The kinetics of estrogen binding to rat α-fetoprotein

Summary Fluid obtained from rat fetuses was utilized to characterize the affinity, number of binding sites, and the association and dissociation rate kinetics of the binding of estradiol and estrone to AFP. Statistical analysis demonstrated no differences when the values for the AFP-estradiol interaction were compared with those obtained for the AFP-estrone interaction. These data demonstrate that rat AFP specifically binds estradiol and estrone with a high capacity, high affinity, and similar binding kinetics.This work was supported by NSF grant PCM-8109847 and by a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society.     

Sertoli cells of adult rats in vitro. II. Effect of different steroid precursors on estradiol 17 beta-synthesis.

The estradiol 17 beta-synthesis by delta 4 pathway has been studied in homogenous cultures of Sertoli cells isolated from adult rat testes. The data reported clearly demonstrate that progesterone, androstenedione, testosterone and estrone induce an increase of the estradiol 17 beta-production.     

Binding of estradiol to synaptosomal mitochondria: physiological significance

The subsynaptosomal distribution and specific binding of 17beta-estradiol in vitro to mitochondria isolated from presynaptic nerve endings of female rat brain were examined. 17Beta-estradiol is (i) distributed unequally in synaptosomes and mitochondria posses the highest capacity to bind estradiol with respect to the available amount of the hormone. (ii) Estradiol binds specifically to isolated synaptosomal mitochondria. A Michaelis-Menten plot of specific binding was sigmoidal within a concentration range of 0.1-5 nM of added estradiol, with a saturation plateau at 3 nM. Binding of higher estradiol concentrations demonstrated an exponential Michaelis-Menten plot, indicating non-specific binding to mitochondria. Vmax and Km for the sigmoidal-shape range were estimated as 46 +/- 6 fmol of estradiol/mg of mitochondrial proteins and 0.46 +/- 0.07 nM free estradiol respectively. (iii) Estradiol binding is not affected by the removal of ovaries. The results show that inhibition of Na-dependent Ca2+ efflux from mitochondria by estradiol occurs according to an affinity change of the translocator for Na+, at the same estradiol concentrations that show specific binding to mitochondrial membranes. These data imply that physiological concentrations of estradiol, acting on mitochondrial membrane properties, extragenomically modulate the mitochondrial, and consequently the synaptosomal content of Ca2+, and in that way exert a significant change in nerve cell homeostasis.     

Noninvolvement of a rat intestinal folate binding protein in physiological folate absorption

Summary Folic acid and its derivatives are observed to be instantaneously bound to rat intestinal mucosal cell curface binding protein. Various parameters were measured such as the optimal pH for binding (6.5), the saturation kinetics observed for folate binding and the avid affinity with which polyglutamyl folates are preferentially bound to the binder. A comparison of the observed results with the conditions reported as favorable for intestinal folate absorption in the rat precludes the involvement of this folate-binding protein in the physiological absorption of folates.     

Noninvolvement of a rat intestinal folate binding protein in physiological folate absorption

Folic acid and its derivatives are observed to be instantaneously bound to rat intestinal mucosal cell curface binding protein. Various parameters were measured such as the optimal pH for binding (6.5), the saturation kinetics observed for folate binding and the avid affinity with which polyglutamyl folates are preferentially bound to the binder. A comparison of the observed results with the conditions reported as favorable for intestinal folate absorption in the rat precludes the involvement of this folate-binding protein in the physiological absorption of folates.     

The role of sialic acid in 5-HT binding to synaptic membranes.

The high affinity binding of [14C]5-HT to nerve ending membranes isolated from rat brain is not affected by neuraminidase treatment. The specificity of ligand receptor interaction was demonstrated by displacement studies with tryptamine derivatives, noradrenaline, and acetylcholine.     

The role of sialic acid in 5-HT binding to synaptic membranes

Summary The high affinity binding of [¹⁴C]5-HT to nerve ending membranes isolated from rat brain is not affected by neuraminidase treatment. The specificity of ligand receptor interaction was demonstrated by displacement studies with tryptamine derivatives, noradrenaline, and acetylcholine.The expert technical assistance of Miss Christiane von Winterfeld is appreciated. This work was supported by a grant of the Deutsche Forschungsgemeinschaft.     

Demonstration of high affinity binding of estradiol in adrenal cortex tissue]

Evidence is presented demonstrating the presence of a high affinity (Ka10(8)M-1), limited capacity (3-4 pmoles/mg protein) estradiol binder in the soluble fraction of the Bovine, Rat and Human adrenal cortex. The binding appears specific to the estrogen structure whereas C19 and C21 steroids do not bind. Upon sucrose density gradient centrifugation, the estradiol binder sedimented at 9 S at low ionic strength and was shifted to 4.5 S in the presence of 0.5 M KCl. This demonstration of a receptor-like moiety for estradiol in the adrenal cortex lends biochemical support to previous observations suggesting that adrenal cortex functions may be modulated by a direct effect of gonadal steroid hormones.     

Regulation of estrogen "receptors" in mammary tumors: action of prolactin in vivo

The effect of prolactin on estradiol receptors was determined in cytosol of estrogen-dependent mammary tumors of Sprague Dawley rats. Tumors were induced with 20 mg dimethylbenzanthracene, rats were ovariectomized, and the rats showing regression in tumor weight were given 1 mg sheep or rat prolactin for 5 days. Receptors were separated by dextran-charcoal and analyzed by sucrose gradient. Prolactin, milk proteins, serum proteins, and 1-alpha-feto-proteins were excluded immunologically. The estradiol receptors resembled uterine receptors in affinity, specificity, sedimentation, and nuclear transfer under estradiol influence. Castration diminished receptor content exponentially 170 f moles to 16 f moles/mg protein within 10 days; prolactin increased the number of receptors to 87 f moles compared with 15 in controls. Affinity, cellular location, and uterine receptor content were unaffected. Sheep and rat prolactin acted similarly. These results justify hypophysectomy as therapy for certain estrogen-dependent breast tumors.     

Microtubule targeting agents: from biophysics to proteomics

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug–tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule–tubulin equilibrium and tubulin–drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton.     

Effect of lipophilic cations on thiamine transport system in isolated rat hepatocytes

The effect of lipophilic cations such as triphenylmethylphosphonium, tetraphenylphosphonium and tetraphenylarsonium in addition to dibenzyldimethylammonium on thiamine transport in isolated rat hepatocytes was studied. Lipophilic cations at the concentration 10 microM almost completely inhibited thiamine uptake. Kinetic studies showed that these compounds were competitive inhibitors with a very high affinity. These results suggest that lipophilic cations in addition to quaternary ammonium compounds also share a common binding site for thiamine in isolated rat hepatocytes.     

Specific in vivo binding of d-LSD in rat brain

A reproducible in vivo d-LSD binding method in rat brain is described, with high affinity (Kd of 5 pmoles/g wet wt), stereospecificity (d- vs. 1-LSD) and regional selectivity. It may be a useful adjunct to in vitro methods for measuring changes in turnover at the synaptic level related to the intact receptor.     

Study by a rapid mixing method of the interaction between a fluorescent cholinergic agonist and membrane fragments rich in cholinergic receptors from Torpedo marmorata]

The analysis by stopped-flow of the interaction of fluorescent agonist (C5DACho1) with the acetylcholine receptor in its membrane-bound form reveals several kinetic steps: a fast one, in the millisecond range, associated with the binding of C5DACho1 to a high affinity state and a "medium" and "slow" one, the last one representing possibly an isomerisation of the receptor molecule towards the high affinity state.     

Sertoli cells of adult rats in vitro. II. Effect of different steroid precursors on estradiol 17β-synthesis

Summary The estradiol 17-synthesis by ⁴ pathway has been studied in homogenous cultures of Sertoli cells isolated from adult rat testes. The data reported clearly demonstrate that progesterone, androstenedione, testosterone and estrone induce an increase of the estradiol 17-production.This work was partially supported by grants No. 77.01322.04 and No. 77.01958.04 from the Consiglio Nazionale delle Ricerche.The excellent technical assistance of Antonio Muller and Massimo Rosati is deeply appreciated.     

Specific in vivo binding of d-LSD in rat brain

Summary A reproducible in vivo d-LSD binding method in rat brain is described, with high affinity (K_d of 5 pmoles/g wet wt), stereospecificity (d-vs, vs. l-LSD) and regional selectivity. It may be a useful adjunct to in vitro methods for measuring changes in turnover at the synaptic level related to the intact receptor.     

High affinity binding of 5-hydroxytryptamine to some membrane preparations from cattle brain. Relationship to lysergic acid diethylamide (LSD)]

5 hydroxytryptamine binds to crude brain membrane preparations with two different affinities (KD = 1 to 2 X 10(-9) M for the highest, 1 to 2 X 10(-8) M for the lowest). LSD also binds with two affinities (KD = 3 to 4 X 10(-9) M and KD = 2 to 3 X 10(-8) M). Subcellular distribution of these sites shows that binding involves the two binding affinities in microsomal membranes but solely the high affinity binding sites are present in purified synaptosomal membranes. High affinity sites for 5 HT and for LSD are different as no direct competitive inhibition is observed in that case. On microsomal membranes, direct relationship occurs between low affinity binding for 5 HT and high affinity binding for LSD.     

Steroid hormones enhanced sister-chromatid exchange in cultured CHO cells

The influence of steroid hormones on the induction of sister-chromatid exchange (SCE) in cultured CHO cells was studied. It was observed that estradiol-17 beta, estriol, estrone and ethynyl estradiol treatments enhanced SCE rates compared to the controls. Overall, these compounds produced a dose response effect. The importance of a detailed study on the long-term genetic effects of steroids on mammalian cells is emphasized.     

Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.     

Luteolytic potency of 16-phenoxy-derivatives of prostaglandin F2 alpha

The binding of 16-phenoxy derivatives of prostaglandin (PG) F2 alpha to rat luteal membranes, and also their abortifacient potency in pregnant rats, have been studied. Competitive binding studies with various PG-analogues were performed in ovaries of juvenile rats pretreated with PMSG and HCG, and in parallel studies the abortifacient potency of these substances was tested in pregnant rats. It was observed that this class of derivatives bound to the PGF2 alpha receptor as well as, or even better than the parent compound PGF2 alpha. Modifications in the carboxyl group at C-1 yielded derivatives with a higher affinity for the receptor, in decreasing order of effectiveness as follows: -COOR greater than COOH greater than OH. The data obtained from the binding studies also compared well with data on the abortifacient potency in pregnant rats. It is concluded that the addition of a phenoxy group to either the lower or upper side chain of PGF2 alpha may augment the binding to the receptor as well as the biological responses induced by the post receptor effect.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.