Search

A considerable body of evidence has been accumulated showing the interrelationship between zinc and the plasma thyroid hormone (TH) distributor protein, transthyretin (TTR). TTR is a multi-functional protein, which emerged from 5-hydroxyisourate hydrolase (HIUHase) by neo-functionalization after gene duplication during early chordate evolution. HIUHase is also a zinc-binding protein. Most biochemical and molecular biological findings have been obtained from mammalian studies. However, in the past two decades, it has become clear that fish TTR displays zinc-dependent TH binding. After a brief introduction on plasma zinc, THs and their binding proteins, this review will focus on the role of zinc in TTR functions of various vertebrates. In particular primitive fish TTR has an extremely high zinc content, with an increased number of histidine residues which are involved in TH binding. However, zinc-dependent TH binding may have been gradually lost from TTRs during higher vertebrate evolution. Although human TTR has a low zinc content, zinc plays an essential role in TTR functions other than TH binding: the stability of TTR-holo retinol binding protein 4 (holoRBP4) complex, TTR amyloidogenesis, the sequestration of amyloid β (Aβ) fibrils and cryptic proteolytic activity. The interaction of TTR with metallothioneins may be a critical step in the exertion of some of these functions. Evolutionary and physiological insights on zinc-dependent functions of TTRs are also discussed. [ABSTRACT FROM AUTHOR]

Incretins, endogenous polypeptide hormones released in response to food intake, potentiate insulin secretion from pancreatic β cells after oral glucose ingestion (the incretin effect). This response is signaled by the two peptide hormones glucose-dependent insulinotropic polypeptide (GIP) (also known as gastric inhibitory polypeptide) and glucagon-like peptide 1 through binding and activation of their cognate class 2 G protein-coupled receptors (GPCRs). Because the incretin effect is lost or significantly reduced in patients with type 2 diabetes mellitus, glucagon-like peptide 1 and GIP have attracted considerable attention for their potential in antidiabetic therapy. A paucity of structural information precludes a detailed understanding of the processes of hormone binding and receptor activation, hampering efforts to develop novel pharmaceuticals. Here we report the crystal structure of the complex of human GIP receptor extracellular domain (ECD) with its agonist, the incretin GIP1-42. The hormone binds in an α-helical conformation in a surface groove of the ECD largely through hydrophobic interactions. The N-terminal ligand residues would remain free to interact with other parts of the receptor. Thermodynamic data suggest that binding is concomitant with structural organization of the hormone, resulting in a complex mode of receptor-ligand recognition. The presentation of a well structured, α-helical ligand by the ECD is expected to be conserved among other hormone receptors of this class. [ABSTRACT FROM AUTHOR]

Kene PS, Dighe RR, Mahale SD. Delineation of regions in the extracellular domain of follicle-stimulating hormone receptor involved in hormone binding and signal transduction. AJRI 2005; 54:38–48 © Blackwell Munksgaard, 2005 To use antipeptide antibodies to potential surface-oriented regions of the extracellular domain (ECD) of the human follicle-stimulating hormone receptor (hFSHR) to delineate regions involved in FSH binding and FSH-induced signal transduction. We developed and characterized antipeptide antibodies to different, potentially surface-oriented regions of the ECD of hFSHR. The ability of these antibodies to recognize the receptor was studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western blotting. The ability to modulate FSH binding and cAMP generation was studied by the radioreceptor assay and in vitro FSH bioassay respectively. Antipeptide antibodies to regions 15–31, 216–235, 285–300 and 327–341 hFSHR inhibited both FSH binding and cAMP production. Regions 15–31 and 216–235 were accessible to their cognate antipeptide antibodies both before and after FSH binding, while regions 285–300 and 327–341 hFSHR were accessible only prior to FSH binding. Based on the observations made with respect to accessibility to antipeptide antibodies, ability of antibodies to inhibit FSH binding and the subsequent cAMP generation and kinetics of antibody binding, regions 285–300 and 327–341 hFSHR appear to be the chief FSH-binding sites, while regions 15–31 and 216–235 hFSHR serve as ancillary FSH-binding sites. [ABSTRACT FROM AUTHOR]

A fragment of a cytosolic thyroid-hormone-binding protein from Xenopus liver was reported to be 92–100% identical to residues 236–258 in several cytosolic aldehyde dehydrogenases [Yamauchi, K. & Tata, J. R. (1994) Eur. J. Biochem. 225, 1105–1112], which have been proposed to form part of the hinge region necessary to bind the adenosine moiety of NAD. Here we investigated the effects of two thyroxine analogs, 3,3′,5-triiodo-L-thyronine and 3,3′,5-triiodothyroacetic acid, on purified human liver mitochondrial and cytosolic aldehyde dehydrogenases. The compounds were found to be competitive inhibitors against NAD and uncompetitive inhibitors with respect to aldehyde. At pH 7.4, the apparent Ki values were in the micromolar range when the concentration of NAD was varied. The inhibition against recombinantly expressed mutant forms of aldehyde dehydrogenase, which possessed diminished NAD binding, was determined. Essentially no differences were found between the native enzyme and the mutants, showing that the analog binding was not affected by altering the NAD-binding site. Furthermore, the analogs could displace NAD but not NADH from the enzyme. These findings indicated that the binding of NAD differed from that of NADH, and that aldehyde dehydrogenases, like other dehydrogenases, can be inhibited by thyroxine analogs. [ABSTRACT FROM AUTHOR]

Studies on the subcellular location of ethylene binding activity from developing cotyledons of Phaseolus vulgaris L. are described. Binding activity has been shown to be predominantly membrane bound. When separated by rate-zonal centrifugation more than 70% of this activity was of low sedimentation rate. The slowly sedimenting band of activity was further fractionated into three bands by isopycnic density gradient centrifugation. The three bands occur at sucrose densities of 1.125 g cm-3, 1.155 g cm-3 and 1.175 g cm-3, corresponding to the distribution of putative marker enzymes for the cell endomembrane system and to protein body membranes. Further circumstantial evidence was obtained by electron microscopy and sucrose step gradient centrifugation. [ABSTRACT FROM AUTHOR]

Many methods are available for determining binding of small-molecular ligands to macromolecules. In studies on plant hormone binding, precipitation of putative hormone-protein complexes with ammonium sulphate is often used as the sole method of assay. Several such reports on auxin binding have been re-examined, using additional assay procedures. The results show that apparent binding activity measured by ammonium sulphate precipitation may be undetectable by independent, less ambiguous methods. It is concluded that hormone-binding studies that rely solely on the precipitation assay can be grossly misleading, and that this assay, if used at all, should always be validated against a procedure less likely to generate artefacts. An example of such validation for a plant protein is shown. [ABSTRACT FROM AUTHOR]

The preparation is described of a cell-free system from developing cotyledons of Phaseolus vulgaris cv. Canadian Wonder which is capable of binding ethylene. The binding is saturable and the apparent dissociation constant for ethylene is 6.4·10 M in solution. The binding site is associated with subcellular particles and treatment with Triton X-100 results in substantial solubilisation of the activity. The kinetics of association and dissociation of the ligand and the binding site are described. The system is heat labile and binding activity is diminished by treatment with some proteolytic enzymes. [ABSTRACT FROM AUTHOR]

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [H]benzyladenine (BA) to a 13,000-80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10 and 10M. [H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000-80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption. [ABSTRACT FROM AUTHOR]

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco ( Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known. [ABSTRACT FROM AUTHOR]

Human serum in a concentration of 10% in the perfusion medium failed to increase glucose uptake by the isolated perfused rat heart, indicating that nonsuppressible insulin-like activity (NSILA) in whole serum was inactive in this system. When NSILA-carrier protein was added to partially purified NSILA-S, its biological activity on the rat heart disappeared. In contrast, the action of insulin was not affected by the presence of NSILA-carrier protein. Binding ofI-labelled NSILA-S to rat heart was inhibited by NSILA-carrier protein.I-labelled insulin binding was not inhibited. These results support the hypothesis that NSILA-S bound to serum carrier protein is a large molecular compound which does not readily diffuse out of the capillary bed and therefore does not exert insulin-like effects in vivo. [ABSTRACT FROM AUTHOR]

• Insulin mimetics could represent an alternative strategy for treatment of diabetes. • Multipodal compounds based on adamantane or proline scaffolds were synthesized. • Adamantane-based compound displaced insulin from insulin receptor with K d of 500 nM. • Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (K d value of 0.5 μM) than proline-derived compounds (K d values of 15–38 μM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics. [ABSTRACT FROM AUTHOR]