The effect of heterogeneous structural diffusion on ligand binding to heme proteins

The kinetics of ligand binding to heme proteins studied by flash photolysis display an algebraic time dependence at low temperatures in contrast exponential recombination observed under physiological conditions. This result shows that protein structures should be viewed as a time average of interconverting microstates which are frozen in at low temperatures. We propose a quasi-one-dimensional model of heterogeneous structural diffusion coupled to ligand binding which describes freezing transition as an inherent property of protein fluctuations. The structural hopping rates are derived from a temperature invariant spectrum of activation energies. The model predicts power law kinetics of the form t ^- at long times. The exponent is constant (0.5) at high temperatures but decreases below a critical temperature in the frozen regime. These results are compared to experiments performed with myoglobin and -chains of hemoglobin.     

Do Antiepileptics Phenytoin,Carbamazepine, and Loreclezole Show GABAA Receptor Subtype Selectivity in Rat Brain Sections?

[³⁵S]t-Butylbicyclophosphorothionate ([³⁵S]TBPS), a convulsant site ligand of GABA_A receptors, was used in autoradiography with rat brain sections to test suggested receptor subtype-selective actions of antiepileptics phenytoin, carbamazepine and loreclezole on native GABA_A receptors. At maximal 100 M concentration, both phenytoin and carbamazepine decreased [³⁵S]TBPS binding only by 20%, indicating that their low potency and efficacy prevents their use as 1 subunit-identifying compounds. Ten M loreclezole did not affect the binding, but a further increase in loreclezole concentration strongly decreased it. The action of loreclezole, assumed to reflect 2/3 subunit-containing receptors, varied from brain region to region, but the effects were unrelated to the regional expression profiles of subunit variants. We conclude that in autoradiographic [³⁵S]TBPS binding assay neither carbamazepine, phenytoin nor loreclezole are useful tools in characterizing brain regional heterogeneity of GABA_A receptors in rats and that only loreclezole exhibits high, pharmacologically relevant efficacy.     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.     

Steroid hormone binding receptors in the rat kidney

Summary The histochemical localization of ⁵-3-HSDH in individual follicles isolated from the adult mouse ovary and in ovulated cumulus cell-oocyte masses recovered from the oviduct was examined using a new embedding technique. The procedure employed involves the histochemical staining of such tissue for ⁵-3-HSDH with subsequent embedding in GMA (glycol methacrylate). This method not only permits the acquisition of sections as thin as 3 m but also preserves the histological detail of the tissue allowing for the specific cellular localization of the enzyme. Results obtained from this technique far surpass those obtained from frozen material. Virgin female mice were injected with PMSG and sacrificed either 10 or 17 h later in order to acquire preovulatory or ovulated oocyte-cumulus cell masses, respectively. The sites of localization of ⁵-3-HSDH corresponded to sites demonstrated by histochemical studies on frozen tissue sections; however, the present study revealed that not all cells of a specific type within the same follicle reacted with the same intensity. Granulosa cells lining the walls of vesicular follicles displayed different degrees of enzyme activity based on their distance from the basement membrane. Intrafollicular transformed cumulus masses and cumulus cells of ovulated masses within the oviduct did not react uniformly in that some were positive for the enzyme and others were not. Such results indicate that not all cells of a given type in the ovary possess similar ⁵-3-HSDH activity at a particular time. Thus, the cells comprising a specific cellular component of the ovary should be treated as individual entities and not as a homogeneous group with respect to their metabolic activities.This project was supported by Grant 1 RO1 OH 00835-01 awarded by the National Institute for Occupational Safety and Health and by a grant from the Edward G. Schlieder Foundation awarded to W.J.S. and NIH Grant 5 RO1 HD08041-03 awarded to A.W.S.     

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE⁺ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a sup-Estrain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel -strands organized into two orthogonally situated -sheets. The overall conformation of the protein resembles that of a clam — hence the term -clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg¹⁰⁶ and Gln¹¹⁵. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the -clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations pf several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism. Comparison of the C coordinates of apo- and holo-I-FABP with those of other proteins indicates it is a member of a superfamily that currently includes (i) 10 mammalian intracellular lipid binding proteins, (ii) the photoactive yellow protein from the purple photoautotrophic bacterium Ectothiorhodospira halophila and (iii) a group of extracellular lipid binding proteins from a diverse number of phyla that have a common barrel consisting of 8 anti-parallel -strands stacked in two nearly orthogonal sheets. In summary, E. coli-derived I-FABP not only represents a useful model for assessing the atomic details of fatty acid-protein interactions and the mechanisms which regulate acquisition and release of this type of ligand, but also structure/function relationships in other superfamily members.Abbreviations I-FABP Intestinal Fatty Acid Binding Protein - r.m.s root mean square     

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

-Endorphin and the synthetic -endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met⁵]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of -endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of ¹²⁵I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K _d = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits ¹²⁵I-labeled -endorphin specific binding to naloxone insensitive receptors on T lymphocytes (K _i = 0.6 ± 0.1 nM)). Thus, -endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.     

Endogenous sugar receptor pattern in human glioblastomas and gangliocytomas studied by histochemical application of biotinylated (neo)glycoproteins and affinity chromatography

Summary Biotinylation of chemically glycosylated bovine serum albumin, yielding a panel of neoglycoproteins, and of desialylated, naturally occurring glycoproteins allowed to systematically evaluate presence and distribution of various types of endogenous sugar receptors in the sections of human glioblastomas and gangliocytomas by a routine histochemical procedure. Pronounced cytoplasmic staining with markers, carrying constituents of natural glycoconjugates, e.g. for -galactoside-specific receptors, contrasted with the different intensities, noticed for - and -glucosidespecific receptors. Significant qualitative differences between the two tumor types were detected with N-acetyl-D-galactosamine-and sialic acid-carrying probes. Nuclear staining with only a part of the applied panel underscored the specificity of the protein-carbohydrate interaction. Fine structural features of the synthetic neoglycoproteins, e.g. the mode of coupling of the carbohydrate moiety to the protein, were found to exert a significant influence on their suitability as histochemical markers. On the basis of the histochemical results, exemplary biochemical analysis of certain classes of endogenous sugar receptors by affinity chromatography and subsequent gel electrophoresis, namely of -galactoside-, -fucoside-, -mannoside- and -glucoside-specific proteins, revealed presence and characteristics of respective sugar receptors that can contribute to the histochemical staining. Similar extent of histochemical staining with the respective probes notwithstanding, the different tumor types exhibited qualitative differences in the expression of individual endogenous sugar receptors. The combined histochemical and biochemical analysis is supposed to be of conspicuous value for biological and clinical investigations on endogenous sugar receptors.     

In vitro binding of Helicobacter pylori to monohexosylceramides

H. pylori is the major cause of human gastritis, duodenal ulcer and thus gastric adenocarcinoma. Many glycosphingolipid species have been postulated as receptors for H. pylori and it is likely that H. pylori attachment requires multiple, perhaps sequential receptor/ligand interactions. In this study, the binding of a number of H. pylori clinical isolates, as well as stock strains, to acid and neutral glycosphingolipids separated on thin-layer chromatograms was characterized under microaerobic conditions. All H. pylori clinical isolates, laboratory strains and type culture collection strains recognized galactosylceramide (Gal1Cer) with ceramide containing sphingosine and hydroxylated fatty acid (type I), or non-hydroxylated fatty acid (type II), on thin-layer chromatograms and when incorporated into liposomes. The clinical isolates bound stronger to Gal1Cer (type II) than Gal1Cer (type I) on TLC, whereas lab and culture collection strains showed the opposite binding preference. A clear preference in binding to Gal1Cer (type I) incorporated into liposome was shown by most tested strains. Clinical isolates bound well to glucosylceramide (Glc1Cer) with hydroxylated fatty acid, whereas weak binding to this glycolipid was detected with the lab and type collection strains. None of the tested strains bound Glc1Cer with non-hydroxylated fatty acid on the solid surface, but some strains of both clinical or type collection origins showed weak or very weak binding in the liposome assay. A clear distinction between the binding specificity of living organisms (under microaerobic conditions) as opposed to dying organisms (under normoxic conditions) illustrates the importance of cellular physiology in this process.These studies illustrate lipid modulation of the potential receptor function of monohexosylceramides and the distinction between the receptor repertoire of H. pylori clinical isolates and cultured strains commonly used to study host-cell adhesion.     

Autoradiographic localization of sex steroid hormones in the lymphatic organs of baboons

Summary The localization of radiolabeled estradiol and dihydrotestosterone was examined in the lymphatic organs of both male and female baboons. A total of 12 baboons were divided into two groups, each containing three males and three females. Each animal in one group, both males and females, was injected intravenously with 1 g/kg body weight of ³H-estradiol while those in the second group were each injected with 1 g/kg body weight of ³H-dihydrotestosterone. As controls, one male and one female from each group also received a dose of 100 g/kg body weight of the corresponding unlabeled steroid. One and a half hours after the injections, the animals were sacrificed and the spleen, thymus, and inguinal lymph nodes removed and processed for autoradiography. The localization of ³H-estradiol was similar in both males and females. In the thymus fibroblasts and epithelio-reticular cells, but not thymocytes, localized ³H-estradiol. In lymph node and spleen, nonlymphoid tissue concentrated the labeled estrogen. Additionally, in the paracortical region of the lymph node, an unknown cell type was labeled with estrogen. Only one male baboon demonstrated nuclear localization of ³H-dihydrotestosterone. This was observed in the reticular cells in the spleen and lymph nodes. The same cell type in the organs of the remaining animals was unlabeled.     

Binding of [125I]iodocyanopindolol by rat Harderian gland crude membranes: Kinetic characteristics and day—night variations

The Harderian glands are innervated by sympathetic fibers originating in the superior cervical ganglia. The aim of this study is to characterize the -adrenergic receptors in the rat Harderian gland. The characteristics of -adrenergic receptors were determined in crude membrane preparations from rat Harderian gland, using [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) as radioligand. The binding of the ligand to the receptor is rapid, reversible, saturable, specific and dependent on time, temperature and membrane concentration. At 30 °C, stoichiometric data suggest the presence of one binding site with a K_d value of 0.29 nM and B_max of 32 pmol/L. The interaction shows a high degree of specificity for -adrenergic agonists and blockers, as suggested by competitive displacement experiments with isoproterenol (IC₅₀=19.1 nM), propranolol (IC₅₀=28.1 nM), and norepinephrine (IC₅₀=96.3 nM). Clonidine, yohimbine, methoxamine, and prazosin are ineffective at concentrations up to 1 M. In the other hand, binding of [¹²⁵I]CYP by Harderian gland membranes exhibits day—night variations. Binding values are low during the daytime and increase progressively late in the evening to reach a maximum at 2200 h (2 h after the onset of dark period), but decreased to the end of the dark period (0600 h). In conclusion, the results presented in this paper show the functional and pharmacological characterization of -adrenergic receptors in the rat Harderian gland. This neurotransmitter may play a physiological role at this level regulating, at least, processes such as a thyroid hormone metabolism.     

Discrepancy between the histochemically and biochemically determined 3β-hydroxysteroiddehydrogenase activity in the calf adrenal cortex

Summary In sections of the calf adrenal cortex the histochemically determined3-hydroxysteroiddehydrogenaae activity is lower in the zona glomerulosa than in the zona fasciculata. Biochemically the activity of this enzyme was found in mitochondrial as well as in microsomal fractions. The mitochondrial, respectively the microsomal fractions of the two zones showed identical enzyme activities. So there is a discrepancy between the histochemical results on tissue sections and biochemical results obtained with isolated subcellular fractions.However, if the histochemical determination of 3-hydroxysteroiddehydrogenase activity is carried out on sections of mitochondrial and microsomal pellets of the two zones the results are in agreement with the biochemical findings. Therefore the observed discrepancy rather seems to be related with the state of tissue—intact or cell fractions—than with the used histochemical method.     

Recruitment of beta-2-glycoprotein 1 to cell surfaces in extrinsic and intrinsic apoptosis

Apoptotic cells and phagocytes have developed a diverse array of distinct ligand-receptor systems that drive the recognition and uptake of dying cells. Phagocytes recognize apoptotic cells either directly, by binding to specific ligands at their cell surface, or indirectly, by binding to bridging proteins that bind these ligands. Previous observations showed that the plasma bridging protein 2GP1, binds PS containing vesicles, and enhances their binding and engulfment by phagocytes in vitro. In this study we show that apoptotic cells injected intravenously and intraperitonealy into syngeneic mice recruited the PS binding protein, 2GP1. Examination of peritoneal exudates and spleen thin sections showed that only the injected apoptotic cells picked up endogenous 2GP1. Recovery of cells from the peritoneum showed that apoptotic cells bearing 2GP1 were clustered around host peritoneal phagocytes. In addition, tissue sections from mice injected with Fas antibody showed colocalization of 2GP1 with TUNEL-positive apoptotic cells. These results provide evidence that endogenous 2GP1 binds apoptotic cells in vivo, suggesting that the protein plays an important physiologic role in the recognition of dying cells.     

Effects of β-Amyloid-(25-35) Peptides on Radioligand Binding to Excitatory Amino Acid Receptors and Voltage-Dependent Calcium Channels: Evidence for a Selective Affinity for the Glutamate and Glycine Recognition Sites of the NMDA Receptor

The neurotoxic fragment corresponding to residues 25-35 of the -amyloid (A) peptide [A-(25-35)] has been shown to exert effects on (+)-[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([³H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A-(25-35) [A-(25-35-NH₂) and A-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A-(25-35-NH₂) and A-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [³H]glutamate and [³H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten M A-(25-35-NH₂) and A-(25-35-COOH) gave 25% and 20% inhibitions of [³H]glutamate binding and 75% and 70% inhibitions of [³H]glycine binding, respectively. A-(25-35-NH₂), but not A-(25-35-COOH), gave a small (ca. 17% at 10 M) statistically significant increase of [³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) binding. [³H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC₅₀ value for calcium stimulation of [³H]nitrendipine binding. It is concluded that A-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels.     

Beta-adrenergic ([3H]CGP-12177) binding to brain slices and single intact pineal glands

We have characterized and quantified the binding of [³H]CGP-12177 to -adrenergic receptor sites in slices (300 microns) of rat cerebral cortex. The receptors are stereospecific, saturable and of high affinity. Binding of [³H]CGP is readily reversible and demonstrates appropriated drug specificty. This assay method allows the demonstration of isoproterenol-induced down-regulation (internalization) of -adrenoreceptors. Receptor recycling is observed at 37°C in the absence of -agonist but can be blocked by low temperature (0°C) or by monensin. -Adrenoreceptors can also be labeled and quantified in intact, single pineal glands of rat, mouse and hamster. Rat pineals contain approximately 10 times more binding sites than do hamster or mouse pineals and up to 8 times more sites than found in rat cerebral cortex. Rat pineal [³H]CGP binding can be up- and down-regulated but not to the same degree as seen in brain slices. This assay method is simple, rapid and provides new opportunities for the study of other receptor types in intact tissue.     

Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

We studied ³H-glycine and ³H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a K_D of 2.5 and 2.0 M, respectively. Specific binding of glycine was displaced by -alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.     

Detection of mRNA and hnRNA using a digoxigenin labelled cDNA probe byin situ hybridization on frozen tissue sections

Summary A full-length cDNA clone encoding the constant region of T cell receptor chain was labelled by random priming DNA with digoxigenin-dUTP. The probe was used to estimate the relative amount of the receptor chain mRNA byin situ hybridization on frozen sections from human thymus and lymph nodes. The hybridization was visualized in blue using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a subsequent enzyme-catalysed colour reaction. The distributions of the signal in tissue sections were as expected. Moreover, labelled cells showed hybrids both in the cytoplasm and in the nucleus, and strongly and weakly stained cells were clearly distinguishable. The results indicate that this method ofin situ hybridization should be useful in the detection of specific mRNA in frozen sections.     

Membrane associated estrogen receptors and related proteins: Localization at the plasma membrane and the endoplasmic reticulum

The female sex steroid, estradiol 17, mediates its effect through its association with estrogen receptor present in the target cell. So far the major emphasis has been given to the genomic actions of the hormone mediated by the nuclear estrogen receptors. Recent years have seen a shift in the ideas revealing the existence of estradiol binding entities both in the plasma membrane and the endoplasmic reticulum. Though the true identity of this membrane associated receptors is far from being known, a functional role for the same have been implicated both at the genomic as well as the non-genomic level. The major focus of the review is to highlight the existence of membrane associated estrogen receptors and receptor-related proteins and the functional roles played by some of them. The signalling events exerted by this class of membrane associated estrogen receptor could partly explain the physiological significance of estrogen in cardiovascular disease, osteoporosis and breast cancer as well as the molecular mechanism associated with xenoestrogen action.     

Phorbol ester receptors in cerebral cortex of cats with GM1 gangliosidosis

The pathogenesis of neuronal dysfunction in the gangliosidoses is poorly understood. Studies of the feline gangliosidoses and in vitro experiments implicate ganglioside inhibition of protein kinase C (PKC) in the pathogenesis of these neurological diseases. Therefore, in the present study, the binding of [³H]phorbol-12, 13 dibutyrate was measured to determine the levels of PKC in cerebral cortex of cats with GM₁ gangliosidosis (mutant) and age matched normal siblings. This binding of ([³H]PDB) to cerebral cortex homogenates in both normal and mutant cats was highly specific. The specificity of receptors was ascertained also from displacement studies using nonradioactive phorbol ester analogues to displace [³H]PDB bound to its receptors. In both mutant and normal cat brain, phorbol 12, 13-dibutyrate (PDB), 4--phorbol 12,13-didecanoate (-PDA) and 4--phorbol 12,13-dibenzoate (-PDBz) were highly potent (approximately to same degree) and effective in displacing [³H]PDB. On the other hand, 4- phorbol 12,13-diacetate (-PDA) was a weak displacer and 4--phorbol did not displace the bound [³H]PDB in either normal or mutant brain. Scatchard analysis of the binding data indicated a homogenous single class of binding sites in normal and mutant brain (Normal: K_d=1.42×10^–7 M, B_max=8.40 pmoles/mg protein. Mutant: K_d=1.60×10^–7 M, B_max=10.00 pmoles/mg protein). Sphingosine inhibited the binding to approximately the same extent in normal and mutant cortex. These studies demonstrate the presence of highly specific, homogenous, single type phorbol ester receptors in cerebral cortex of cats with GM₁ gangliosidosis which are qualitatively and quantitatively similar to normal cat brain.     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization