Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium

11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25 degrees C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9 X 10(-7) M. A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled 'trans' and 'cis' isomers of vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.     

Effects of chronic lithium administration on concanavalin A binding to plasma membranes from the corpus striatum of rat brain

Summary The effects of chronic in vivo lithium administration on mannose-containing components of plasma membranes from rat corpus striatum were examined by a³H-concanavalin A binding displacement method. No difference in Con A binding was observed between sodium or lithium-treated rats during a 1-month period.We thank Dr S. Gershon of this research unit for his encouragement and support during these studies.     

Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted)Tetrahymena pyriformis

Receptor-mediated endocytosis byTetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin recptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals andTetrahymena, and galactose, did not influence the uptake of TRITC-Con A.     

Binding of (125I) triiodothyronine to human peripheral leukocytes and its internalization

Ultrastructural autoradiography showed high specific binding of (125I) triiodothyronine, as confirmed by a competition test, to plasma membranes, nuclei and mitochondria of human peripheral leukocytes. A high level of binding was also noted on the granulocytes' granules, especially in eosinophils.     

A comment on the estimation of times required for the attainment of equilibrium by noncooperative,single site ligand-receptor systems

Summary A table presents the number of hours required for binding to reach 80% and 95% of the equilibrium value for a noncooperative, single site ligand binding system. A 2nd table provides the fraction of binding sites occupied and the fraction of the total ligand bound at equilibrium under the same conditions.     

Demonstration of specific receptors for angiotensin III in rat adrenal glands]

Distinct and specific binding sites for 3H-angiotensin II (A II) AND 3H-angiotensin III (A III) have been demonstrated in Rat adrenal gland preparations. A III binding sites have the highest affinity (KD29C1-2.10(-10)M) for the 2-8 heptapeptide. This suggests the possibility of a separate and distinct physiological role for the 2-8 heptapeptide, mediated via such A III binding sites.     

Binding of (125I) triiodothyronine to human peripheral leukocytes and its internalization

Summary Ultrastructural autoradiography showed high specific binding of (¹²⁵I) triiodothyronine, as confirmed by a competition test, to plasma membranes, nuclei and mitochondria of human peripheral leukocytes. A high level of binding was also noted on the granulocytes' granules, especially in eosinophils.     

Human platelet 5-hydroxytryptamine receptors: binding of [3H]-lysergic acid diethylamide (LSD). Effects of chronic neuroleptic and antidepressant drug administration

Chronic treatment with phenothiazines and thioxanthenes has been found to enhance 5-HT-induced aggregation of human platelets. A method has been developed to study 5-HT2 receptor binding sites on platelets utilising [3H]-LSD and more recently 125I/LSD. Results are presented which suggest that the LSD binding site is indeed the 5-HT2 binding site and that the LSD binding characterises the specific receptor responsible for 5-HT-induced shape change and aggregation. In a group of patients receiving phenothiazines or thioxanthenes, the Bmax of LSD binding was increased. The mean binding affinity was decreased possibly due to a persistence of neuroleptic in the platelet membrane preparation. Analysis showed that this was not the reason why the mean binding capacity was increased. The results show that chronic phenothiazine and thioxanthene delta treatment 'up-regulates' platelet 5-HT2 binding sites and that this may be accompanied by increased sensitivity to platelet aggregation by 5-HT. In normal subjects desipramine treatment increased the Bmax of platelet LSD binding and this was accompanied by an increased prolactin response to tryptophan which is thought to be mediated by central 5-HT function.     

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors

Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A2 and C strongly (ED50 congruent to 4 X 10(-2) and 4 X 10(-1) micrograms/ml, respectively) and rapidly reduced 125I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.     

Differential binding of conA and WGA on the cell surface, the role of sialic acid in their expression and the increased activity of sialidase after cis-Platin treatment

It is reported that concanavalin A (conA) and wheat germ agglutinin (WGA) have a differential binding pattern on normal mouse spleen lymphocytes and the surface of Dalton's lymphoma cells. It is suggested that sialic acid on the cell surface controls the expression of lectin binding sites. Further, it has been observed that the increased release of sialic acid from cell surfaces after cis-dichlorodiammine platinum (II) (cis-Platin) treatment is due to the increased activity of sialidase.     

Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium

Summary 11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25°C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9×10^–7 M.A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled trans and cis isomers of Vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.     

Differential binding of conA and WGA on the cell surface,the role of sialic acid in their expression and the increased activity of sialidase after cis-Platin treatment

Summary It is reported that concanavalin A (conA) and wheat germ agglutinin (WGA) have a differential binding pattern on normal mouse spleen lymphocytes and the surface of Dalton's lymphoma cells. It is suggested that sialic acid on the cell surface controls the expression of lectin binding sites. Further, it has been observed that the increased release of sialic acid from cell surfaces aftercis-dichlorodiammine platinum (II) (cis-Platin) treatment is due to the increased activity of sialidase.To whom reprint requests should be addressed.     

A daily rhythm in hCG binding to ovarian follicles of the cyclic hamster

Summary On each day of the estrous cycle hCG binding to follicle increased from 09.00 to 21.00 h; then hCG binding was static until 09.00 h of the next day. FSH binding did not exhibit rhythmicity. This pattern of hCG binding may be related to the pulsing of LH on each cycle day.Acknowledgments. This work was supported by grants from NICHD (15 526, KO478) to P.F.T. and Center grant to the Kansas Center of Mental Retardation and Human Development (HD 02528). A. K. was supported in part by the Council for International Exchange of Scholars (Fulbright Foundation).     

The interface of protein-protein complexes: Analysis of contacts and prediction of interactions

Specific protein-protein interactions are essential for cellular functions. Experimentally determined three-dimensional structures of protein-protein complexes offer the possibility to characterize binding interfaces in terms of size, shape and packing density. Comparison with crystal-packing interfaces representing nonspecific protein-protein contacts gives insight into how specific binding differs from nonspecific low-affinity binding. An overview is given on empirical structural rules for specific protein-protein recognition derived from known complex structures. Although single parameters such as interface size, shape or surface complementary show clear trends for different interface types, each parameter alone is insufficient to fully distinguish between specific versus crystal-packing contacts. A combination of interface parameters is, however, well suited to characterize a specific interface. This knowledge provides us with the essential ingredients that make up a specific protein recognition site. It is also of great value for the prediction of protein binding sites and for the evaluation of predicted complex structures. Received 1 October 2007; received after revision 9 November 2007; accepted 9 November 2007     

Strategies for the design of inhibitors of aldose reductase, an enzyme showing pronounced induced-fit adaptations

Aldose reductase is involved in the polyol pathway, catalyzing the reduction of glucose to sorbitol. However, due to pronounced binding site adaptations, the enzyme can operate on a broad palette of structurally diverse substrates ranging from small aliphatic and aromatic aldehydes up to steroid-type ligands. A comparative analysis of the presently accessible crystal structures of aldose reductase complexes reveals four binding-competent protein conformations. Additional relevant conformers are detected through molecular dynamics simulations. They indicate an equilibrium of several conformers which is shifted towards the binding-competent geometries upon ligand binding. Such a manifold system with several alternative binding site conformers requires some tailored concepts in virtual screening. We followed two strategies, both successfully suggesting new micromolar inhibitors. In a first attempt, we concentrated on one preferred conformer and performed a virtual screening, assuming that the binding pocket of aldose reductase adopts only this conformation. In a second approach, we followed a ligand superpositioning method. Ligands were extracted in their bound conformations from three different crystal structures, all accommodating the ligands with different active site conformations. After merging these ligands into one supermolecule, mutual alignments were computed, taking candidate ligands from a screening database. The latter strategy also retrieved several structurally new inhibitors of micromolar potency.     

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.     

Presence of the H blood group antigen on a carcinoembryonic antigen (CEA). Enzymatic transformation of H to A and B specificities]

The carcinoembryonic antigen (CEA-M) was purified from a hepatic metastasis obtained from a blood group O patient with a cancer of the rectum. Using 125I-labelled-CEA and blood group antisera, H specificity was found on the CEA-M; the addition of anti-H to anti-CEA does not modify the binding of labelled-CEA-M to its antibodies (86%), this result leads us to conclude that H and CEA determinants are carried by the same molecule. However the low percentage of binding (30% with 1/10 anti-H) suggests that only a few CEA-M molecules do carry the H antigenic determinant. Finally, glycosyltransferases were used to modify the H specificity into blood group A and B specificities.