Intraassay and interassay variations were 4.6 and 8.9% for ER and 7.9 and 12.0% for AR, respectively.

Steroid receptors in the pathogenesis of chronic subdural hematoma.

Chronic subdural hematoma (CSDH), located between the dura mater and the arachnoid and usually characterized by a well-vascularized external capsule (HEM), has a higher incidence in male patients with elevated urinary estrogens than in female patients. In an attempt to increase our understanding of the physiopathogenesis of CSDH, total estrogen receptor (ER) was measured in HEM specimens from four male patients by a sodium thiocyanate exchange assay and cytosol progesterone receptor (PRc) in three specimens by a dextran-coated charcoal adsorption assay. Although no nuclear ER could be detected, ERc and PRc were found in all three specimens examined. The presence of ER in a mesenchymal tissue like HEM could suggest that, in addition to inducing vascular changes, estrogens might act directly on HEM through a receptor-mediated mechanism more pronounced in men than in women, whose vascular network is adapted to high estrogen values.

Efficacy of five methods for the bound-free separation of gluco- and mineralocorticoids from type I, II and III receptors found in HEPES- and TRIS-buffered mouse brain cytosol.

We compared the efficacy of G-25 and LH-20 column chromatography, dextran-coated charcoal adsorption, and DEAE-cellulose and glass fiber filter disc assays to separate unbound steroids from three classes of brain cytosolic receptors prepared in HEPES and TRIS buffers and labeled selectively as follows: Type I = [ 3H]aldosterone + unlabeled RU26988, Type II = [ 3H]triamcinolone acetomide and Type III = [ 3H]corticosterone + unlabeled Prorenone and RU26988. Prorenone and RU26988 were added to reduce unwanted [ 3H]steroid binding to Type I and Type II receptors, respectively. In each case total, non-specific and specific binding and free steroid were compared individually. No single assay was found to be best for all three receptor classes, but both buffers and most assays could be used with appropriate correction factors. Variations between the results with different assays suggest fundamental differences between the three classes of adrenosteroid receptors and their ligands.

High affinity binding of [3H]R5020 and [3H]progesterone by putative progesterone receptors in cytosol and nuclear extract of turtle oviduct.

The purpose of this investigation was to identify and characterize putative cytosolic and nuclear forms of progesterone receptor in the female reproductive tract of a turtle, Chrysemys picta. A dextran-coated charcoal adsorption assay and DNA-cellulose affinity chromatography were used as the primary methodologies with [3H]R5020 [3H-labeled 17 alpha-dimethyl-19-norpregna-4,9-diene-3,20-dione) and [3H]progesterone (P4) as the ligands. The receptor was of high affinity (Kd = 4.7 X 10(-10) M for [3H]P4; 2.2 X 10(-10) M for [3H]R5020) and limited capacity (500-6000 fmol/g tissue wet wt). Association was rapid (apparent equilibrium being reached in 30-40 min), as was dissociation (t1/2 = 45 min for [3H]P4 and 180 min for [3H] R5020). The putative receptor demonstrated strict steroid specificity, binding progestins but not estrogens, androgens, or glucocorticoids. Heterogeneity of the cytosolic receptor was demonstrated as two forms eluting off DNA-cellulose columns at 0.2- and 0.3-M salt concentrations. Binding of cytosolic receptor to DNA-cellulose was not increased by preexposure of cytosol to 25 C for 30 min. Some variations in cytosolic, but not nuclear, receptor were associated with different stages of the reproductive cycle and were positively correlated with body weight. Preliminary studies using an explant culture system suggest that the progesterone receptor in turtle oviduct may be maintained by estrogen and translocated from the cytosol to the nucleus by P4. In summary, we have partially characterized a putative P4 receptor in the oviduct of the turtle that is similar to mammalian and avian P4 receptors in specificity, affinity, and other physicochemical properties, supporting the idea that steroid receptor proteins have been highly conserved in vertebrate evolution. However, temperature sensitivity of activation and DNA affinity are different in the turtle and suggest modifications that may be related to physiological adaptation in such a poikilothermic species.

Estrogen receptor in malignant melanoma: Fact or artefact?

A model system is presented to explain how tyrosinase, an enzyme unique to pigmented cells such as normal and malignant melanocytes can oxidize [ 3 H]-estradiol to radiolabeled products which closely resemble the tight binding of [ 3 H]-estradiol to estrogen receptor. In the model system studied, tyrosinase oxidized [2,4,6,7- 3 H]-estradiol to [ 3 H]-water and [ 3 H]-estradiol metabolites, the latter of which formed ring-substituted conjugates with nucleophiles like monothioglycerol and BSA. Radiolabeled estradiol without tritium in the C-2 position (i.e. [6,7- 3 H]-estradiol) failed to liberate [ 3 H]-water when exposed to tyrosinase but, nevertheless, did form ring-substituted [ 3 H]-estradiol adducts with nucleophiles. The [ 3 H]-water and the ring-substituted radiolabeled products possessed several characteristics of a genuine estrogen receptor protein in that they were resistant to dextran-coated charcoal (DCC) adsorption and their enzymatic formation was inhibited with non-steroidal estrogens like diethylstilbestrol. Other natural (estradiol) and synthetic (hydroxytamoxifen) estrogens which contain the phenol grouping also inhibited the enzymatic oxidation of [ 3 H]-estradiol. Although it was difficult to differentiate estrogen receptor from tyrosinase using the conventional DCC assay system, several differences in these two proteins permitted a distinction to be made between them. First, tyrosinase oxidation of [ 3 H]-estradiol was markedly inhibited by sulfhydryl reducing agents (monothioglycerol) that stabilize [ 3 H]-estradiol binding to estrogen receptor. Second, estrogen receptor adsorbed by hydroxylapatite whereas tyrosinase did not, thus permitting the separation of these two proteins prior to incubation with [ 3 H]-estradiol. We conclude that the [ 3 H]-estradiol binding components in melanoma previously reported to be estrogen receptor probably represent instead the radiolabeled products of the tyrosinase-catalyzed oxidation of [ 3 H]-estradiol.

Isolation, characterization, and synthesis of alpha-fetoprotein from neonatal rat brain.

Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was observed to bind estradiol with Ka = 5.8 X 10(8) M -1 and 1.3 X 10(8) M -1 by dextran-coated charcoal adsorption and Sephadex gel filtration techniques, respectively. Newborn rat brain cells linearly incorporated [14C]leucine into immunoprecipitable AFP during 6 h in culture. It is, therefore, concluded that feto-neonatal rat brain contains AFP similar to that present in fetal serum and that it may arise in brain as a result of its in situ synthesis.

Ex vivo binding to glucocorticoid receptors in the thymus of the adrenalectomized rat

The interaction of glucocorticoids with thymic cytosol receptors in the adrenalectomized rat was studied by a method based on their capacity to deplete cytosol receptors when administered in vivo. Three h after a single oral administration of reference steroids at various dose levels, thymus cytosol aliquots were incubated with a saturating concentration of [ 3H]dexamethasome (30 nM) for 24 h at 0–2°C with and without 2 000 nM unlabeled dexamethasone. Bound radioactivity was determined by dextran-coated charcoal adsorption. The depletion of cytosol receptors was dose-dependent for each glucocorticoid, with the following ED 50 (mg/kg): fluocinolone acetonide 0.032, dexamethasone 17.0. A striking correlation (r 0.991) between ex vivo receptor binding and thymolytic effect was observed. When data from in vitro competition studies were compared with those obtained in ex vivo experiments, the latter were correlated more tightly with the biological response.

High-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cell nuclei from rat liver.

The intranuclear binding of radioactive 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver has been studied both in vivo and in vitro. Following the intravenous administration of [1,6-3H]TCDD, a maximum uptake by cell nuclei could be observed at 2 h after injection with a concurrent decrease in the cytosolic uptake. Using linear sucrose density gradient centrifugation, dextran-coated charcoal adsorption assay, DEAE-Sepharose ion-exchange chromatography, competition, enzymatic and saturation studies, a high-affinity binding protein for TCDD in liver cell nuclei could be demonstrated both in vivo and after an exchange in vitro of intravenously administered unlabelled 2,3,7,8- tetrachlorodibenzofuran (TCDBF) for [3H]TCDD. Sucrose density gradient analysis showed a size of 4-5 S for both the cytosolic and nuclear TCDD binding entity. The specific binding of [3H]TCDD to nuclear components was heat labile and saturable and had an equilibrium dissociation constant of 1.05 nM. Based on a differential susceptibility to specific hydrolases, i.e. DNAase, RNAase, trypsin and pronase, the binding entity appears to be a 4-5 S salt-extractable protein.

Spontaneous canine mammary tumors: A model for human endocrine therapy?

Animal models for the study of mammary tumors usually involve the artificial induction of tumors by chemical or viral agents. Canine mammary tumors are, however, spontaneous. A comparison of the incidence, pathology and endocrine properties of dog and human breast tumors is therefore of interest in determining whether canine tumors can constitute a useful model for the study of clinical response to hormone therapy and whether, in recognized cases of hormone-dependence, pet bitches with mammary tumors should undergo endocrine therapy with synthetic hormones following mastectomy. Estrogen (ER) and progestin (PR) receptor concentrations were therefore assayed in mammary tumours from 144 bitches and 2 male dogs by a dextran-coated charcoal adsorption technique using [ 3H]-R 2858 and [ 3H]-R 5020 as radioligands. The incidence of ER and PR was analyzed according to the race, age, case history (number of pregnancies…) of the dogs and according to tumor size and type. About 1 3 rd of the tumors were classified as benign and 2 3 rds as malignant (WHO classification). Of the benign tumors, 32% had no receptor, 56% had both ER (10–124 fmol/mg protein) and PR (10–259 fmol/mg protein), 6% ER only and 6% PR only. Of the malignant tumors, 39% had no receptor, 44% had both ER (10–33 fmol/mg protein) and PR (10–90 fmol/mg protein), 9% ER only and 8% PR only.

DNA measurement by mithramycin fluorescence in chromatin solubilized by heparin

The mithramycin fluorescence procedure described by B. T. Hill and S. Whatley (1975, FEBS Lett., 56, 20–23) for DNA measurement tends to underestimate DNA concentrations in biological samples as compared to the results obtained by the diphenylamine reaction. This discrepancy disappears when DNA is first solubilized, by buffer containing heparin, from either cell homogenates or nuclear preparations. The optimal conditions for maximal fluorescence are 8 m m Mg 2+, 10 μg/ml mithramycin, and heparin to DNA ratios ≥0.15 ( w w ). Background fluorescence is reduced 90% by dextran-coated charcoal adsorption of unbound mithramycin. The limit of sensitivity of the assay is 0.3 μg/ml and fluorescence is linear up to 30 μg DNA/ml.