Addition of a rat CBG preparation to a progesterone solution abolished the effect of the hormone on the ligated uterus of the ovariectomized mouse. Inactivation was also observed when rat serum albumin was used as the progesterone-binding protein. These results seem to show that the biological activity of progesterone is suppressed by complex formation with CBG or with serum albumin. (Endocrinology85: 778, 1969)
This chapter discusses oestradiol-binding and protein phosphorylation. Partly purified oestradiol binding protein prepared by sodium chloride extraction of nuclei and hydroxylapatite chromatography formed insoluble complexes with polycations, such as histone and protamine. The polycations precipitated the cytoplasmic 8 and 4S receptors. The interaction between the acidic nuclear proteins and polycation is ionic and this raised the possibility that oestradiol might exert its effect on chromatin by altering the ionic interaction between histone and acidic protein. This could be achieved either by methylation or acetylation of lysine amino groups in histone or phosphorylation of serine and threonine hydroxyl groups in either histone or acidic protein. No histone acetylase or methylase activity was detected in a crude nuclear oestradiol-binding fraction from uterus or 7,12 dimethyl-benzanthracene (DMBA)-induced mammary tumour but an active protein phospholcinase was present. Histone was a poor phosphate acceptor.
This chapter discusses the antibodies to oestrogens. It was demonstrated that the attachment of small molecules to protein might lead to the formation of immunogenic macromolecules, capable of eliciting the production of antibodies whose specificity is directed towards the small molecule or “hapten”. Production of specific antibodies to oestrogens helps in the development of radioimmunoassay. The phage inactivation assay procedures were modified; for establishing the role of endogenous oestrogen in ovum implantation in the rat; and for possible medical and veterinary applications. It has been possible to render oestrogens antigenic by coupling them to synthetic macromolecules or protein at sites remote from the hydroxyl functions or the phenolic ring of the hormone molecule, and to devise specific immunoadsorbants to isolate the oestrogen-directed immunoglobulins so produced. These should be useful for the determination of oestrogens by immunoassay. Isoflavone-induced antibodies do not cross-react with oestradiol.
The cyclic variations in the rat mammary gland seem to be of no significance for the induction of breast cancer by dimethylbenzanthracene (DMBA) nor for the development of hormone sensitivity in these tumours. The uptake studies give no indication of any competition between oestradiol and DMBA with regard to intracellular binding. The occasional occurrence of one hormone‐sensitive and one hormone‐resistant cancer in the same animal points to the significance of local factors for the development of hormone‐sensitivity in breast cancers.
This chapter presents the study of C19O2 steroids in human plasma by competitive protein binding. The steroid binding β-globulin is neither specific for testosterone nor for estradiol and binds a number of other steroids, particularly those possessing a 17β-hydroxyl group. In the present study, the presence of some of these steroids in plasma extracts of normal men and women were studied by means of a competitive protein binding technique. It has been shown that the plasma extracts of normal men and women contain significant amounts of neutral steroids, different from testosterone, which produce a displacing effect in the binding assay. The measurement of testosterone by competitive protein binding to the steroid binding β-globulin should involve a chromatographic purification step.
This chapter discusses the binding of steroids by tissue proteins steroid hormone "receptors". Estrogens are among the biologically active substances that act at the cellular level in low concentrations. They have been compared earlier with cardiac glycosides for which pharmacologists proposed a sort of hit and run mechanism of action, because the minute amounts present could not be detected with the techniques available. With the advent of radioactive compounds of high specific activity, this concept had to be revised. The experiments of Jensen and Jacobson extended and confirmed by numerous laboratories, leave no doubt that estradiol is selectively accumulated and retained by the target cells.
Journal Article Solid-Phase Radioimmunoassay of Estradiol-17β Get access GUY E. ABRAHAM GUY E. ABRAHAM 1Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545 Search for other works by this author on: Oxford Academic Google Scholar The Journal of Clinical Endocrinology & Metabolism, Volume 29, Issue 6, 1 June 1969, Pages 866–870, https://doi.org/10.1210/jcem-29-6-866 Published: 01 June 1969 Article history Received: 16 December 1968 Published: 01 June 1969
Oestrogen or androgen administration results in a variety of alterations in plasma protein concentrations and their physico-chemical properties. In normal subjects, short- and long-term oestrogen therapy produces an increase in corticosteroid-binding globulin (CBG) or transcortin concentration. The transcortin changes resulting from androgen treatment have not been elucidated; however the data available, at present, indicates that the CBG concentration tends to decrease. Three anabolic steroids have been used in studies: the 17α-alkyl derivatives of testosterone (methyl-testosterone) and of 1-dehydrotestosterone and the 17β-cyclopent-1-enyl ether of 1-dehydrotestosterone. The oral activity is due in the first two compounds to a methyl-group in 17α-position, while in the latter to a 17β-enol-ether linkage. The effects of orally active anabolic steroids on cortisol-binding capacity of plasma transcortin, on plasma 11-hydroxycorticosteroids and on non-protein-bound cortisol are different from those of testosterone. The 17α-alkyl derivatives—methyl-testosterone and methandrostenolone—have no effect, while the action of quinbolone resembles that of natural and synthetic oestrogens.
This chapter discusses the interaction of diethylstilbestrol with uterine receptor sites. Progress towards the understanding of the action of oestrogens has been made at the molecular level since it was demonstrated that hormone responsive tissues, such as uterus, vagina, and anterior pituitary contain specific oestradiol binding substances called "oestrogen receptors". While knowledge is becoming available on the interaction of oestradiol with uterine receptor sites, not much is known about the influence of stereochemistry and the structural requirements for the steroid in this interaction. Oestradiol-17β is biologically a potent oestrogen, while its corresponding 17α isomer. However, some of the non-steroidal stilbene compounds represented by diethylstilbestrol and hexestrol possess marked oestrogenic activity. In biological property, diethylstilbestrol behaves like oestradiol-17β except that in the spayed rat uterus, diethylstilbestrol or hexestrol are not effective in priming the uterus for deciduoma formation, whereas natural oestrogen is effective.
This chapter presents a method for the estimation of plasma testosterone using a technique depending on competitive protein binding. The application of the technique of competitive protein binding analysis to measure the hormone testosterone necessitates the study of many parameters. On the basis of such an investigation, a method has been proposed. The parameters that were studied, included: (1) preparation of the specific binding protein (SBP) for testosterone, (2) establishment of the dilution curve, (3) storage of SBP, (4) search for convenient SBP for testosterone in species other than man, (5) mode of separation of protein bound testosterone from unbound hormone, (6) establishment of standard curve for testosterone, (7) affinities of SBP with various C-18, C-19 and C-21 steroids, and (8) effects of various chromatographic materials and solvents on the blank values.
Publisher Summary Specific steroid-binding plasma proteins, as well as specific steroid-binding cellular proteins, can be used to measure unknown amounts of steroids by saturation analysis in the way as specific antibodies are used to quantitate antigenic substances in radioimmunoassay. The determination of steroids by the competitive binding of their labeled and unlabeled forms to plasma proteins called the principle of the assay competitive protein binding. The ideal assay system consists of a diluted solution of the binding protein and of a known amount of the labeled form of the steroid to be quantitated or of another labeled steroid binding to the protein. The binding protein should have a high association constant for these steroids and should also be specific. It should bind only a limited number of related compounds. When increasing amounts of the non-labeled steroid are added to this solution, they occupy a part of the binding sites of the protein and the fraction of the radioactive steroid bound to the specific protein diminishes. The relation between the bound fraction and the total concentration of the steroid is not linear, but can be used to quantitate unknown amounts of the latter.
Publisher Summary This chapter provides an account of a 4S oestradiol-binding protein in uteri from mature rats that is controlled by endogenous hormones. Most experiments on the binding of oestradiol have used tissues taken from immature or long term ovariectomised animals and the relevance of such studies to the situation in mature, unovariectomised animals is not certain. The observation that protamine precipitated protein-bound oestradiol from the 4S as well as the 8S region of sucrose gradients carried out on cytoplasm derived from mature uteri prompted a study of estradiol-binding in uteri from mature animals. Analysis of the binding data obtained by the protamine method using the Scatchard plot indicated that cytoplasm from mature uteri contained one tight-binding component. This suggested that the 8S and 4S receptors detected by gradient analysis might be related to each other. If this was true, any factor influencing the 8S receptor would also affect the 4S one.
Clomiphene citrate (Clomid) is chemically related to the non-steroid oestrogen chlorotrianisene (TACE). This compound has a weak oestrogenic activity, but it is considered as an anti-oestrogen because it inhibits oestrogen action. Some results have been reported, establishing a new effect of clomiphene: modifications of the binding affinities of testosterone, oestradiol, and Cortisol in the serum of the normal adult male. Clomiphene acts at the level of the hypothalamic and/or pituitary receptors by a mechanism of competition with oestradiol at the cellular- binding sites. Luteinizing hormone (LH) is released and gives rise to an increased oestrogen and testosterone production. The effect observed on the binding index cannot be explained by the elevation of LH, because the administration of human chorionic gonadotropin (HCG) to man reduces the binding affinity of testosterone. As a non-steroid synthetic compound with poor oestrogenic activity, clomiphene could act in the same manner as oestradiol on the sexual hormones and cortisol binding proteins.
A cortisol-binding protein was found in extracts of lymphosarcoma Ρ 1798 in the BALB/C mouse. This tumor is grown subcutaneously in the mice, is transplantable, and occurs in two varieties: corticoid-sensitive, which regresses after three daily injections of Cortisol or 9α-fluoroprednisolone; and corticoid-resistant, which either remains the same size or continues to grow under the same treatment. This chapter reviews some of the properties of the cortisol-binding protein of the lymphosarcoma. It is shown that the extracts obtained from certain lymphoid tissues of the mouse and rabbit have similar properties. These properties differ from those of serum corticosteroid-binding protein (CBG). The presence of a cortisol-binding protein was demonstrated in the high-speed supernatant fraction of mouse P 1798 lymphosarcoma. Corticosteroid-binding protein from serum has a higher dissociation constant with increase in temperature.
The existence of a testosterone binding plasma protein (β—globulin) has been shown. It was confirmed by the observation that the testosterone binding affinity was increased during pregnancy. The inactivation of testosterone by this protein has been proposed as an explanation for the anomalies of testosterone metabolism observed in thyrotoxicosis and hirsutism. To test this biological role in vitro, a testosterone binding fraction was purified and the behavior of testosterone—free or bound to this fraction—was compared when submitted to aromatization by placental enzymes. The inhibiting effect of the protein fraction and of albumin at physiological concentrations on the aromatization of testosterone has been demonstrated in vitro, but from these in vitro results, definitive conclusions about the respective roles of the two proteins in vivo cannot be drawn.
