Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs,monensin, low temperature,and chloroquine

We studied the effects of low temperature (20–37°C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37°C, hepatocytes were incubated at 37°C, 31°C, 25°C, or 20°C or treated with or without inhibitors at 37°C in the absence of ligand. The cells were then assayed at 4°C for ¹²⁵I-asialo-orosomucoid binding, to measure receptor activity, or ¹²⁵I-anti-Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin-permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub-37°C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50–65% of their surface Gal receptor activity but only ? 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and75%, respectively, were present intracellularly in monensin-and chloroquine-treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.     

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes

The influence of a mild heat shock on the fate of the insulin-receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330–340]. After exposure from 15 min to 2 hr at 42.5°C, the amount of ¹²⁵I-insulin associated with cells at 37°C was progressively decreased (by 35% after 1 hr), while the release of ¹²⁵I-insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin-induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37°C of receptors previously internalized at 42.5°C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37°C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock-stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between ¹²⁵I-insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone-like action in fetal hepatocytes submitted to heat stress. © 1996 Wiley-Liss, Inc.     

Uptake of 125I-labelled α2-macroglobulin and albumin by human placental syncytiotrophoblast in vitro

We have investigated the binding and internalization of α₂-macroglobulin and serum albumin by human placental syncytiotrophoblast cells in vitro. The time course (obtained at 4°C) of α₂-macroglobulin binding indicated that an equilibrium was reached after 4 h. The binding of ¹²⁵I-labelled α₂-macroglobulin to syncytiotrophoblast cells was competitively reduced in the presence of excess unlabelled α₂-macroglobulin. When the concentration-dependence of binding was examined over a wide concentration range, non-linear regression analysis yielded a K_d of 6.4 nM. In the case of albumin, binding was weak and ligand dissociated from the cell surface during aqueous washing making it impractical to analyze the binding reaction. In other experiments, syncytiotrophoblast cells were incubated with ¹²⁵I-labelled α₂-macroglobulin at 37°C. Under these conditions, trypsin-resistant cell-associated radioactivity increased with time consistent with ligand internalization. ¹²⁵I-Labelled-ligand was internalized with a t_1/2 of about 5 min. After a lag period some radioactivity was released back into the incubation medium. When measured at times up to 210 min, this was found to consist of mostly TCA-precipitable material that had been lost from the cell surface. However, when the incubation was extended to 24 h, almost 15% of the initial cell-associated radioactivity was released to the extracellular medium as TCA-soluble material, consistent with a slow rate of ligand degradation. The specific binding of ⁶⁵Zn-labelled α₂M was similar to that of the ¹²⁵I-labelled ligand and trypsin-resistance measurements provided evidence of α₂M-mediated ⁶⁵Zn uptake. These results support a role for syncytiotrophoblast in the metabolism of α₂-macroglobulin during pregnancy and are also consistent with a role for α₂-macroglobulin in the maternal-fetal transport of zinc. J. Cell. Biochem. 68:427–435, 1998. © 1998 Wiley-Liss, Inc.     

Temperature shifts induce the selective loss of alveolar-macrophage plasma membrane components

A shift in the incubation temperature of rabbit alveolar macrophages (0 degree C leads to 37 degrees C leads to 0 degree C) resulted in a 40-60% reduction in the ability of cells to bind alphamacroglobulin. 125I-trypsin complexes (alphaM. 125I-T). The reduction in binding activity did not reflect a disruption of cell integrity since the levels of intracellular components (lactate dehydrogenase, beta-N-acetyl-hexosaminidase) or other plasma membrane components (alkaline phosphodiesterase) were unaltered. Analysis of receptor-ligand interaction indicated that the temperature shift effected a decline in receptor number rather than an alteration in ligand-receptor affinity. Studies indicated that a temperature shift resulted in the loss of unoccupied receptors, and that ligand bound to receptors was not lost. However, after ligand internalization, receptors were removed by the temperature shift. The rate of receptor loss was maximal when cells were incubated at temperatures greater than 24 degrees C. Receptor loss was not prevented by treatment of cells with colchicine, cytochalasin B, or N-ethylamaleimide, but was prevented by treatment with the cross-linking agent paraformaldehyde. Data indicate that the reduction in alphaM. 125I-T binding activity resulted from shedding of receptors into the media since media obtained from temperature-shifted cells contained material that competed with cell-bound receptors for alphaM. 125I-T. Additionally, binding of alphaM. 125I-T was diminished on membrane fragments obtained from temperature-shifted cells. Incubation with Triton X-100, of cells whose receptors were occupied with alphaM. 125I-T, led to the extraction of 40% of cell-bound activity. However, no radioactivity was extracted from cells labeled with alphaM. 125I-T after a temperature shift. Measurement of ligand accumulation by control and temperature-shifted cells incubated at 20 degrees C indicated that control cells exhibited a subpopulation of receptors capable of binding ligand but only slowly internalizing it. This subpopulation was not present on temperature-shifted cells. These results indicate that surface receptors for alphamacroglobulin . protease complexes are heterogeneous and that the temperature shift resulted in the selective loss of membrane components.     

Small amount of concanavalin A modifies radiation-induced alteration in cell-surface charge depending on its binding condition

Cell electrophoretic mobility of cultured melanoma cells or rat erythrocytes decreased with time after X-irradiation. Addition of tetravalent concanavalin A or divalent succinyl-concanavalin A before (not after) irradiation, completely blocked the mobility reduction in greater concentrations than 5 μg/l.At 5 μg/1 only 3.7 · 10³ concanavalin A molecules bound to receptors per cell, while 4.18 · 10⁷ molecules/cell bound at saturating concentrations. Preincubation with concanavalin A at 37°C was effective even when the cells were treated with α-methylmannoside immediately after irradiation. At low temperature, however, concanavalin A was not effective despite a sufficient amount of bound ¹²⁵I-labelled concanavalin A. Treatment with α-methylmannoside following the binding of concanavalin A at 37°C before irradiation inhibited the concanavalin A effect depending on temperature. The residual amount of bound lectin could not account for the temperature dependence. The amount of sialic acid (the main charged substance) was not altered by X-irradiation with or without the lectin. Divalent succinyl-concanavalin A was also effective in blocking the radiation effect on electrophoretic mobility. These results seem to suggest that binding of a very small amount of concanavalin A without causing cell agglutination or clustering of its receptors, induces some alteration in the conformation of receptor glycoprotein, which blocks the internalization of acidic sugar residues by subsequent irradiation.     

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of ¹²⁵I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of ¹²⁵I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin.Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10^?11 mol/l to 10^?6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10⁷ l/mol with a total receptor concentration of 3.4 · 10⁶ sites per cell.The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10^?9 mol/l), both, under conditions of low and high fractional saturation of receptors.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of ¹²⁵I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a K_d of 8·10^?9 M. Binding is reduced 90% by 10^?5 M (+)- tubocurarine chloride and 10^?4 M nicotine, whereas concentrations of 10^?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with ¹²⁵I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ¹²⁵I-labeled α-bungarotoxin with a K_d of 5·10^?9 M. Nicotine and α-bungarotoxin at concentrations of 10^?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10^?5 M.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormones and labelled antibody binding techniques

The parathyrin receptor in renal cortex has been investigated by studying the binding of ¹²⁵I-labelled parathyrin, or of unlabelled parathyrin detected with ¹²⁵I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 °C but this phenomenon was not detectable at 37 °C. Specific displacement of lactoperoxidase labelled ¹²⁵I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was $\text{2.3 · 10}{}^8\text{M}{}^{\mathrm{?1}}$ and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0–7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1–34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glueagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of ¹²⁵I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of ¹²⁵I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K_d of 1·10^?8 M. Nicotine and acetylcholine iodide at concentrations of 10^?5 M inhibit ¹²⁵I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10^?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10^?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

Analysis of the effect of amines on inhibition of receptor-mediated and fluid-phase pinocytosis in rabbit alveolar macrophages

Incubation of rabbit alveolar macrophages in vitro with methyl amine led to a time- and concentration-dependent inhibition of uptake of alpha macroglobulin-125I-trypsin complexes (alpha M-125I-T). Upon addition of methyl amine (50 mM) to cells prelabeled with alpha M-125I-T there was a rapid inhibition of lysosomal catabolism of internalized ligand. In the absence of ligand, incubation of cells with 50 mM methyl amine led to a 40-70% decrease in surface-receptor number. The methyl amine-induced decrease in surface-receptor number only occurred in metabolically active cells since cells incubated at 0 degrees C, or treated with N-ethyl maleimide and incubated at 37 degrees C, did not show the effect. Incubation of cells at 37 degrees C with methyl amine also effected a 40-70% decrease in fluid-phase pinocytosis. Although there was a decline in surface-receptor number, the remaining population of receptors were capable of mediating (at least) one round of ligand internalization. However, further ligand uptake was prevented. Data demonstrate that although receptors were present on cell surfaces, they were incapable of mediating ligand internalization. Incubation of macrophages with chloroquine at 37 degrees C for 60 min also led to a disappearance of receptors, and a concomitant reduction in fluid-phase pinocytosis.     

Differences in time course of internalization of receptors of insulin and insulin-like growth factor (IGF-1) in isolated rat hepatocytes

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of ¹²⁵I-insulin and ¹²⁵I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular ¹²⁵I-IGF-I correlated with a decrease in the ¹²⁵I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.     

Glioblastoma targeting via integrins is concentration dependent

A novel approach to treat cancer more selectively is achieved by targeting drugs to cells via conjugating the drug or imaging agent to an antibody or ligand for a cell surface receptor that is over‐expressed by the target cell population. Previous work by us has suggested that enhanced specificity can be obtained by multivalency of binding moieties. In this study we investigated the binding specificity of a multivalent construct including three peptides segments (TWYKIAFQRNRK), which bind the α₆β₁‐integrin, linked by poly(ethylene glycol) spacers. The binding specificity of the constructs was calculated by quantifying their binding to target cells (glioma cells, SF 767) relative to non‐targeted cells (normal human astrocytes, NHA). Dodecapeptide constructs (monovalent) exhibit specificity equal to the ratio of receptor expression at all concentrations. However, trivalent constructs demonstrated a sharp increase in specificity at concentrations less than the affinity of the receptor–ligand bond (4.28 µM). These experiments (conducted at 4°C) were consistent with the theoretical prediction and indicate that the biophysical model captures the basic trend of the data in the absence of receptor internalization, although the concentration at which increased specificity is observed is greater than predicted. The biophysical model does not predict the results of 37°C experiments, and this is shown to be due to internalization which occurs at 37°C but not at 4°C. Biotechnol. Bioeng. 2009; 104: 408–417 © 2009 Wiley Periodicals, Inc.     

Kinetics of 125I-PDGF binding and down-regulation of PDGF receptor in arterial smooth muscle cells derived from patients with moyamoya disease

Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. We recently found that cultured smooth muscle cells (SMC) derived from arteries of patients with moyamoya disease responded poorly to serum mitogens, especially to platelet-derived growth factor (PDGF). In the present study, we investigated further the binding and processing of ¹²⁵I-PDGF, as well as down-regulation of the PDGF receptors in arterial SMC derived from patients with moyamoya disease. The specific binding sites of ¹²⁵I-PDGF were reduced significantly at both 4°C and 22°C on SMC from moyamoya disease compared with those from controls (4.78 vs. 11.92 × 10⁴/cell at 4°C), though the apparen dissociation constant (Kd) were the same. Kinetics of ¹²⁵I-PDGF binding at 37°C in cells from moyamoya disease showed fewer binding sites (less than 1/3 of controls) and lower degradation per cell than in those from controls, though no difference was observed in either internalization or degradation of each receptor. When SMC were exposed to lower concentrations of nonlabeled PDGF at 37°C, the percentage of remaining binding sites on cells from moyamoya disease was significantly less than that from controls. This excess down-regulation of PDGF receptor in SMC from moyamoya disease may be interpreted as insufficent recycling or a decreased intracellular pool of the PDGF receptor. These results are closely correlated with the diminished proliferation responses to PDGF in SMC from moyamoya disease and provide evidence that functional alterations in vascular cells are involved in the mechanism of development of intimal thickening in moyamoya disease. © 1993 Wiley-Liss, Inc.     

Testicular membranes with improved stability of the gonadotropin receptor

A plasma membrane fraction has been prepared from rat testis using an aqueous double-phase polymer system containing dextran, poly(ethylene glycol) 6000 and Zn²⁺. The membrane-associated gonadotropin receptor for lutropin and human choriogonadotropin can be markedly stabilized by a thawing-washing step of frozen membranes which prolongs the apparent half-life of the unoccupied membrane-associated receptors from less than 1 h at 37°C to greater than 5 h. Also, no degradation of ¹²⁵I-labeled human choriogonadotropin was detected following incubation with the membrane fraction. The equilibrium binding was characterized by an apparent association constant of 1.6 · 10¹⁰ M^?1 and a receptor content of 33 fmol/mg protein. Binding kinetic yielded an association rate constant of 1.0 · 10⁸ M^?1, while the dissociation rate constant for human choriogonadotropin was too low to be accurately determined under the conditions used. In contrast, ovine lutropin could be reversibly bound to the membranes leaving the previously occupied receptors available for binding by ¹²⁵I-labeled human choriogonadotropin.     

Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of ¹²⁵I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with ¹²⁵I-NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of ¹²⁵I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.