Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10−8 M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using 35S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [35S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent Kd values for [35S]PSK-α binding were found to be 1 × 10−9 M for the high-affinity type and 1 × 10−7 M for the low-affinity type, with maximal numbers of binding sites of 1 × 104 sites per cell and 1 × 105 sites per cell, respectively. Competition studies with [35S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Location and properties of metal-binding sites on the human prion protein

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a Kd for copper(II) of 10−14 M, with other metals (Ni2+, Zn2+, and Mn2+) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The Kd for copper(II) at this site is 4 × 10−14 M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized α-helical (α-PrP) or reduced β-sheet (β-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

N-acetoxy-N-acetyl-2-aminofluorene binding sites on [phi] X174 and SV40

Restriction enzyme inhibition and lambda exonuclease studies indicate that carcinogen N-acetoxy-N-acetyl-2 aminofluorene (AAAF) binds to sequences on ɸX174 RF and SV40 plasmids DNA that are similar to the eight preferred binding sites previously located on pBR 322. Both DNAs were digested with enzyme Hinf I and resultant fragments 32P end-labeled. Labeled fragments were reacted with the carcinogen to give one to sixteen bound moieties per DNA. Fragments were isolated and restriccion enzyme and lambda exonuclease inhibition assays were performed. Inhibition detected occurred at selected sites and was not specific for a certain enzyme or certain size of recognition sequence. Results of these assays allow mapping of the location of high affinity binding sites of the carcinogen on both DNAs. All sites have common sequence elements: the presence of either the sequence T(G/C)TT(G/C) or the sequence T(G/C) CTT(G/C).

CONCERTED ACTION OF THE HIGH-AFFINITY CALCIUM-BINDING SITES IN SKELETAL-MUSCLE TROPONIN-C

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect, Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2 . TnC complex is more tightly bound to the filaments than the Mg2 . TnC form, In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Smart Plastic Antibody Material for Hemoglobin Tailored by Silica Surface Imprinting and with Charged Binding Sites: Its use as Ionophore in Potentiometric Transduction

JORNADAS DE ELECTROQUÍMICA E INOVAÇÃO 2013

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder.

In the urinary bladder of the toad Bufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na+ transport. We have investigated whether T3 might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T3 (60 nM) for up to 20 hr of incubation; ii) the kinetics of uptake of 3H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T3. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0 degrees C) did not vary significantly after 18 hr of exposure to T3, while aldosterone-dependent Na+ transport was significantly inhibited. In addition, T3 did not modify the kinetics of uptake (90 min) of 3H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubated in vitro at 25 degrees C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80 nM). T3 slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T3 does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Novel Zn(2+)-binding Sites in Human Transthyretin IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn(2+) enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn(2+) at pH 4.6-7.5. All four structures reveal three tetra-coordinated Zn(2+)-binding sites (ZBS 1-3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR.Zn(2+) binding perturbs loop E-alpha-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases similar to 5-fold in the presence of Zn(2+). Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn(2+). HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn(2+) binding, although the alpha-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn(2+), which is consistent with the tertiary structural perturbation provoked by Zn(2+) binding, tetramer stability is only marginally affected by Zn(2+). These data highlight structural and functional roles of Zn(2+) in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.