Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

Interaction of cryptogein with its binding sites in tobacco plasma membrane studied using the piezoelectric biosensor

Elicitins are low-molecular-weight proteins representing the elicitor family secreted by many species of the oomycete Phytophthora. Elicitins induce a hypersensitive reaction in tobacco, a process that is triggered by binding of elicitin to the high-affinity site on the plasma membrane. Specific interaction of cryptogein with the binding sites on tobacco plasma membranes was studied using the piezoelectric biosensor in real time in a flow-through mode. Cryptogeins (wild-type and mutant forms) were covalently immobilized on the sensing surface, and membrane vesicles containing receptors were in solution. Kinetic characterization of the interaction provided values of kinetic rate association (k_a) = 5.74 · 10⁶ M¹ s⁻¹ and kinetic rate dissociation (k_d) = 6.87 10⁻⁴ s⁻¹ constants, respectively. The kinetic equilibrium dissociation constant was calculated as K_D = 12.0 nM. The piezoelectric biosensor appeared to be a convenient tool for studying interactions of receptors embedded in membrane vesicles.     

Characterization of platelet-activating factor binding sites on uterine membranes from pregnant rabbits

One of the earliest signs of endometrial preparation for blastocyst implantation is a localized increase in capillary permeability, an event that is essentially inflammatory in character and thought to be a prerequisite for subsequent decidual tissue formation. Platelet-activating factor (PAF), chemically identified as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, is a very potent vasoactive compound that recently has been implicated in the implantation process. In the present study, PAF binding sites are characterized in the rabbit uterus. A specific, reversible, saturable, and thermally labile binding of [3H]PAF to uterine membranes has been demonstrated, exhibiting multiple binding sites. The equilibrium dissociation constant (Kd) of the higher affinity binding site (type 1) was 3.6 +/- 0.4 nM (mean +/- SD) with a binding capacity (Bmax) of 3.4 +/- 1.6 pmol/mg protein. The second (lower affinity) binding site (type 2) had an apparent Kd of 114.6 +/- 13.5 nM and a Bmax of 164.3 +/- 17.6 pmol/mg membrane protein, under the conditions of maximal [3H]PAF binding, 25 degrees C, 150 min. Incubations at 4 degrees C for up to 3 h yielded only 30% of the Bmax observed at 25 degrees C. In crude and purified endometrial membrane preparations in which the PAF binding was predominantly located, the affinity of the binding for PAF was significantly higher than for the whole uterus, giving Kds of 1.5 +/- 0.8 and 0.8 +/- 0.5 nM; these latter values were not significantly different. However, the Bmax values of 3.9 +/- 0.9 pmol/mg protein and 376.8 +/- 163.3 fmol/mg protein for the two endometrial preparations, respectively, did differ significantly. Kinetic analysis at 25 degrees C resulted in a calculated Kd of 3.28 +/- 1.14 nM, which did not differ from the value for for the whole uterus at the same temperature, but was greater than for the endometrial preparations. Using 4 nM [3H]PAF to selectively label only the type 1 binding sites, the relative potencies of PAF and its antagonists in displacing [3H]PAF were lyso-PAF greater than CV3988 greater than PAF greater than U66985 greater than A02405 greater than BN52021 greater than U66982. The antagonists SRI 63,441 and L652,731 were ineffective in displacing [3H]PAF at up to 5000-fold molar excess of [3H]PAF. [3H]Lyso-PAF binding at 4 nM was displaceable by PAF. All cations tested, i.e. Ca2+, Mg2+, K+, Na+, and Li+, inhibited [3H]PAF binding. Serine hydrolase inhibitors, diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), inhibited binding, but bacitracin, leupeptin, and antipain stabilized it.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

Enhanced number of actin binding sites on plasma membranes of polyoma virus-transformed fibroblasts

Plasma membranes, isolated from normal (C13) and polyoma virus-transformed (J1) cultured BHK cells were incubated with G-actin under polymerizing conditions, followed by a low-speed centrifugation. The amount of actin attached to the pelleted BHK-J1 plasma membranes was at least twice that on BHK-C13 membranes, indicating a greater number of actin attachment sites on the former. This result was confirmed by the observation that the plasma membranes from the transformed cells were also more active in nucleating polymerization of pyrene-labelled actin. Most of the actin attachment sites could be solubilized by Triton or low-salt extraction treatment.     

Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes

Neuropeptide Y (NPY) binding sites in rat cardiac ventricular membranes have been characterized in detail. 125I-NPY bound to the membranes with high affinity. Binding was saturable, reversible and specific, and depended on time, pH and temperature. Analysis of the binding data obtained under optimal conditions, 2 hr, 18 degrees C and at pH 7.5, revealed the presence of low and high affinity binding sites. The high affinity binding sites had an apparent dissociation constant (Kd) of 0.38 nM and a binding capacity (Bmax) of 7.13 fmol/mg protein. The apparent Kd and Bmax for low affinity binding sites were 22.34 nM and 261.25 fmol/mg protein, respectively. Peptides unrelated to NPY did not compete with 125I-NPY for the binding sites even at 1 microM concentrations, whereas homologous peptides, peptide YY (PYY) and pancreatic polypeptide (PP), and NPY(13-36) inhibited 125I-NPY binding but with lower potency compared to NPY. 125I-NPY binding was sensitive to the nonhydrolyzable GTP analog, Gpp(NH)p, suggesting that the NPY receptor is coupled to the adenylate cyclase system. The ventricular membrane receptor characterized in this study may play an important role in mediating the physiological effects of NPY in the heart.     

Characterization of cortisol binding sites in chicken liver plasma membrane

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.     

High affinity thyroid hormone binding sites on purified rat liver plasma membranes.

Two orders of saturable binding sites for L-T₃ were detected on purified rat liver plasma membranes--a high affinity, low capacity binding site with a Kd of 3.2 ± 0.5 nM, and a lower affinity, higher capacity site with a Kd of 220 ± 50 nM. Competition-inhibition studies revealed that both D-T₃ and L-T₄ (two compounds with lower biological potencies than L-T₃) were also less potent than L-T₃ in competing for these binding sites. The present studies demonstrate, therefore, the presence of specific thyroid hormone binding sites on rat liver plasma membranes. In addition, they suggest that these sites may have a role both in mediating the known effects of thyroid hormones on membrane functions, and in regulating the entry of thyroid hormones into target cells.     

Characterization of fusicoccin binding to receptor sites on cell membranes of maize coleoptile tissues

Abstract Radioactive dihydrofusicoccin (³H-FC), known to have the same biological activity as fusicoccin on plant tissues, has a specific affinity in vitro for sites localized on subcellular, postmitochondrial particles from maize coleoptiles. The analysis of the kinetics of dihydrofusicoccin binding suggests the presence of two classes of sites, one class with a high affinity and a second class with a lower affinity. The high affinity class of sites has a dissociation constant (K_d) of 1.2 × 10^?9 mol dm^?3, and an apparent pH optimum at 5.5. Binding is antagonized by non-physiological pH, high temperatures and protein-reactive substances like HgCl₂, p-chloromercuribenzensulphonate and glutaraldehyde. Treatment of dihydrofusicoccin-bound membrane preparations with Triton X-100 leads to the solubilization of a protein fraction associated with dihydrofusicoccin. These data suggest a protein nature for the receptor sites.     

Hemoglobin binding sites on renal brush-border membranes

Prolonged exposure of renal tubules to hemoglobin markedly reduces kidney function and eventually leads to acute renal failure called pigment nephropathy. Intracellular hemoglobin toxicity is one of main pathomechanisms involved in the disease development. However, the process in which hemoglobin is taken up by renal tubular epithelium has not been characterized so far. Isolated renal brush-border membranes of the rat and radioiodinated rat and human hemoglobins were used. Binding properties were examined by the use of rapid filtration technique. Partial isolation of hemoglobin binding proteins was achieved by affinity chromatography. Our experiments showed that both human and rat hemoglobins can be specifically bound to renal brush-border membranes by one class of low affinity (Kd, 7.7 microM) and high capacity (Bmax, 0.18 nmol/mg protein) binding sites. The sites were relatively selective for hemoglobin. Albumin did not compete with hemoglobin. Cationic molecules cytochrome C and lysine exhibited some competition while strong competition of myoglobin was observed. The binding was affected by EGTA indicating a Ca2+ requirement for the interaction. There was a rise in binding in pH 5.4. Fall in binding activity after preincubation of the membranes with peptidases suggested the proteinaceous nature of the binding sites. Affinity chromatography of membrane proteins extract yielded heterogeneous preparation consisting of proteins with molecular masses of 110, 72, 38 and 27 kDa respectively. The existence of binding sites for hemoglobin in renal brush-border membranes strongly suggests that uptake of the protein by tubular epithelia occurs via adsorptive endocytosis. Increased binding of hemoglobin to the membranes under acidic conditions may explain exacerbation of hemoglobinuric acute renal failure in aciduric states.     

Characterization of naphthaleneacetic Acid binding to receptor sites on cellular membranes of maize coleoptile tissue

Characteristics of and optimum conditions for saturable ("specific") binding of [(14)C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a K(D) of 5 to 7 x 10(-7)m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg(2+) or Ca(2+) above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor ("supernatant factor") found in maize tissue.     

GABAA type binding sites on membranes of spermatozoa

The binding of [3H] gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABAA type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa.     

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes

Summary The specific binding of [³H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding areK _D=4.4nm andB _max=685 fmol/mg protein in presence of 1 m of corticosterone. With respect to the binding of 4nm [³H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone>corticosterone>progesterone>cortisol >prednisolone>testosterone>20-hydroxyprogesterone >cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca²⁺ nor Mg²⁺ affected the binding of [³H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [³H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.     

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.     

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.     

Evidence for the presence of specific binding sites for transcortin in human liver plasma membranes

Binding sites which recognize and bind specifically asialotranscortin and the native transcortin-cortisol complex have been found in plasma membranes of human liver cells. The native conformation of transcortin is an absolute requirement for the binding reaction of the transcortin-hormone complex. Sex-hormone-binding globulin and thyroxine-binding globulin from human serum do not bind to this binding sites.     

Characterization of high-density lipoprotein binding to rat adipocytes and adipocyte plasma membranes

The interaction of high-density lipoproteins (HDL) with adipocytes is important in the regulation of cellular cholesterol flux. To study the mechanisms of HDL binding and cellular processing, we incubated adipocytes isolated from epididymal and perirenal adipose tissue of male Wistar rats (300 g) with HDL1 (1.07-1.10 g/mL) and HDL2 (1.10-1.14 g/mL) fractions separated from rat plasma by gradient ultracentrifugation. Freshly isolated adipocytes were incubated with 125I-labeled HDL for 2 h at 37 degrees C to determine cell-associated uptake and degradation. Adipocytes from both fat regions showed significant cell-associated HDL1 and HDL2 uptake and very high medium degradation (2- to 6-fold higher than uptake). To assess 125I-labeled HDL binding independent of cellular metabolism, we purified adipocyte plasma membranes from isolated adipocytes and used them in binding assays. Binding of HDL1 and HDL2 in the membrane system was 85-95% specific, sensitive to high NaCl concentrations, and abolished by pronase treatment. In contrast to HDL2 binding, the maximum HDL1 binding to perirenal plasma membranes was significantly higher than its binding to epididymal membranes (7.2 +/- 1.3 vs. 4.4 +/- 0.2 micrograms/mg, n = 6, p less than 0.05). This increment in HDL1 binding to perirenal membranes represented an EDTA- sensitive, calcium-dependent component. These results indicate that HDL binding to adipocyte plasma membranes depends on both adipose tissue region and HDL subtype. The membrane binding characteristics, taken together with the cellular uptake results, suggest that adipocytes bind and metabolize HDL and that this interaction may involve a protein receptor.     

Polyamine binding sites in the rat brain hippocampus plasma membranes: MK 801 does not influence the binding process

The study was undertaken to characterize the polyamine binding sites in rat brain hippocampus plasma membranes. There were two types of binding sites for putrescine, Bmax 650 and 100 pmol/mg protein, with Kd1 = 39.2 and Kd2 = 6.7 microM, respectively, while those for spermidine (Spd) and spermine (Spm) represented only one type of population with Bmax 2.55 and 15 nmol/mg protein, respectively. The Kd values for Spd and Spm were 34 and 30.3 microM, respectively. The maximum binding of polyamines was found at pH 8.0. The binding capacity of these molecules was curtailed at 4 degrees C, indicating that the binding is an energy-dependent phenomenon. The specific binding was not appreciably influenced by the addition of MK 801, an antagonist of NMDA receptor, indicating that there are polyamine-specific binding sites that are different from those for MK 801. Glycine also did not significantly influence the binding of these biogenic amines. Interestingly, the addition of polyamino acids (polylysine, polyornithine, and polyglutamic acid) inhibited the polyamine binding to their receptor sites, supporting the notion that positive charge of polyamines could be important factor in the binding process.     

Location of terbium binding sites on acetylcholine receptor-enriched membranes

Acetylcholine receptor-enriched membranes bind 45 terbium cations per receptor. The Tb(III) X-ray scattering factor changes by as much as 30% over a 50 eV range about the L3 absorption edge. We exploit these changes to modulate the contribution of these ions to the X-ray diffraction pattern of oriented receptor-enriched membranes by varying the incident X-ray energy. Difference Fourier analysis of the meridional diffraction amplitudes at two X-ray energies revealed six localized regions of Tb(III) density across the membrane. Most significant is the finding of 18 Tb(III) ions near the entrance and 11 ions near the exit of the ion channel as well as 4 or 5 Tb(III) ions localized in the channel itself. This evidence strongly suggests the presence of anionic carboxylate side-chains on the channel lining.