Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Hormonal regulation of tumor suppressor proteins in breast cancer cells

This laboratory is studying hormonal regulation of tumor suppressor proteins, p53 and retinoblastoma (pRB). Estrogen receptor and progesterone receptor positive human breast cancer cell lines, T47D and MCF-7, were utilized for determining influence of hormonal and antihormonal agents on the level of expression of p53, state of phosphorylation of pRB, and rate of cell proliferation. The expression of p53 in T47D cells grown for 4–5 days in culture medium containing charcoal-treated (stripped) fetal bovine serum declined gradually to 10% of the level seen in control (whole serum, non charcoal-treated) groups. Supplementation of culture medium containing stripped serum with 0.1–1 nM estradiol (E₂) restored p53 to its level seen in the control within 6–24 h. Under above conditions, treatment of cells with R5020 or RU486 reduced (15–30%) the level of p53. Incubation of cells in E₂-containing growth medium caused cell proliferation and hyperphosphorylation of pRB; the latter effect was seen maximally between 24–72 h. The E₂-induced hyperphosphorylation of pRB and increase in the level of p53 were sensitive to the presence of ICI and 4-hydroxy tamoxifen (OHT). T47D and MCF-7 cells were also transiently transfected with a P1CAT reporter plasmid containing c-Myc responsive element and the levels of chloramphenicol acetyltransferase (CAT) activity were observed in response to various treatments. E₂ and OHT caused P1CAT induction as seen by increased CAT activity: E₂ caused an endogenous increase in the expression of an ICI-sensitive c-Myc form. These data suggest that estrogen upregulates p53 expression while progesterone downregulates this process. Further, E₂ regulates p53 level and pRB activity in a coordinated manner.     

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate.

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.     

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

Glucagon-like peptide-1 (7–36)amide (GLP-1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP-1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-β-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7–36)amide to the solubilized receptor was inhibited conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K_a= 0.26 ± 0.03 nM and B_max= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and K_a= 22.54 ± 4.42 μM and B_max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-γ-S nor GDP-β-S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.     

Dopamine receptors in human foetal brains: Characterization, regulation and ontogeny of [H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [³H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC₅₀ values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC₅₀ values of 2.5 and 10 μM, respectively suggesting a D₂ type receptor.

Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 10⁸ M⁻¹ min⁻¹ and 5.0 × 10⁻² min⁻¹, respectively. Equilibrium dissociation constant was 87 pM and the K_D obtained by saturation binding was 73 pM.

During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [³H]spiperone binding sites exhibited a heterogeneity with a high (K_D, 13–85 pM) and a low (K_D, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.

GTP decreased the agonist affinity as observed by dopamine competition of [³H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC₅₀ values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.     

Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Characterization of glucagon-like peptide-I(7–36)amide receptors of rat lung membranes by covalent cross-linking

¹²⁵I-labelled GLP-I(7–36)amide was cross-linked to a specific binding protein in rat lung membranes using disuccinimidyl suberate. A single radio-labelled band at M_r 66000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex which is consistent with the presence of a single class of binding sites on rat lung membranes. The band was undetectable when 1 μmol/1 GLP-I(7–36)amide was included in the binding assay. No change in the mobility of the band was observed under reducing conditions suggesting that the binding protein in the receptor is not part of a larger disulphide-liked protein. The intensity of the radiolabelled protein band was reduced when the incubation with ¹²⁵I-labelled GLP-I(7–36)amide was carried out in the presence of guanine nucleotides suggesting that the GLP-I(7–36)amide receptor is coupled to the adenylate cyclase system.     

Progestin binding in mammary tissue of prepartum,nonlactating and postpartum,lactating cows

Binding of [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [³H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [³H]R5020 binding to progestin binding sites, while estradiol-17β, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), dexamethasone (9-fluoro-11β, 17, 21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for specifically bound [³H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 ( ± 0.3) and 0.8( ± 0.2) × 10?⁹M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 ± 53) than postpartum, lactating (41 ± 15) cows. Specific binding components in cytosol from lactating cows sedimented in the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.     

Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein

Interaction of tetracycline (TC) derivatives viz. oxytetracycline, doxycycline, demeclocycline and chlorotetracycline with bovine serum albumin (BSA) and concomitant changes in protein conformation were studied using fluorescence quenching and circular dichroism measurements. Fluorescence data revealed the presence of one to three binding sites on BSA for different TC derivatives. Binding studies with the marker ligands, warfarin and bilirubin, elucidated site-I as a primary binding site for TCs on albumin. Scatchard analysis revealed the binding affinity (K_a) and capacity (n) for these derivatives vary in the range from 0.8 to 3.2×10⁶ l/mole and 1.3–3.4, respectively. Significant reduction (60–45%) in secondary structure (-helical content) of BSA was noticed upon interaction with different TC derivatives in presence of Cu (II) ions. High affinity binding of TCs with BSA signifies drug stability. However, excessive binding at higher TC concentrations in combination with Cu (II) induces conformational change in protein structure, which may exert detrimental effect on cellular protein.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

The F1-ATPase beta-subunit is the putative enterostatin receptor

It has been suggested that the F₁-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin_1–7, in the absence and presence of cold enterostatin. ¹²⁵I-β-casomorphin_1–7 weakly binds to the rat F₁-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin_1–7 binding to the F₁-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin_1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin_1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F₁-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F₁-ATPase complex. Western blot analysis showed the F₁-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F₁-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin_1–7 bind to distinct sites on the protein.     

Comparative binding specificity of methyltrienolone in human and rat prostate.

The binding of methyltrienolone (R 1881) in crude human hyperplastic prostate cytosol was determined by a charcoal assay. Maximum binding was observed after 2-3 h of incubation at 0 degrees C. This binding decreased steadily thereafter and reached 41% of the 2-hour values after 96 h of incubation. In human hyperplastic prostate, the binding of 3H-R 1881 was competed by low concentrations of R 1881, R 5020 and progesterone and by high concentrations of dihydrotestosterone (DHT) and 17 alpha-methyl-DHT. In the rat prostate, on the other hand, this binding was competed by low concentrations of DHT and 17 alpha-methyl-DHT and only by high concentrations of progesterone and R 5020. The apparent association constant (Ka) of R 1881 was determined in three human prostates and found to be 0.2-0.4 X 10(9) liters/mol; the number of binding sites ranged from 101 to 158 fmol per mg of protein. These findings constitute further evidence for the existence of relatively large amounts of a progesterone-binding component in human hyperplastic prostate.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

The presence of progesterone receptors in the sexual skin of the monkey

R5020(17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dlone)-binding components with sedimentation coefficient of 8S were detected in sexual skin cytosols from estrogen-primed ovarlectomized Japanese monkey ($\text{Macaca fuscata fuscata}$). In contrast, little 8S binding was found In similar preparations from the abdominal skin. The dissociation constants and the number of binding sites of the components were l.6×10^?10M and 36 fmoles/mg cytosol protein, respectively. The 8S binding components were specific for progestational compounds. Incubation with pronase abolished the 8S binding. Thermal experiments revealed the thermolabile nature of the components. Moreover, the concentration of the R5020-binding components was markedly increased by estradiol-17β 3-benzoate injections. We conclude from these results that the cytosols from the sexual skin of estrogen-primed female monkeys contain progesterone receptors.     

Probing the Binding Sites of Antibiotic Drugs Doxorubicin and N-(trifluoroacetyl) Doxorubicin with Human and Bovine Serum Albumins

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K _DOX-BSA = 7.8 (±0.7)×10³ M⁻¹, K _FDOX-BSA = 4.8 (±0.5)×10³ M⁻¹ and K _DOX-HSA = 1.1 (±0.3)×10⁴ M⁻¹, K _FDOX-HSA = 8.3 (±0.6)×10³ M⁻¹. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.     

Parathyroid hormone binding sites in the brain

Parathyroid hormone (PTH) has been shown to have actions within the brain, suggesting the presence of central PTH receptors. This possibility was examined by determining the binding of ¹²⁵I-labeled [Nle^8,18,Tyr³⁴]bovine PTH to the plasma membranes of rat and rabbit brains. Specific binding of the tracer to membranes of the whole brain was time and tissue dependent, and was greater with membranes from the hypothalamus than with membranes from the cerebellum, cerebrum, or brain stem. The binding of the tracer to rat hypothalamic membranes was saturable and competitively displaced by unlabeled PTH(1–34), PTH(3–34), [Nle^8,18,Tyr³⁴]PTH(1–34), and by PTH-related protein, indicating the presence of a single class of high-affinity (dissociation constant = 2–5 nM), low-capacity (maximum binding capacity, B_max = 110–250 fmol/mg protein) binding site. The binding of radiolabeled PTH to these sites was not displaced by unrelated peptides of comparable molecular size (calcitonin, calcitonin-gene related peptide, adrenocorticotropin). The binding of PTH to these sites did not, however, appear to stimulate adenylate cyclase activity, as in peripheral PTH target sites. Thus, although these results indicate the presence of PTH receptors in the brain, these binding sites have a lower affinity than those in peripheral tissues and may utilize a different signal transduction system.     

Isolation and characterization of an estrogen binding protein which may integrate the plethora of estrogenic actions in non-reproductive organs

A putative estrogen receptor (pER) from mouse liver has been characterized. The heterodimer protein (81–84 kDa) consists of two covalently bound subunits (61–67 and 17–27 kDa) with following characteristics: sedimentation constant — 4.9 S; IP — 4.8; dissociation constant (K_d) for estradiol-17β binding — 0.7 nmol; binding sites — 0.746 pmol/mg protein; relative binding affinity — estradiol-17β — 100, estrone — 80 and estriol — 30; specificity — does not bind, other natural steroids, synthetic estrogens, antiestrogens and bioflavonoids. Importantly, immunosuppressants, neuroleptic and carcinogens influence ³H-estradiol-17β binding to pER. Interestingly, pER is a serine phosphatase and this may have relevancy to estrogen action in Alzheimer's disease. The polyclonal anti-pER antibody does not react with estrogen receptors (ER). ER antibody does not react with pER. Remarkably, anti-pER antibody reacts with calcineurin, a brain phosphatase and anti-calcineurin antibody reacts with pER. Immunohistochemical analyses showed that pER is undetectable in reproductive organs (except ovary). It is localized on the plasma or the nuclear membranes in some, in cytoplasm and/or nucleus in other cells of non-reproductive organs (skeletal, neural, vascular, hair and retina), and in tumors (mammary, endometrial and prostate cancers, and prostatic hyperplasia). The information presented justifies the proposition that pER may mediate the estrogenic actions in non-reproductive organs.     

Molecular modelling studies unveil potential binding sites on human serum albumin for selected experimental and in silico COVID-19 drug candidate molecules

Human serum albumin (HSA) is the most prevalent protein in the blood plasma which binds an array of exogenous compounds. Drug binding to HSA is an important consideration when developing new therapeutic molecules, and it also aids in understanding the underlying mechanisms that govern their pharmacological effects. This study aims to investigate the molecular binding of coronavirus disease 2019 (COVID-19) therapeutic candidate molecules to HSA and to identify their putative binding sites. Binding energies and interacting residues were used to evaluate the molecular interaction. Four drug candidate molecules (β-D-N4-hydroxycytidine, Chloroquine, Disulfiram, and Carmofur) demonstrate weak binding to HSA, with binding energies ranging from −5 to −6.7 kcal/mol. Ivermectin, Hydroxychloroquine, Remdesivir, Arbidol, and other twenty drug molecules with binding energies ranging from −6.9 to −9.5 kcal/mol demonstrated moderate binding to HSA. The strong HSA binding drug candidates consist of fourteen molecules (Saquinavir, Ritonavir, Dihydroergotamine, Daclatasvir, Paritaprevir etc.) with binding energies ranging from −9.7 to −12.1 kcal/mol. All these molecules bind to different HSA subdomains (IA, IB, IIA, IIB, IIIA, and IIIB) through molecular forces such as hydrogen bonds and hydrophobic interactions. Various pharmacokinetic properties (gastrointestinal absorption, blood-brain barrier permeation, P-glycoprotein substrate, and cytochrome P450 inhibitor) of each molecule were determined using SwissADME program. Further, the stability of the HSA-ligand complexes was analyzed through 100 ns molecular dynamics simulations considering various geometric properties. The binding free energy between free HSA and compounds were calculated using Molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) approach. The findings of this study might be useful in understanding the mechanism of COVID-19 drug candidates binding to serum albumin protein, as well as their pharmacodynamics and pharmacokinetics.Keyword: Human serum albumin, HAS, Serum protein, COVID-19, Molecular docking, Molecular dynamics simulation, Pharmacokinetics, Pharmacodynamics