Biological effects of electromagnetic fields

Life on earth has evolved in a sea of natural electromagnetic (EM) fields. Over the past century, this natural environment has sharply changed with introduction of a vast and growing spectrum of man-made EM fields. From models based on equilibrium thermodynamics and thermal effects, these fields were initially considered too weak to interact with biomolecular systems, and thus incapable of influencing physiological functions. Laboratory studies have tested a spectrum of EM fields for bioeffects at cell and molecular levels, focusing on exposures at athermal levels. A clear emergent conclusion is that many observed interactions are not based on tissue heating. Modulation of cell surface chemical events by weak EM fields indicates a major amplification of initial weak triggers associated with binding of hormones, antibodies, and neurotransmitters to their specific binding sites. Calcium ions play a key role in this amplification. These studies support new concepts of communication between cells across the barriers of cell membranes; and point with increasing certainty to an essential physical organization in living matter, at a far finer level than the structural and functional image defined in the chemistry of molecules. New collaborations between physical and biological scientists define common goals, seeking solutions to the physical nature of matter through a strong focus on biological matter. The evidence indicates mediation by highly nonlinear, nonequilibrium processes at critical steps in signal coupling across cell membranes. There is increasing evidence that these events relate to quantum states and resonant responses in biomolecular systems, and not to equilibrium thermodynamics associated with thermal energy exchanges and tissue heating.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca²⁺ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb²⁺, a structural and functional surrogate of Ca²⁺, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb²⁺-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.     

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth

We have quantitated by autoradiography the binding of [¹²⁵I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G₁ phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [¹²⁵I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Changes in cell surface glycoproteins on non-differentiating L6 rat myoblasts selected for resistance to concanavalin A

Four independently selected conA-resistant, non-differentiating rat L6 myoblast cell lines and their parental wild-type populations were examined for cell surface alterations. [³H]conA-binding studies indicated that the variant myoblasts bound significantly less lectin than wild-type cells at 4 and at 37 °C. Scatchard analysis revealed two general types of binding sites (high and low affinity sites) on wild-type cells; the variants appeared to be deficient in the high affinity sites. These changes in conA binding probably play an important role in determining the conA-resistant phenotype. Lectin-binding results could be significantly modified by altering the composition of the serum in the growth medium used to culture myoblasts prior to performing binding experiments, suggesting the existence of productive and non-productive lectin-binding sites on the cell surface. SDS slab gel electrophoresis of [³H]mannose-labelled surface membranes prepared from variant and wild-type cells showed that several glycoproteins of the conA-resistant myoblasts were defective in mannosylation. The conA-binding abilities of a pronase digest of one of these altered regions from variant separations, with a molecular weight of 44 500 D, was found to contain glycopeptides with reduced affinity for the lectin, supporting the idea that variant membranes are deficient in a set of high affinity lectin-binding sites. Studies on [GDP-¹⁴C]-mannose incorporation into lipid by membranes from variant and wild-type myoblasts indicated that the biosynthetic lesion likely involved a mannosyl transferase enzyme directly, rather than a lack of free dolichol-PO₄. These studies link conA resistance, cell surface glycoprotein alterations, and defective mannosyl transferase activity with the inability to carry out normal cellular differentiation to form multinucleated myotubes.     

Calcium-binding properties and ATPase activities of rat liver plasma membranes

Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a K_D of 3 µM. Adrenalectomy increases the number of the higher affinity sites and the corresponding K_D. Plasma membranes exhibit a (Na⁺-K⁺)-independent-Mg²⁺-ATPase activity which is not activated by calcium between 0.1 µM and 10 mM CaCl₂. Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na⁺-K⁺)-independent ATPase. Both Mg²⁺- and Ca²⁺-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [γ-³²P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca²⁺-activated-Mg²⁺-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level.     

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [³H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K_0.5 = 2.8 × 10⁻⁷ molar. The K_0.5 for NPA under these conditions is 2 × 10⁻⁸ molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [³H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [³H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.     

1H and 13C nuclear magnetic resonance spectra of the lipids in normal and SV 40 virus-transformed hamster embryo fibroblast membranes

Well resolved ¹H and ¹³C NMR spectra were obtained with normal and SV 40-transformed cell membranes. Estimation of the ratio of ¹³CT₂ values of the normal to transformed cell membranes showed an increased intermolecular motion in the transformed cell membranes. The temperature dependence of the (CH₂)_n line in the ¹H spectra in the temperature range 298–343 °K shows an activation energy for the lateral diffusion of the fluid phospholipid regions in the normal cell membranes while the transformed ones show practically no temperature dependence in this temperature range. The fluidity of the phospholipid region in the transformed cell membrane seems to be significantly higher than that observed in the normal cell material. These data support and extend the findings concerning the mobility of the concanavalin A binding/agglutinating sites on the surface of normal and virus-transformed cells and suggest further approaches to the study of the membrane alterations in tumor cells.     

Molecular characteristics of glutamate receptors in the mammalian brain

Summary The strong excitatory activity of L-glutamic acid on central nervous system neurons is thought to be produced by interaction of this amino acid with specific neuronal plasma membrane receptors. The binding of L-glutamate to these surface receptors brings about an increase in membrane permeability to Na⁺ and Ca²⁺ ions presumably through direct activation of ion channels linked to the membrane receptors. The studies described in this paper represent attempts to define the subcellular distribution and pharmacological properties of the recognition site for L-glutamic acid in brain neuronal preparations, to isolate and explore the molecular characteristics of the receptor recognition site, and, finally, to demonstrate the activation of Na⁺ channels in synaptic membranes following the interaction of glutamate with its receptors.Radioligand binding assays with L-[³H] glutamic acid have been used to demonstrate a relative enrichment of these glutamate recognition sites in isolated synaptic plasma membranes. The specific binding of L-[³H] glutamate to these membrane sites exhibits rapid association and dissociation kinetics and rather complex equilibrium binding kinetics. The glutamate binding macromolecule from synaptic membranes has been solubilized and purified and was shown to be a small molecular weight glycoprotein (M_T 13 000). This protein tends to form aggregates which have higher specific activity at low concentrations of glutamate than the M_T 13 000 protein has. The overall affinity of the purified protein is lower than that of the high affinity sites in the membrane. Nevertheless, the purified protein exhibits pharmacological characteristics very similar to those of the membrane binding sites. On the basis of its pharmacological properties this protein belongs in the category of the physiologic glutamate preferring receptors.By means of differential solubilization of membrane proteins with Na-cholate, it was shown that this recognition site is an intrinsic synaptic membrane protein whose binding activity is enhanced rather than diminished by cholate extraction of the synaptic membranes. The role of membrane constituents in regulating the binding activity of this protein has been explored and a possible modulation of glutamate binding by membrane gangliosides has been demonstrated. Finally, this glutamate binding glycoprotein is a metalloprotein whose activity is dependent on the integrity of its metallic (Fe) center. This is a clear distinguishing characteristic of this protein vis-à-vis the glutamate transport carriers.The presence of functional glutamate receptors in synaptosomes and resealed synaptic plasma membranes has also been documented by the demonstration of glutamate-activated Na⁺ flux across the membrane of these preparations. The bidirectionality, temperature independence, and apparent desensitization of this stimulated flux following exposure to high concentrations of glutamate are properties indicative of a receptor-initiated ion channel activation. It would appear, then, that the synaptic membrane preparations provide a very useful system for the study of both recognition and effector function of the glutamate receptor complex.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.     

Effect of phagocytosis on guinea pig granulocyte membrane markers

The internalization of membrane markers during phagocytosis was followed in guinea pig granulocytes as a function of the extent of particle ingestion. The plasmalemma was carefully labeled with diazotized ³⁵S-sulfanilate, or by treatment with periodate and sodium ³H-borohydride. These treatments provided general membrane markers. More specific markers used were 5′-nucleotidase, neuraminidase-releasable membrane sialate, and concanavalin A binding sites. In all cases except the last, internalization of membrane was directly determined on isolated phagosomes; disappearance of binding sites from the cell surface was followed in the last instance. Both phagosomal levels and disappearance from the surface were measured in the case of 5′-nucleotidase, permitting balance studies. Phagocytosis was determined as uptake of paraffin emulsions labeled with Oil-Red 0. “Marker/particle” ratios were determined as the percent external marker internalized per mg paraffin ingested. The “marker/particle” ratios for cells with chemically labeled membranes were considered to reflect random internalization of membrane entities. Colchicine had no effect on those ratios. Internalization of 5′-nucleotidase and neuraminidase-releasable sialate gave “marker/particle” ratios similar to those of the random markers, and these increased in the presence of colchicine. Concanavalin A binding sites did not appear to be removed from the cell surface in the absence of colchicine, but disappearance was observed in its presence. Control experiments indicated that these changes due to colchicine must be very cautiously interpreted. Our results differ from those obtained by others, and the reasons due to species, cell type, experimental design, etc. are discussed. Maximal particle uptake was computed to require internalization of one-fifth to one-third of the total external membrane of the cells—based on the assumption of random internalization of markers.     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

Coexistence of central and peripheral benzodiazepine binding sites in the human pineal gland

The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [³H] flunitrazepam and [³H] PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [³H] flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [³H] flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [³H] PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [³H] PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [³H] PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Photo-activated inhibition of sulfate equilibrium exchange in human erythrocyte ghosts by a 4-azido-2-nitrobenzoate derivative of phlorizin

Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO₄^2? equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO₄^2? exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular tritiated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccessibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.     

Beta-adrenoceptors of human lymphocytes: decrease in affinity of agonist for (-)3h-dihydroalprenolol binding sites by cytoplasmic fractions

The beta-adrenoceptors of intact human lymphocytes were investigated by binding assays with a radiolabeled β-adrenergic antagonists, (?)³H-dihydroalprenolol (DHA). Results shown are 1.48 ± 0.57 × 10⁴ binding sites for the ligand per cell. Binding of (?)³H-DHA to the membrane fractions of human lymphocytes was studied for comparison. The affinity of (?)³H-DHA for membrane was similar to that for whole lymphocytes, but the binding to the membranes was inhibited more strongly by β-adrenergic agonist. Moreover, the cytoplasmic fraction of human lymphocytes decreased the inhibition by (?)-isoproterenol of (?)³H-DHA binding to the membrane fraction. The results imply that the cytoplasmic fraction of human lymphocytes lowered the affinity of (?)-isoproterenol to (?)³H-DHA binding sites and left no effect on the binding of the β-adrenergic antagonist to the sites.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes

The binding of hemoglobins A, S, and A₂ to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with ¹⁴C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A₂, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4–22 °C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied.An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites.These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Specific 3,3′,5′-Triiodothyronine (Reverse T3) Binding Sites on Rat Liver Plasma Membranes: Comparison with Thyroxine (T4) Binding Sites

Abstract

The binding characteristics of thyroxine (T4), triiodothyronine (T3), and reverse T3 (rT3) to rat liver plasma membranes (RLPM) were examined to explore the interactions of thyroid hormones with cell surface receptors. Scatchard analysis suggested that all three ligands bound to two classes of binding sites. The high affinity rT3 binding sites appeared to be distinct from the high affinity T4 sites, on the basis of differing optimum physicochemical conditions for binding, and analog displacement studies. The higher affinity constant for T4 was 1.7 ± 0.2 × 10⁹ M^-1 (mean ± SEM) and binding capacity was 3.1 ± 0.3 pmol mg ^-1 protein whereas for rT3 binding the Ka was 2.5 ± 0.4 × 10⁸ M^-1 and capacity was 6.2 ± 0.9 pmol mg ^-1. (¹²⁵ I) T3 bound with lower affinity and T3 tracer was readily displaced by T4. Moreover, comparatively higher concentrations of T3 were needed to displace either radiolabeled T4 or rT3, suggesting that T3 was binding to both the T4 and rT3 sites with lower affinity. Marker enzyme studies on RLPM, of varying purity prepared by different methods, showed a positive correlation between the activity of the plasma membrane enzyme magnesium-stimulated ATPase and high affinity rT3 and T4 binding. Column chromatography of the radioligands, after dissociation from membrane binding sites, confirmed that the integrity of the hormones was not altered during association or dissociation. Our results raise the possibility that the high affinity T4 and rT3 binding sites on RLPM may be hormone receptors mediating biological actions at the membrane level.