Reassessment of plasma angiotensins measurement: effects of protease inhibitors and sample handling procedures.

Characterization of C- and N-terminal forms of angiotensin (Ang) peptides mandated assessment of methods to determine plasma levels. 125I-Ang I, 125I-Ang II, and 125I-Ang(1-7) were added to blood samples in the presence of protease inhibitors. Ethylenediaminetetraacetic acid (EDTA) inhibited the conversion of 125I-Ang I to 125I-Ang II. o-Phenanthroline and EDTA (EDTA + o-Ph) did not eliminate [des-Asp1] fragments or 125I-Ang(1-7). The combination of EDTA + o-Ph and pepstatin A or 4-(chloromercuri) benzoic acid (PCMB) significantly reduced 125I-Ang(1-7) generation. Only PCMB plus EDTA + o-Ph eliminated [des-Asp1] fragments. Authentic plasma values of Ang peptides require the correct choice of protease inhibitors.     

Salt stimulation of serum insulin-like growth factor binding protein activity

Insulin-like growth factors (IGF)-I and -II are bound to carrier or binding proteins in serum. There are at least two classes of binding protein: a high molecular weight complex and a low molecular weight species that is relatively unsaturated. Total binding capacity in serum generally is determined by incubating [125I]IGF with protein that has been stripped of IGF by acid gel filtration. We found that addition of NaCl to the assay increased binding to stripped guinea pig binding protein to about two to four times the level measured in the absence of salt. Stimulation by NaCl was optimal between concentrations of 0.6 and 1.4 M and also was observed when fetal calf or human sera were used as sources of stripped binding protein or when IGF-II was the ligand. Using chloride salts, the order of activity with respect to cations was Na+ greater than K+ greater than Li+. Na2HPO4 at 0.6 M was as stimulatory as 1.2 M NaCl but 0.6 M Na2SO4 was less effective. NH4HCO3 was as effective as NaCl at 0.6 M. Scatchard plots of data from competitive dilution experiments with [125I]IGF-I and unlabeled IGF-I showed that binding was heterogeneous in the absence of 0.6 M NaCl but linear in its presence. NaCl did not stimulate binding when whole serum was used, but after gel filtration of serum on Sephacryl 200 at pH 8, which does not dissociate IGFs from binding protein, binding to individual fractions was stimulated three- to fourfold by NaCl. Fractions stimulated included those containing the large complex or the unsaturated binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-dependent AT(4) receptor internalization in bovine aortic endothelial cells

Recent studies have characterized a specific binding site for the C-terminal 3-8 fragment of angiotensin II (Ang IV). In the present study we looked at the internalization process of this receptor on bovine aortic endothelial cells (BAEC). Under normal culture conditions, BAEC efficiently internalized (125)I-Ang IV as assessed by acid-resistant binding. Internalization of (125)I-Ang IV was considerably decreased after pretreatment of cells with hyperosmolar sucrose or after pretreatment of BAEC with inhibitors of endosomal acidification such as monensin or NH(4)Cl. About 50% of internalized (125)I-Ang IV recycled back to the extracellular medium during a 2 h incubation at 37 degrees C. (125)I-Ang IV remained mostly intact during the whole process of internalization and recycling as assessed by thin layer chromatography. As expected, internalization of (125)I-Ang IV was completely abolished by divalinal-Ang IV, a known AT(4) receptor antagonist. Interestingly, (125)I-divalinal-Ang IV did not internalize into BAEC. These results suggest that AT(4) receptor undergoes an agonist-dependent internalization and recycling process commonly observed upon activation of functional receptors.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Interaction of gastrin with transferrin: effects of ferric ions

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.     

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.     

Characterization of soluble fibronectin binding to Bacille Calmette-Guérin

Fibronectin (FN), a 420 kDa glycoprotein, consists of two similar subunits linked by a disulphide bond near the C-terminal end. FN is present in soluble and matrix forms in various body fluids and tissues and has been shown to bind to variety of organisms. We characterized the conditions required for 125I-FN binding to Bacille Calmette-Guérin (BCG). The binding was dose-dependent, reached saturation within 3 min, and was essentially irreversible for at least 24 h under optimal binding conditions at pH 6.0. In contrast, the binding was reversible (greater than 90% in 24 h) when the pH was increased to 10.0. Scatchard analysis of the dose-response experiments produced a straight line, suggesting the presence of a single class of FN receptor on BCG. 125I-FN binding was trypsin-sensitive, suggesting that the BCG-binding molecule is a protein. The number of FN receptors was determined to be 8000-15,000 per bacterium. 125I-FN binding was pH dependent, with maximal binding at acidic pH. 125I-FN binding was sensitive to the presence of NaCl, with 0.08 M-NaCl inhibiting binding by 85%. These data demonstrate that soluble FN binds to a trypsin-sensitive cell-surface component of BCG in an essentially irreversible manner.     

The interaction of radioiodinated thyrotropin with plasma membranes: Evidence for high affinity binding sites in the thyroid

The binding of biologically active [¹²⁵I]thyrotropin to purified plasma membranes prepared from bovine thyroid glands was studied. At 4°C, specific binding reached a maximum after 2 h of incubation and a plateau was maintained for up to 20 h. Degradation of [¹²⁵I]thyrotropin was undetectable after 2 h of incubation and was only 10% of the total after 20 h.At pH 6.0, at which binding was maximal, a single class of binding sites, having a dissociation constant of approx. 25 nM, was evident. Dissociation studies revealed first order kinetics with a half-time of 2–3 min. At pH 7.5, binding curves were complex, suggesting two orders of binding sites with dissociation constants of approx. 200 nM and 80 pM. Further, at this pH, dissociation of the thyrotropin from its receptor was also complex, suggesting the presence of two first order reactions, one with a half-time similar to that seen at pH 6.0 and another with a half-time of 4 h. At both pH 6.0 and 7.5, insulin, glucagon, growth hormone, and prolactin were without effect on [¹²⁵I]thyrotropin binding.Similar high affinity and low affinity binding sites were seen with porcine thyroid membranes, but only low affinity sites were seen with either rat liver membranes or human cultured lymphocytes.     

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Processing of angiotensin peptides by NG108-15 neuroblastoma x glioma hybrid cell line

The metabolism of angiotensin (Ang) peptides was studied in NG108-15 neuroblastoma x glioma hybrid cells which express Ang II receptors, renin, dipeptidyl carboxypeptidase A (converting enzyme), as well as Ang I and Ang II. In these experiments, 0.2 nM of either 125I-Ang I or 125I-Ang II was incubated with intact cell monolayers and the medium was analyzed for 125I-products by high performance liquid chromatography. The major product generated from the metabolism of labeled Ang I or Ang II was identified as the amino-terminal heptapeptide Ang-(1-7). N-benzyloxycarbonyl-prolyl-prolinal (ZPP), a specific inhibitor of prolyl endopeptidase, inhibited the formation of Ang-(1-7) from Ang I by 35%. Complete inhibition of Ang-(1-7) generation was attained with p-chloromercuriphenyl-sulfonate, which suggests that a sulfhydryl-containing peptidase other than prolyl endopeptidase is also involved in Ang-(1-7) formation. Ang II was observed to be a minor product resulting from Ang I metabolism. Although the converting enzyme inhibitor enalaprilat (MK-422) significantly reduced Ang II formation, it had no effect on the levels of Ang-(1-7). These findings demonstrate a preferential processing of Ang I into Ang-(1-7) which is not dependent on the prior formation of Ang II.     

Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.     

Interaction between Alzheimer's disease βA4 precursor protein (APP) and the extracellular matrix: Evidence for the participation of heparan sulfate proteoglycans

The interaction between the Alzheimer amyloid precursor protein (APP) and an intact extracellular matrix (ECM), matrigel, obtained from Engelbreth-Holm-Swarm tumors was evaluated. Based on quantitative analyses of the binding data obtained from solid phase binding assays, two binding sites on the ECM were identified for [¹²⁵I]-APP (with apparent Kd₁ of 1.0 × 10 ⁻¹¹ M and Kd₂ of 1.6 × 10 ⁻⁹ M respectively). Over 70% of [¹²⁵I]-APP was displaced by heparin and N-desulfated heparin but not by chondroitin sulfate. Pretreatment of matrigel with heparitinase decreased the binding of [¹²⁵I]-APP by 80%. β-amyloid peptides (residues 1–40, 1–28, and 1–16) containing a heparin binding domain also displaced 80% of bound [¹²⁵I]-APP, which was totally displaced by intact APP. The binding of [¹²⁵I]-APP to matrigel increased by 210% with a decrease in the pH. These observations suggest that [¹²⁵I]-APP interacts mainly with heparan sulfate proteoglycan present in the ECM. The binding of [¹²⁵I]-APP to individual ECM components was also analyzed. [¹²⁵I]-APP was found to bind laminin and collagen type IV but not fibronectin. However, when these ECM constituents were combined, the extent of APP-binding decreased significantly, to levels comparable to those obtained with intact matrigel, suggesting that multiple interactions may occur between ECM constituents and [¹²⁵I]-APP. The results are discussed in terms of APP function and amyloidogenesis. J. Cell. Biochem. 65:145–158. © 1997 Wiley-Liss, Inc.     

Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Effect of prolonged high salt intake on atrial natriuretic factor's kinetics in rats

Plasma atrial natriuretic factor (ANF) paradoxically decreases after 5 weeks (but not after 3 weeks) of 8% NaCl intake in normotensive rats. As this phenomenon remains unaccounted for by changes in ANF production, we studied the disappearance of [125I]ANF(99-126) from the circulation as an alternative explanation of plasma ANF decline. Following 5 weeks (but not 3 weeks) of an 8% NaCl diet, plasma concentrations of [125I]ANF were significantly decreased and metabolic clearance rate and volume of distribution were increased compared to control rats fed a 0.8% NaCl diet. By studying [125I]ANF tissue uptake we noted significantly greater peptide uptake after 5 weeks (but not after 3 weeks) of high salt consumption in several tissues. We hypothesize that prolonged (at least 5 weeks) 8% NaCl ingestion increases the density and/or affinity of ANF binding sites. These changes may be responsible for the previously observed decline in plasma ANF concentrations after a prolonged high salt intake.     

Effects of polyelectrolyte chain stiffness, charge mobility, and charge sequences on binding to proteins and micelles

The binding affinities of polyanions for bovine serum albumin in NaCl solutions from I = 0.01-0.6 M, were evaluated on the basis of the pH at the point of incipient binding, converting each such pH(c) value into a critical protein charge Zc. Analogous values of critical charge for mixed micelles were obtained as the cationic surfactant mole fraction Yc. The data were well fitted as Yc or Zc = KI a, and values of K and a were considered as a function of normalized polymer charge densities (tau), charge mobility, and chain stiffness. Binding increased with chain flexibility and charge mobility, as expected from simulations and theory. Complex effects of tau were related to intrapolyanion repulsions within micelle-bound loops (seen in the simulations) or negative protein domain-polyanion repulsions. The linearity of Zc with radicalI at I < 0.3 M was explained by using protein electrostatic images, showing that Zc at I < 0.3 M depends on a single positive "patch"; the appearance of multiple positive domains I > 0.3 M (lower pH(c)) disrupts this simple behavior.     

Affinity Labeling Mu Opioid Receptors With Novel Radioligands

1. A series of novel opiate ligands based upon 6α-naloxamine have been examined in opioid receptor binding assays.2. Coupling an ethylamine spacer alone to 6-α-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa₁ opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives.3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [¹²⁵I]NalAmiBen and the azido derivative [¹²⁵I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [¹²⁵I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [¹²⁵I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [¹²⁵I]NalAziBen binding showed a band at approximately 70–80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [¹²⁵I]NalAmiBen.     

125I-Ifenprodil: Synthesis and Characterization of Binding to a Polyamine-Sensitive Site in Cerebral Cortical Membranes

Abstract: The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by ¹²⁵I-ifenprodil, a noncompetitive NMDA receptor antagonist, are described. ¹²⁵I-ifenprodil was synthesized using Na¹²⁵I in the presence of chloramine-T and purified by paper chromatography. Binding of the ¹²⁵I-ligand was optimal at pH 7.7 in 5 mM Tris · HCl buffer. Equilibrium binding of ¹²⁵I-ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 μM). Zn²⁺, Ca²⁺, and Mg²⁺ inhibited specific binding of ¹²⁵I-ifenprodil in a concentration-dependent manner, with IC₅₀ values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (K_D) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single-binding-site model (Hill coefficient = 0.95), with a density of binding sites (B_max) of 141 pmol/mg of protein. Binding of ¹²⁵I-ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3-diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine-sensitive binding of ¹²⁵I-ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that σ/antitussive agents partially inhibited specific binding, but inclusion of the σ drug GBR 12909 had little effect on the binding of ¹²⁵I-ifenprodil, suggesting this site was not involved. The binding site labeled by ¹²⁵I-ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the σ site.     

Response of corn to micronutrients (Zn and Cu) on a saline soil. I. Growth and ionic relationships

Summary Response of corn to Zn and Cu on a salinized soil in pots has been studied. Zinc increased the dry weights of tops and roots at all levels of NaCl+CaCl₂. Increasing Zn level increased the weights considerably at 10 mM NaCl+CaCl₂. Copper increased the weight of tops at 10 mM NaCl+CaCl₂: it had little effects on plant weights at 50–125 mM NaCl+CaCl₂. The growth response of plant to low Cu was somewhat similar to that of higher Zinc.NaCl+CaCl₂ treatments, in general, increased Zn concentrations in tops as well as roots. At low Zn application, Zn concentrations in the tops were higher than those in the roots but at high Zn application, the concentrations of Zn in tops were similar to those in the roots.NaCl+CaCl₂ treatments increased Cu concentrations in the tops to a slight extent but had a depressive effect on those in the roots. Copper concentrations in the tops were, however, much below those in the roots. The greater retention of Cu in the roots remains to be explained.     

Multivalent cations depress ligand affinity of insulin-like growth factor-binding proteins-3 and -5 on human GM-10 fibroblast cell surfaces

The effect of multivalent cations on [¹²⁵I]-IGF binding to cell-associated IGFBPs was investigated using human fibroblasts. The major cell-associated binding site for [¹²⁵I]-IGF-I is IGFBP-3 and for [¹²⁵I]-IGF-II are IGFBP-3 and IGFBP-5. Lanthanum and chromium did not affect either [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II binding to cell-associated IGFBPs. By contrast, zinc (Zn²⁺), gold (Au³⁺), and cadmium (Cd²⁺) depressed binding of both ligands. Ligand binding resulted in nonlinear Scatchard plots. Assuming a pre-existent asymmetric model with high- (K_aHi) and low- (K_aLo) affinity sites, Zn²⁺ lowered both K_aHi and K_aLo. Au³⁺ eliminated K_aHi. Assuming that the nonlinear plots were caused by ligand-induced negative cooperativity, Zn²⁺ and Cd²⁺ lowered both K_e and K_f (affinity of unoccupied and saturated IGFBPs, respectively). Au³⁺ eliminated K_e and reduced K_f. Zn²⁺ was active at serum levels in lowering IGF binding. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) indicating similar mechanisms of action. A zinc-binding motif is present in the IGFBPs, but not in the IGFs. We demonstrate for the first time that the trace nutrient zinc and related multivalent cations decrease IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF–IGFBP interaction. J. Cell. Biochem. 69:364–375, 1998. © 1998 Wiley-Liss, Inc.