Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Evidence for Receptor Function of Auxin Binding Sites in Maize : RED LIGHT INHIBITION OF MESOCOTYL ELONGATION AND AUXIN BINDING

When 3- to 4-day-old dark-grown maize (Zea mays L. WF9 × Bear 38) seedlings are given red light, auxin-binding activity localized on endoplasmic reticulum membranes of the mesocotyl begins to decrease after 4 hours; by 9 hours, it falls to 50 to 60% of that in dark controls, on either a fresh weight or total particulate protein basis. Endoplasmic reticulum-localized NADH:cytochrome c reductase activity decreases in parallel. Loss of binding is due to decrease in number of sites, with no change in their affinity for auxin (K_d 0.2 micromolar for naphthalene-1-acetic acid). Elongation of mesocotyl segments in response to auxin decreases with a similar time course. Elongation of segments from irradiated plants shows the same apparent affinity for auxin as that of the dark controls. Auxin-binding activity and elongation response also decrease in parallel down the length of the mesocotyl. These observations are consistent with a role of endoplasmic reticulum-localized auxin binding sites as receptors for auxin action in cell elongation.     

Characteristics of [3H](+)-amphetamine binding sites in the rat central nervous system

Recent studies in our laboratory have demonstrated the presence of specific binding sites for [³H](+)-amphetamine in crude membrane preparations derived from rat brain. In this report we have further characterized the specific binding of [³H](+)-amphetamine in various subcellular fractions of rat brain and demonstrate a greater than five-fold enrichment in the crude synaptosomal (P₂) fraction compared to a crude membrane preparation. Specific [³H](+)-amphetamine binding in crude synaptosomal membranes in saturable and stereospecific with an apparent dissociation constant, K_d, of 2.8 ± 0.5 μM and an estimated maximum number of binding sites, B_max, of 60.4 ± 8.4 pmoles/mg protein derived by Scatchard or Klotz analysis of binding data using filtration assays. Centrifugation assays yield a similar K_d though the apparent B_max is higher. In addition specific [³H](+)-amphetamine binding is: rapidly reversible, temperature sensitive, labile to preincubation in Tris buffer, inhibited by sodium ions and unevenly distributed in various brain regions. Specific [³H](+)-amphetamine binding sites are found almost exclusively in the rat central nervous system (the brainstem, hypothalamus, and striatum exhibiting relatively high levels of binding), whereas peripheral tissues such as liver, kidney and heart have very low to undetectable levels of specific binding.     

Solubilization and Characterization of a Membrane-Bound Auxin-Binding Protein from Cell Suspension Cultures of Nicotiana tabacum

A membrane-bound auxin-binding protein (MABP) was solubilized by Triton X-100 from cell suspension cultures of Nicotiana tabacum L. Solubilization of MABP was dependent on the detergent concentration and more than 80% of naphthalene-1-acetic acid (NAA)-binding activity was recovered by an optimum concentration of 0.2%. The solubilized MABP was highly heat-unstable and sensitive to protease. The properties of MABP (affinity, temperature dependence, pH optimum, and analog specificity for auxin binding) did not significantly change after solubilization, e.g. the solubilized MABP showed no or very low levels of NAA-binding at 0 to 4°C but showed a high-affinity binding (dissociation constant K_d = 2.7 ± 0.3 × 10⁻⁷m) at 25°C at an optimum pH of 5.0. NAA-binding of the solubilized MABP proceeded very slowly, i.e. a time of half-maximum binding was at least 15 minutes, although the solubilized MABP showed higher rates of association (k₁ = 1.3 versus 0.9 × 10⁵m⁻¹ min⁻¹) and dissociation (k₋₁ = 2.2 versus 1.6 × 10⁻² min⁻¹) with NAA than the bound MABP. These results show that specific, saturable, and reversible auxin binding to MABP from dicotyledonous N. tabacum differs from that from monocotyledonous Zea mays, and confirm that MABP is distinct from a soluble auxin-binding protein which also is present in N. tabacum.     

Binding of Escherichia coli heat-stable enterotoxin and rise of cyclic GMP in COLO 205 human colonic carcinoma cells

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [¹²⁵I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a K_d of 0.5×10⁻¹⁰ M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [¹²⁵I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Sulphydryl groups in the maintenance of the activity of binding protein for N-1-naphthylphthalamic acid fromAcer pseudoplatanus cells

Binding protein for N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, was studied by analysis of the effects of reactions which modify particular amino acid side chains upon their binding activity. Na₂SO₃, N-ethylmaleimide (NEM) and dithiobisnitrobenzoic acid all inhibited the specific binding of NPA to its binding protein fromAcer pseudoplatanus L. cells. The presence of 10^-6 M Na₂SO₃ in the binding assay reduced the affinity of the binding protein to NPA from K_d of 1.5 ￡ 10^-8 M to K_d of 2.1 ￡ 10_-8 M, while concentration of the binding protein was not significantly changed. When the same analysis was applied to NPA binding to the NEM-treated membrane particles, it was found that NEM inactivated binding without changing the affinity for NPA. This study revealed the importance of sulphydryl group(s) in the maintenance of NPA binding protein activity.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Inactivation by phenylglyoxal of the specific binding of 1-naphthyl acetic Acid with membrane-bound auxin binding sites from maize coleoptiles

The specific binding of 1-[³H]naphthyl acetic acid (NAA) to membrane-bound binding sites from maize (Zea mays cv INRA 258) coleoptiles is inactivated by phenylglyoxal. The inactivation obeys pseudo first-order kinetics. The rate of inactivation is proportional to phenylglyoxal concentration. Under conditions at which significant binding occurs, NAA, R and S-1-naphthyl 2-propionic acids protect the auxin binding site against inactivation by phenylglyoxal. Scatchard analysis shows that the inhibition of binding corresponds to a decrease in the concentration of sites but not in the affinity. The results of the present chemical modification study indicate that at least one arginyl residue is involved in the positively charged recognition site of the carboxylate anion of NAA.     

Azido auxins: quantitative binding data in maize

The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N₃IAA), and of the photoproducts of 5-N₃IAA to the naphthalene-1-acetic acid (NAA) binding sites of Zea mays L. WF9 × BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4- and 5-N₃IAA bind to these sites with affinities similar to that of IAA, while 6-N₃IAA and the photoproducts of 5-N₃IAA bind less tightly. This binding is fully reversible in the dark. Binding of 5-N₃IAA becomes covalent and irreversible upon UV irradiation, as evidenced by a 30% loss in NAA binding at sites pretreated with 5-N₃IAA and UV irradiation, then washed extensively. IAA or NAA, included with this 5-N₃IAA pretreatment, can protect the sites from blockage, whereas benzoic acid and tryptophan are unable to protect the site, indicating that 5-N₃IAA specifically labels the auxin sites.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Specific and saturable binding of albumin to rat adipocytes: Modulation by epinephrine and possible role in free fatty acid transfer

Specific and saturable binding of ¹²⁵I-bovine albumin to rat adipocytes in suspension was observed (apparent K_d 2.09 ± 0.52 × 10^?6 M; 8.58 ± 2.49 × 10⁶ sites per cell; mean ± SEM). The binding was rapid and reversible for at least 10 min, suggesting that endocytosis of albumin was minor under assay conditions. Pre-incubation of cells with epinephrine bitartrate caused an apparent increase in number and decrease in affinity of the adipocyte binding sites for albumin. These findings suggest that a specific and saturable interaction of albumin with the adipocyte surface may play a role in the cellular uptake and release of free fatty acids.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

Stereospecific,reversible binding of D-[3H] glucose to crude membrane fractions prepared from rat brain

To a crude preparation of synaptic membranes prepared from rat brain, stereospecific, saturable, reversible binding was described of D-[³H]glucose. Binding showed a K_d = 0.45 μM and the fractional rate of dissociation was approximately eight times the fractional rate of association. D-[³H]glucose binding was displaced by 2-deoxyglucose and 3-0-methylglucose and it was abolished when membranes were denatured by heating.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

K+ channels of stomatal guard cells: bimodal control of the K+ inward-rectifier evoked by auxin

The influence of the auxins indole-3-acetic acid (IAA) and 1-napthylene acetic acid (NAA) on K⁺ channels and their control was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes to record membrane potentials and to monitor K⁺ channel currents under voltage clamp during exposures to 0.1–100 µM IAA and NAA. Following impalements, challenge with either IAA or NAA in the presence of 10 mM KCl resulted in the concerted modulation of at least four different currents with distinct kinetic characteristics and concentration dependencies. Equivalent concentrations of benzoic acid were wholly without effect. Most striking, current carried by inward-rectifying K⁺ channels (I_K,in) exhibited a bimodal response to both IAA and NAA which was reversed on washing the auxins from the bathing medium. The steady-state current was augmented 1.3- to 2-fold at concentrations between 0.1 and 10 µM and antagonized at concentrations near 30 µM and above. Auxin agonism of I_K,in was time- and voltage-independent. By contrast, I_K,in inactivation at the higher auxin concentrations was marked by a voltage-dependence and slowing of the kinetics for current activation. Inactivation of I_K,in by the auxins was relieved when cytoplasmic pH (pH_i) was clamped near 7.0 in the presence of 30 mM Na⁺-butyrate. In addition to the control of I_K,in, current carried by a second class of (outward-rectifying) K⁺ channels rose in a monotonic and largely voltage-independent manner with auxin concentrations about 10 µM and above, and IAA and NAA also activated an inward-going current with a voltage dependence characteristic of guard cell anion channels. Further changes in background current were consistent with a limited activation of the H⁺-ATPase. Over the concentration range examined, the auxins evoked membrane hyperpolarizations and depolarizations of up to ±12–19 mV, depending on the free-running membrane potential prevailing before auxin additions. Prolonging exposures to 100 µM auxin beyond 3–5 min frequently elicited rapid transitions to voltages near E_K as well as regenerative action potentials. However, in every case the voltage response was a predictable consequence of auxin action on the K⁺ channels and, at 100 µM auxin, on the anion current. These results demonstrate a control of K⁺ channel activity by auxin, consistent with the roles of these channels in mediating K⁺ flux for stomatal movements; the data associate a bimodal characteristic with the activity of I_K,in, implicating pH_i as a putative intermediate in its control, and offer strong evidence for a multiplicity of signal cascades evoked by auxin; finally, they highlight a coordinate modulation of transport activities by auxin, thereby drawing a close analogy to the pattern of stimulus-response coupling in abscisic acid.     

Localization and pharmacological characterization of nicotinic-cholinergic binding sites in cockroach brain using α- and neuronal bungarotoxin

[¹²⁵I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (K_d = 1.09 nM) having a B_max of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a k_obs = 4.37 × 10⁻³s⁻¹. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10⁻⁵ and 9.94 × 10⁻⁵s⁻¹. The K_ds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized.     

Gravitropism in Higher Plant Shoots : VI. Changing Sensitivity to Auxin in Gravistimulated Soybean Hypocotyls

Although the Cholodny-Went model of auxin redistribution has been used to explain the transduction phase of gravitropism for over 60 years, problems are apparent, especially with dicot stems. An alternative to an auxin gradient is a physiological gradient in which lower tissues of a horizontal stem become more sensitive than upper tissues to auxin already present. Changes in tissue sensitivity to auxin were tested by immersing marked Glycine max Merrill (soybean) hypocotyl sections in buffered auxin solutions (0, 10⁻⁸ to 10⁻² molar indoleacetic acid) and observing bending and growth of upper and lower surfaces. The two surfaces of horizontal hypocotyl sections responded differently to the same applied auxin stimulus; hypocotyls bent up (lower half grew more) in buffer alone or in low auxin levels, but bent down (upper half grew more) in high auxin. Dose-response curves were evaluated with Michaelis-Menten kinetics, with auxin-receptor binding analogous to enzyme-substrate binding. V_max for the lower half was usually greater than that for the upper half, which could indicate more binding sites in the lower half. K_m of the upper half was always greater than that of the lower half (unmeasurably low), which could indicate that upper-half binding sites had a much lower affinity for auxin than lower-half sites. Dose-response curves were also obtained for sections `scrubbed' (cuticle abraded) on top or bottom before immersion in auxin, and `gravitropic memory' experiments of L. Brauner and A. Hagar (1958 Planta 51: 115-147) were duplicated. [1-¹⁴C]Indoleacetic acid penetration was equal into the two halves, and endogenous plus exogenously supplied (not radiolabeled) free auxin in the two halves (by gas chromatography-selected ion monitoring-mass spectrometry) was also equal. Thus, differential growth occurred without free auxin redistribution, contrary to Cholodny-Went but in agreement with a sensitivity model.