Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.     

Specific binding of proinsulin C-peptide to intact and to detergent-solubilized human skin fibroblasts

Proinsulin C-peptide exerts physiological effects on kidney and nerve function, but the mechanisms involved remain incompletely understood. Using fluorescence correlation spectroscopy, we have studied binding of rhodamine-labelled human C-peptide to intact human skin fibroblasts and to detergent-solubilised extracts of fibroblasts, K-562, and IEC-6 cells. Specificity was shown by displacement of rhodamine-labelled human C-peptide with unlabelled human C-peptide. C-peptide was found to bind to the cell membranes of intact fibroblasts with an association constant of 3 x 10(9) M(-1), giving full saturation at about 0.9 nM, close to the physiological C-peptide plasma concentration. Treatment of all investigated cells with the zwitter-ionic detergent Chaps was found to release macromolecules that bind specifically to C-peptide. The binding in Chaps extracts of fibroblasts was sensitive to time but remained reproducible for up to 2 h at room temperature. Lysophosphatidylcholine, Triton X-100, beta-octylglucopyranoside, SDS, or cholate gave extracts with only low or nonspecific binding. It is concluded that C-peptide binding components can be solubilised from cells, and that Chaps appears to be a suitable detergent.     

Platelet-activating factor binding to human platelet membranes

Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor.     

Characterization of the binding of a rat somatomedin to receptors in human placental cell membranes.

The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [¹²⁵I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10^?9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [¹²⁵I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.     

The binding of snake venom cardiotoxins to heart cell membranes

Cobra venom cardiotoxins have the effect, inter alia, of causing systolic arrest of the heart. We have observed significant binding in vitro of ³⁵S-labelled cardiotoxins to mouse heart cell membranes. Part of the binding was saturable and could be displaced with homologous unlabelled cardiotoxins but not by neurotoxins or cardiotoxins inactivated by chemical modification. The specifically bound component represented more than 70% of total binding at saturation. Inclusion of Triton X-100 and NaCl in the phosphate-buffered incubation medium prevented nonspecific adsorption to centrifuge tube walls, and gave lower but more reproducible specific binding results, respectively. An apparent dissociation constant of 5·10^?7 M and a binding density of 500 pmol toxin/mg membrane protein were derived from the saturation isotherms.     

Aluminum binding to phosphatidylcholine lipid bilayer membranes: 27Al and 31P NMR spectroscopic studies

27Al and 31P nuclear magnetic resonance (NMR) spectroscopies were used to investigate aluminum interactions at pH 3.4 with model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). A solution state 27Al NMR difference assay was developed to quantify aluminum binding to POPC multilamellar vesicles (MLVs). Corresponding one-dimensional (1D) fast magic angle spinning (MAS) 31P NMR spectra showed that aluminum induced the appearance of two new isotropic resonances for POPC shifted to -6.4 ppm and -9.6 ppm upfield relative to, and in slow exchange with, the control resonance at -0.6 ppm. Correlation of the (27)Al and (31)P NMR binding data revealed a 1:2 aluminum:phospholipid stoichiometry in the aluminum-bound complex at -9.6 ppm and a 1:1 aluminum:phospholipid stoichiometry in that at -6.4 ppm. Slow MAS 31P NMR spectra demonstrated shifts in the anisotropic chemical shift tensor components of the aluminum-bound POPC consistent with a close coordination of aluminum with phosphorus. A model of the aluminum-bis-phospholipid complex is proposed on the basis of these findings.     

Glucocorticoid binding to solubilized components of human placental trophoblast membranes

We have recently described glucocorticoid uptake by human placental membrane vesicles which is specific, saturable and has a low K (7 nM). This paper describes solubilization of these vesicles with Triton x-100 and successful demonstration of glucocorticoid binding to the putative transport site. This was accomplished by analysis of corticosterone binding to the 140,000 × g supernatant of solubilized vesicles using G-75 Sephadex chromatography. The amount of bound corticosterone present in the void volume was proportional to the concentration of solubilized vesicles. The specificity of binding was tested by coincubation of tritiated corticosterone with 100-fold excesses of various steroids. The relative ability of various steroids to inhibit binding was corticosterone=pregesterone- >aldosterone. Triamcinolone acetonide, and estradiol were ineffective competitors. We conclude from these studies that human placental membranes contain glucocorticoid-specific binding sites which can be solubilized with Triton x-100. It is possible that these sites are involved in membrane mediated transport of glucocorticoids by this tissue.     

GTP regulation of platelet-activating factor binding to human neutrophil membranes

Radiolabeled ligand binding studies showed that specific receptors for platelet-activating factor are present in human neutrophil membranes. GTP at 10(-7) to 10(-3) M decreased the specific binding of platelet-activating factor to neutrophil membranes in a dose-dependent manner. Inhibition of platelet-activating factor binding was also induced by other guanine nucleotides but not by adenine nucleotides. Our results suggest that platelet-activating factor receptor in human neutrophil membranes may be coupled to a guanine nucleotide binding protein.     

Characteristics of calmodulin binding to purified human lymphocyte plasma membranes

We have explored the role of calmodulin in plasma membrane-related phenomena in lymphocyte activation by measurement of [125I]calmodulin binding to highly purified plasma membrane of human peripheral blood lymphocytes. Calcium-dependent calmodulin binding to lymphocyte membrane was found to reach equilibrium within 5 min of incubation at 37 degrees C and to be saturable and specific. A single class of high affinity-binding sites was identified, with a dissociation constant (Kd) of 1 to 3 X 10(-8) M and a total binding capacity (Bt) of 1 to 2 pmol/mg membrane protein. The free calcium concentration necessary for half-maximal binding was 100 to 300 nM. This was strikingly similar to the cytoplasmic-free calcium activity [Ca2+]i measured by the Quin-2 fluorescence technique, particularly after stimulation with phytomitogens. Calmodulin binding was inhibitable by trifluoperazine (TFP), W-7, and chloropramazine, all of which are calmodulin antagonists. The concentration of TFP that caused 50% inhibition of lymphocyte proliferative responses to phytomitogens was found to be identical to the concentration of TFP which causes 50% inhibition of calmodulin binding to lymphocyte plasma membrane. SDS-polyacrylamide gel electrophoresis followed by gel overlay and autoradiography with iodinated calmodulin revealed five calcium-dependent, TFP-inhibitable, calmodulin-binding polypeptides.     

Calcium binding by human erythrocyte membranes

1. The characteristics of Ca²⁺ binding to haemoglobin-free human erythrocyte membranes were investigated by using ⁴⁵Ca and centrifugation partition of `ghosts' from their external incubation medium. Equilibrium of `ghosts' with external Ca²⁺ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 `ghosts' bound a maximum of 283μmol of Ca<sup>2+</sup>/g of `ghost' protein, equivalent to 6.85×10⁷ Ca²⁺ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca²⁺ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca²⁺ bound. 6. Extraction of ⁴⁵Ca-labelled `ghosts' with chloroform–methanol showed that the distribution of Ca²⁺ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca²⁺ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca²⁺: phosphorus ratio of about 1:2.     

Plasmodium falciparum polypeptides interacting with human red cell membranes show high affinity binding to Band-3

P. falciparum proteins were labelled with [35S]methionine and harvested at various asexual stages. A number of parasite proteins bound to uninfected red cell membranes (ghosts). Some of these proteins differentially partitioned when ghosts were extracted with detergent. Several of these proteins bound very strongly to immobilised whole ghost proteins or immobilised purified Band-3 in a stage-specific manner, but not to a sham-coupled matrix or to immobilised Band-3 extract from cells rendered refractory to invasion. Such specific binding of parasite proteins to immobilised Band-3 supports recent conjecture as to its role as a host receptor during parasite invasion. However, our results demonstrate the complex and multifactorial nature of the interaction between parasite and host proteins during invasion and development.     

Deprotonation of Glu9 destabilizes the alpha-helix in C-peptide of RNase A

pK values of Glu2, Glu9, and His12 in the lactone and carboxylate forms of C-peptide at 4.5 and 21 degrees, 0.1 M NACl, have been determined from pH-tritration shifts of n.m.r. signals of the glutamate gamma and histidine ring protons. These have been used in an analysis of the well known pH-induced helix-coil transition in the C-peptide lactone. It has been shown that Glu12 deprotonation results in a twofold increase of the helix content, whereas deprotonation of Glu9 leads to a 28% drop. This latter effect is probably related to Glu-9-His+12 salt bridge formation. The transition within the pH range 5.5-7.5 follows exactly the deprotonation curve of His+12 residue.     

Testosterone-estradiol-binding globulin binds to human prostatic cell membranes

Specific binding sites for human testosterone-estradiol-binding globulin have been found on human prostatic cell membranes. Scatchard analysis reveals both a high and a low affinity binding site for [125I]testosterone-estradiol-binding globulin. The high affinity site is specific for testosterone-estradiol-binding globulin, whereas the low affinity site also binds human corticosteroid-binding globulin and human transferrin.     

Characterization and specificity of lipoprotein binding to term human placental membranes

The binding characteristics of very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoprotein fractions to a purified human term placental microvillous membrane preparation were determined. Binding of LDL was saturable with a maximal binding capacity of 270 ng LDL protein per mg of membrane protein. Scatchard analysis revealed the presence of a single population of 3.4 · 10¹¹ sites per mg of membrane protein and a mean affinity constant of 5.8 · 10⁻⁹ M. Binding of VLDL was also saturable but the maximal capacity was 4.5-times greater than that of LDL. The Scatchard analysis revealed the presence of 2.1 · 10¹¹ binding sites and an affinity constant nearly one order of magnitude greater than that of LDL. Binding of HDL showed less tendency to saturate. Scatchard analysis showed a similar number of receptor sites to that calculated for VLDL and LDL but the affinity constant for HDL was over 100-fold less than that of VLDL. Self- and cross-inhibition studies of VLDL and LDL binding revealed that VLDL was better at blocking the binding of LDL than was LDL itself. This preferential binding of VLDL suggests that this lipoprotein fraction could be an important source of cholesterol for placental progesterone production.     

Specific binding of the C-peptide of proinsulin to cultured B-cells from a transplantable rat islet cell tumor

Specific binding of the C-peptide of proinsulin was evaluated using a transplantable NEDH rat islet cell tumour predominantly composed of insulin-secreting B-cells. Cultured tumour B-cells exhibited greater than 90% viability assessed by trypan blue exclusion, and retained the ability to form tumours with accompanying hypoglycaemia and hyperinsulinaemia after reimplantation. During binding experiments with synthetic rat C-peptide I and iodinated tyrosylated rat C-peptide I, turnout B-cells exhibited 54±6% specific binding. Displacement of tracer increased with increasing concentrations of unlabelled rat C-peptide I (0.25–1,000 ng/ml), and the specificity of binding was substantiated by reduced displacement with human C-peptide. Scatchard analysis of specific C-peptide binding revealed a curvilinear plot with upward concavity. The demonstration of specific C-peptide binding to insulin-secreting B-cells provides evidence for a physiological role of proinsulin C-peptide.     

Calmodulin binding proteins in human erythrocyte membranes

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.     

Muscarinic cholinergic binding in human erythrocyte membranes

Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([³H]QNB). The apparent K_D is 1.3 × 10^?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system.