Charcoal Adsorption Assay 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.

Characterization and properties of steroid binding protein in Bufo arenarum serum.

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

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.

Sex-specific androgen binding in spotted hyaena ( Crocuta crocuta) plasma

1. 1. A charcoal adsorption assay demonstrated a large variance in androgen binding ability in female spotted hyaenas. 2. 2. A positive correlation between plasma androgen binding ability and ovarian steroid concentrations was demonstrated in adult females. 3. 3. The strong plasma binding affinity for testosterone and dihydrotestosterone (DHT) (nM) together with the lack of cortisol and weaker oestradiol-17β binding suggests that a specific androgen binding substance, possibly a protein, is present in adult females of this species. 4. 4. The lack of high affinity binding in male spotted hyaenas is unusual and deserves further investigation. 5. 5. Some androgen binding in all, including males and immature animals suggests that albumin may bind some plasma androgens in this species.

Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Insulin-like growth factor I (IGF-I) stimulates hematopoiesis. We examined whether bone marrow stromal cells synthesize IGF-I. Secretion of IGF-I immunoreactivity by cells from TC-1 murine bone marrow stromal cells was time-dependent and inhibited by cycloheximide. Gel filtration chromatography under denaturing conditions of TC-1 conditioned medium demonstrated two major peaks of apparent IGF-I immunoreactivity with molecular weights of approximately 7.5-8.0 kD, the size of native IGF-I, and greater than 25 kD. Expression of IGF-I mRNA was identified by both RNase protection assay and reverse transcription/polymerase chain reaction. To determine whether the greater than 25-kD species identified by RIA possessed IGF-binding activity, a potential cause of artifactual IGF-I immunoreactivity, charcoal adsorption assay of these gel filtration fractions was performed. The peak of IGF-binding activity coeluted with apparent IGF-I immunoreactivity suggesting that TC-1 cells secrete IGF-binding protein(s). Unfractionated conditioned medium exhibited linear dose-dependent increase in specific binding of [125I]-IGF-I with a pattern of displacement (IGF-I and IGF-II much greater than insulin) characteristic of IGF-binding proteins. Western ligand analysis of conditioned medium showed three IGF-I binding species of approximately 31, 38, and 40 kD. These data indicate that TC-1 bone marrow stromal cells synthesize and secrete IGF-I and IGF-binding proteins and constitute a useful model system to study their regulation and role in hematopoiesis.

Secretion of insulin-like growth factor I and its binding proteins by collecting duct cells

Insulin-like growth factor I (IGF-I) has been found in the kidney, particularly in the collecting duct in the rat. Since cultured rabbit collecting duct cells constitute a convenient system for in vitro studies, we have examined whether these cells secrete IGF-I. Culture medium conditioned by collecting duct cells was concentrated by reverse phase chromatography and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent molecular weights of 7.5 and greater than 25 kilodaltons (kD) were identified by IGF-I RIA. A smaller amount of 10 kD species was also observed. Further characterization of 7.5 kD IGF-I immunoreactive species by reverse phase HPLC showed that it eluted in a single peak. To determine whether the higher molecular weight species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I (thr59) for two hours at 30 degrees C and applied to a Sephadex G100 column equilibrated in a non-dissociating buffer. The major peak of radioactivity was confined to a high molecular weight region; there was no radioactivity in the fractions corresponding to 7.5 kD. Western ligand analysis of unreduced conditioned medium identified two IGF-I binding species of 25 and 30 kD, similar in size to species observed in normal rabbit serum. 125I-IGF-I binding as assessed in a charcoal adsorption assay could be displaced by IGF-I and IGF-II but not by insulin. Further characterization of the 10 kD peak of IGF-I immunoreactivity indicated that it did not possess IGF-I binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Autocrine Regulation of Human Tumor Cell Proliferation by Insulin-Like Growth Factor II: anin-VitroModel*

We have shown that a pleomorphic cell line of abnormal human karyotype derived from a stomach carcinoma (LIM-1839) proliferates in serum-free medium, expresses insulin-like growth factor II (IGF-II) mRNA, and secretes IGF-II (up to 56 ng/ml in serum-free conditioned medium, as measured in a rat liver RRA. No detectable levels of IGF-I can be measured in serum-free conditioned medium by RIA. These cells also secrete IGF-binding proteins, detected by a charcoal adsorption assay. The release of IGF-II and IGF binding proteins into serum-free conditioned medium (1.7 pmol/10(6) cells.24 h and 0.8 pmol binding sites/10(6) cells.24 h for 3 days, respectively) is inhibited 80% by cycloheximide (10 micrograms/ml). The LIM-1839 cells have type I and type II IGF receptors, determined by affinity cross-linking and competition binding studies. These cells proliferated 1.6-fold over 4 days in serum-free medium, with fresh medium changes on days 0 and 2: their growth was inhibited 56% by 40 micrograms/ml Sm 1.2, a monoclonal antibody which recognizes IGF-I and IGF-II. The addition of 20 and 50 ng/ml multiplication stimulating activity (rat IGF-II) caused 1.8- and 1.7-fold increases in cell growth between days 0 and 4 compared to controls, while [Thr59]IGF-I, at 20 and 50 ng/ml, caused 1.6- and 2.0-fold increases. Insulin, at 2 and 10 micrograms/ml, had no significant effect. The stimulatory effects of endogenous and exogenous IGFs on LIM-1839 cell proliferation were inhibited by a monoclonal antibody to the type I IGF receptor, alpha IR-3. These results suggest that the LIM-1839 cells are biologically responsive to endogenously produced IGF-II, and may thereby provide an in vitro model for autocrine regulation of human tumor growth by IGF-II.

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.

GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases.

The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.

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.

Effects of thiocyanate on cytosol androgen receptor from shionogi carcinoma 115

The effects of KSCN and other chaotropic salts on the androgen receptor in cytosol of Shionogi carcinoma 115 were studied by means of charcoal adsorption assay and sucrose gradient centrifugation. When KSCN or NaSCN was added to the [ 3H]-dihydrotestosterone-cytosol mixture at the final concentration of 0.5 M, the androgen binding to the cytosol receptor was considerably inhibited. The inhibition reached maximum within 5 h at 0°C and was dependent on the kind of chaotropic anion added: the potency of inhibitory effect in descending order was KSCN > KI > KBr > KCl. The inhibition was not observed in the estradiol-receptor interaction with KSCN or NaSCN up to 0.5 M. When 0.5 M KSCN-treated androgen-cytosol mixture was subjected to gel nitration or (NH 4) 2 SO 4 fractionation to remove the salt, a partial recovery (30–40%) in specific binding activity was observed. The binding activity of androgen receptor was unaffected by a treatment with KSCN up to 0.1 M and the androgen-receptor complex sedimented in a 5S form in 0.1–0.3 M KSCN, 0.5 M KCl or 0.5 M KBr. These results suggest that the binding activity of androgen receptor is more susceptible than that of estrogen receptor to chaotropic salts which cause impairment in intramolecular hydrophobic interactions.

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.

Binding kinetics and physical properties of androgen receptor in androgen-dependent Shionogi mammary carcinoma 115.

The characteristics of the androgen receptor in the cytoplasmic fraction of Shionogi carcinoma 115 were studied in vitro by means of charcoal adsorption assay, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The equilibrium dissociation constant for [3H]dihydrotestosterone (Kd = (2-5) X 10(-11) M) was estimated from independently determined rates of association and dissociation, and was lower by one order of magnitude than the value obtained by saturation analysis (Kd = (2-8) X 10(-10) M). Evaluation of the effect of temperature on receptor binding of androgen allowed the estimation of several thermodynamic parameters, including activation energies of association (4 kcal/mol) and dissociation (14 kcal/mol), the apparent free energy (-13 kcal/mol), enthalpy (-9 kcal/mol), and entropy (+14 cal/mol per K). The receptor was greatly stabilized when bound with androgen. The results indicate how the lability of the unbound receptor and slow rate of dihydrotestosterone-receptor interaction can influence the estimation of dissociation constants by usual saturation analysis. The sedimentation coefficient of androgen receptor in freshly prepared cytosol was 6S, and became 7S after storage for 2 months at -80 degrees C. The 7S conversion of the receptor was reversed by treatment with heparin. In all cases, a single 5S peak was obtained in the presence of 0.5 M KCl. On electrophoresis in heparin-containing polyacrylamide gel, protein-bound radioactive androgen migrated as a single peak (Rf = 0.5 in 5% gel). Differences in reported values for the sedimentation coefficient of androgen receptor in cytosol of Shionogi carcinoma 115 appear to be derived from aggregation of the receptor protein during the assay procedure.

A sex-steroid-binding protein in the plasma of the freshwater turtle, Chrysemys picta

A sex-steroid-binding protein has been described in the plasma of the freshwater turtle, Chrysemys picta. Binding data for estradiol-17β obtained using a charcoal adsorption assay were analyzed according to Scatchard and by direct linear plot, and indicated a single class of binding sites, an apparent K a of 2.42 ± 0.27 × 10 8 M −1, and a mean binding capacity of 6.01 ± 0.30 × 10 −12 mol/mg. Binding specificity was determined by competitive binding experiments in which turtle plasma was incubated with 1 × 10 −8 M [ 3H]estradiol-17β plus unlabeled competitor steroid. Both testosterone and progesterone displace estradiol-17β, but estrone, estriol, and diethylstilbestrol competed poorly.

Testosterone binding and free plasma androgen concentrations under physiological conditons: chararacterization by flow dialysis technique.

Testosterone binding to plasma has been analyzed under physiological conditions. Flow dialysis technique (FDT) has permitted precise studies of whole 37 C plasma with negligible disturbance of the equilibrium state. The information obtained by FDT was compared with that obtained by a charcoal adsorption assay (CA), which primarily assesses testosterone binding totestosterone-estradiol-binding globulin (TEBG). The results of the two methods correlated closely(r = 0.98). Consequently, the percentage of testosterone free in plasma under in vivo conditions could be readily calculated from data generated by the much easier CA technique. Plasma free androgenconcentrations were then computed for a large variety of sera. For example, the mean (±SDplasma free testosterone concentrations of normal men, hirsute women, normal women, and pregnant women were found to be 128 ± 57, 16.1 ± 11.7, 5.4 ± 2.7, and 4.6 ± 1.7 pg⁄ml, respectively. Theselevels are in close agreement with previous estimates. The plasma free testos...

Juvenile hormone binding proteins in the haemolymph of third instar larvae of Drosophila hydei

Juvenile hormone I (JH I) is bound in vitro by two protein fractions in the haemolymph of mid-third instar larvae of Drosophila hydei. These binding proteins were characterized by gel filtration, density gradient centrifugation, isoelectric focusing, polyacrylamide gel electrophoresis and charcoal adsorption assay. The larger binding protein has a molecular weight of 1.08 × 10 5 , a sedimentation coefficient of approximately 7 S and an isoelectric point at pH 5.1. The equilibrium dissociation constant for JH I is about > 10 −5 M. This protein binds JH I after storage for five days at 0 C, but binding capacity is destroyed by heating to 60°C for 10 min. Proteolytic peptides of this binding protein still possess the capacity to bind JH. The smaller JH-binding protein has a molecular weight of 4.4 × 10 4 , a sedimentation coefficient in the range of 3–4 S, an isoelectric point at approximately pH 8.9 and an equilibrium dissociation constant of approximately 1 × 10 −7 M. Its binding capacity is rapidly lost in total haemolymph and after partial purification at 4°C.

Effect of ionic strength on charcoal adsorption assays of receptor-estradiol complexes.

The effect of increasing ionic strength on the efficacy of the charcoal adsorption assay for estrogen receptors has been examined. Cytosol prepared from immature rat uteri was exposed to variable concentrations of KCl and the saturable or "specific" binding of [3H]estradiol was measured by charcoal and hydroxylapatite adsorption, gel filtration, and/or density gradient centrifugation using excess diethylstilbestrol to correct for non-saturable or "non-specific" binding. As the concentration of KCl was increased, the number of estradiol binding sites as measured via charcoal adsorption decreased in proportion to the conversion of receptor from a low salt (8S) to a high salt (4S) complex. This "stripping" of [3H]estradiol from the 4S receptor species was both time- and charcoal concentration-dependent. It is concluded that the charcoal adsorption procedure quantitatively assesses the binding of [3H]estradiol to low salt, 8S receptor species but is not the assay of choice for salt-extracted or salt-treated receptors in view of the potential for artifactually low estimates of receptor number. Low values for receptor number under high salt conditions may result from exposure of the binding site of the estrogen receptor to charcoal.

Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate.

Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of [3H]-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for [3H] dihydrotestosterone being somewhat higher than for [3H] testosterone. Equilibrium dissociation constants for [3H] dihydrotestosterone and [3H] testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for [3H] dihydrotestosterone and 2 X 10(8) M-1 h-1 for [3H] testosterone) and dissociation (t 1/2 congruent to 40 hr for [3H] dihydrotestosterone and 15 h [3H] testosterone). Evaluation of the effect of temperature on androgen receptor binding of [3H]testosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of [3H] testosterone or [3H] dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S. Differences in sedimentation coefficients were observed following (NH4)2SO4 precipitation which did not influence the binding properties of the receptors. These results, together with measurements of3alpha/beta-hydroxysteroid oxidoreductase activity in vitro, suggest that organ differences in receptor binding of [3H] dihydrotestosterone and [3H] testosterone in vivo result from relative differences in intracellular concentrations of these androgens rather than from differences in receptor affinities.

Charcoal adsorption assay for measurement of colchicine binding and tubulin content of crude tissue extracts

A rapid, reproducible, and quantitative colchicine-binding assay for tubulin content of crude tissue extracts is described and applied to the high speed supernatant fraction of rat liver homogenates. Utilizing Scatchard plots, the colchicine-tubulin association constant is found to be in general agreement with values reported for purified tubulin from other sources.