Properties of the nerve growth factor receptor of a clonal line of rat pheochromocytoma (PC12) cells.

The interaction of nerve growth factor (NGF) with its receptor on cells of the PC 12 cell line was studied. All experiments were done at 0.5 °C to minimize degradation and processes requiring membrane mobility. Under these conditions, a single class of high affinity binding sites with a dissociation constant of 2.9 × 10^?9 M was observed. The number of receptors per cell was 58000. The binding was linear with the number of cells in the assay and was not displaced by proteins other than native nerve growth factor. Trypsin treatment of the cells destroyed the specific binding. The removal of divalent cations had no effect on the binding. Culturing the cells for 2 weeks in NGF prior to assay did not change the receptor number or receptor affinity and there was a similar lack of effect of the density of the culture from which the cells were taken for assay. The present findings are compared with previous studies on the dorsal root ganglia and sympathetic ganglia neurons, and the implication for the use of PC 12 as a model for the study of NGF action are discussed.     

Identification of the cell surface and nuclear receptors for NGF in a breast carcinoma cell line

¹²⁵I-nerve growth factor (NGF) was found to be internalized and translocated to the nucleus of SKBr5 breast carcinoma cells. The cytoplasm and chromatin isolated from nonmitotic cells accumulated two-and five-fold, respectively, more of ¹²⁵I-NGF than the cells undergoing mitosis. MAb 20.4 developed against the NGF cell surface receptor immunoprecipitated the 80,000 M_r receptor from plasma membrane and two protein species from the chromatin; 90,000 M_r (major band) and 200,000 M_r (minor band). In SKBr5 cells, binding of NGF to the chromatin did not affect synthesis of rRNA. Proliferation of SKBr5 cells was slightly stimulated by NGF. In control melanoma A875 cells, which express the 230,000 M_r chromatin receptor, NGF inhibited both rRNA synthesis and cell proliferation. We suggest that the 90,000 M_r chromatin receptor expressed by SKBr5 cells represents a “nonactive”, ligand-binding subunit of the high molecular weight receptor for NGF. The critical role of the chromatin receptor for NGF in rRNA-dependent cell proliferation is discussed. © 1993 Wiley-Liss, Inc.     

Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: dependence on developmental stage

Adrenal medullary cells were cultured in a serum-free medium from fetal, neonatal (calves), and adult bovine animals. Neurite outgrowth in response to nerve growth factor (NGF) was observed in cells obtained from fetuses up to a gestational age of 3 months but not in cultures from older animals. The tyrosine hydroxylase (TH) specific activity was found to depend on the cell density and corresponded, at a density of 2 × 10⁵ cells/cm², to the specific activity found in vivo. The TH specific activity increased about sevenfold from fetuses to adult animals. Administration of NGF in vitro caused an increase of the TH specific activity in fetal cells by up to 140% and in calf cells typically by 70–100%. Cultures from adult animals showed no significant TH increase in response to NGF. Scatchard analysis and kinetic studies of the NGF binding at 0°C to intact adrenal medullary cells cultured from calves or from adult bovine animals revealed the presence of only one class of receptors, having a dissociation constant (K_D) of 1 × 10 ⁹, M. There are 16,000 binding sites per cell. The affinity of the reeptors in vivo (determined in crude membrane preparations) did not alter during development, whereas the receptor density decreased with increasing fetal age, but was the same for calves and adults. Whereas the loss of NGF-mediated fiber outgrowth during development might be related to the reduction of receptor density, the disappearance of the NGF-mediated TH induction does not correlate with changes in the binding characteristics of NGF to the adrenal chromaffin cells.     

Nerve growth factor receptors on PC12 cells: Evidence for two receptor classes with differing cytoskeletal association

PC12, an NGF responsive cell line, exhibits two classes of NGF receptors which we designate “Fast” and “Slow.” Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of ¹²⁵I-NGF. At 37°C, binding of ¹²⁵I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of ¹²⁵I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (about 2 × 10^?10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. ¹²⁵I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while ¹²⁵I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to O°C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF, nor alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.     

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.     

Evidence for the participation of nerve growth factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic program

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75^{neurotrophin receptor} (p75^NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75^NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75^NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion. J. Cell. Physiol. 176:10–21, 1998. © 1998 Wiley-Liss, Inc.     

Interaction of concanavalin A with differentiated and undifferentiated murine neuroblastoma cells.

Tritium-labeled acetyl-concanavalin A (³H-Con A) was used to study its kinetics of binding at 0 °C to murine neuroblastoma cells (clone neuro 2-A) grown in the differentiated (monolayer) and Undifferentiated (spinner) states. The binding of ³H-Con A to both cell types gives sigmoidal saturation curves, suggesting positively cooperative binding of the lectin. The Hill coefficient is 1.75 for differentiated and 1.36 for Undifferentiated cells. The maximal number of ³H-Con A molecules bound per cell is 2.3 × 10⁷ and 3.4 × 10⁷ for differentiated and Undifferentiated cells, respectively, and the apparent rate constants for formation of the lectin-cell complex are 6.13 × 10², m^?1, s^?1 for the Undifferentiated and 6.68 × 10², m^?1, s^?1 for the differentiated cells. The lectin bound to spinner cells does not dissociate spontaneously to any measurable extent over a 60-min period at 0 or 37 °C, but the lectin-cell complex dissociates rapidly after addition of α-methyl-d-mannopyranoside. At 37 °C, this sugar causes virtually complete dissociation of the cell-lectin complex within 30 min. The ³H-Con A dissociated from spinner cells is indistinguishable from the original ³H-Con A by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, gel filtration through Bio-Gels P-30 and P-100, and specific binding to spinner cells. Both the original and the dissociated ³H-Con A are dimers at pH 7.4. The sugar-induced dissociation of the labeled lectin from spinner cells is not accompanied by shedding or inactivation of the lectin binding sites of the cell surface.     

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10⁴ cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

Modification of nerve growth factor receptor properties by wheat germ agglutinin

PC12 is a nerve growth factor (NGF) responsive cell line which exhibits two classes of NGF receptors distinguishable by different kinetic rate constants, sensitivity to trypsin and resistance to Triton detergent solubilization. Whereas incubation of PC12 cells with wheat germ agglutinin (WGA) prior to addition of 125I-NGF inhibits binding of NGF to both classes of receptors, treatment with WGA subsequent to incubation with NGF does not inhibit NGF binding but causes the class of NGF receptors which exhibit rapid or "Fast" dissociation kinetics prior to lectin treatment to be converted to the form which exhibits "Slow" dissociation kinetics. This WGA-mediated receptor conversion is lectin specific, blocked by N-acetyl-D-glucosamine, occurs at similar rates at 4 and 37 degrees C, and is not impaired by a metabolic poison. NGF receptors converted by WGA, like pre-existing Slow receptors, are resistant to trypsinization and remain associated to Triton X-100 extracted "cytoskeletons." Very similar results were obtained for NGF receptors on a human melanoma cell line A875. These results suggest that Fast and Slow receptors are two interconvertible forms of a single protein, rather than distinct proteins. The significance of the generality of these properties for NGF receptors from diverse species and cell types is discussed.     

The interaction of albumin and concanavalin A with normal and sickle human erythrocytes.

The interaction of human albumin and concanavalin A with normal and sickle human red blood cells previously washed in phosphate buffer at pH = 7.4 was studied by titration calorimetry. The amount of albumin bound to normal cells was (6.8 ± 2.2) × 10⁵ molecules/cell. An equilibrium constant of 5 × 10¹⁰ and an enthalpy change of ?(280 ± 90) kcal/mol albumin was determined for albumin interaction with normal cells. The amount of albumin bound to sickle cells was (12.4 ± 1.0) × 10⁵ molecules/cell and the enthalpy change for albumin interaction with sickle cells was ?(390 ± 140) kcal/mol. Normal cells bound (5.7 ± 2.4) × 10⁵ concanavalin A molecules/cell with an enthalpy change of ?(840 ± 200) kcal/mol concanavalin. All experiments were conducted at 25°C.     

Expression of nerve growth factor receptor during human peripheral nerve development

The expression of NGF receptors on human Schwann cells during development and myelination and in culture was analyzed using a murine monoclonal antibody to human NGF receptor. Nonmyelinated femoral nerves from 13- to 14-week fetuses stained strongly for NGF receptor, whereas tissues from later stages of development showed a decrease in the staining intensity. These changes correlated with the initiation of myelination (17-19 weeks), as observed by phase-contrast and electron microscopy, and the reactivity with monoclonal antibody 4C5, a marker of mature Schwann cells. In adult nerves, only the perineurium and few endoneurial cells were stained with anti-NGF receptor antibody. Cultured human fetal Schwann cells were positive for NGF receptor by immunofluorescence irregardless of donor age or length of time in culture. The decreased staining of NGF receptor with nerve maturation may reflect a dependence of antigen expression on Schwann cell differentiation and/or neuron-Schwann cell interaction.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

Biochemical properties of the 1 alpha, 25-dihydroxyvitamin D3 cytoplasmic receptors from human and chick parathyroid glands

Cytoplasmic receptors for 1α, 25-dihydroxyvitamin D₃ from human parathyroid adenoma tissue and rachitic chick parathyroid glands have been characterized with regard to a number of physical, chemical, and ligand binding properties. Both receptors are 3.6–3.7 S proteins with molecular weights of approximately 75,000 and Stoke's molecular radii of 36 Å. It was found that the receptors possess a cysteine residue in or near the 1α, 25-dihydroxyvitamin D₃ binding site which is critical for ligand binding activity. The receptors both have equilibrium dissociation constants for 1α, 25-dihydroxyvitamin D₃ in the range of 2 to 5 × 10^?10m at 4 °C and second-order association rate constants for their seco-steroid ligand of 1 × 10⁷, m^?1 min^?1 (0 °C). The dissociation rate constants were found to be 5.3 × 10^?4 min^?1 (4 °C) for the human receptor and 1.3 × 10^?5 min^?1 (4 °C) for the chick receptor. The great deal of similarity which exists between the cytoplasmic 1α, 25-dihydroxyvitamin D₃ receptors from avian and mammalian parathyroid glands suggests a homologous function for these molecules in the two tissues.     

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.     

The influence of temperature upon polysomes of spermatids of rat testes

Incorporation of [³H]phenylalanine into protein by a reconstituted lysate subcellular system (ribosomes plus high-speed supernatant) from rat spermatids was measured at 34°C after 5 minutes preincubation of one component at 0°C while the other component was incubated at temperatures from 30°C to 40°C. Preincubation at temperatures above 34°C inhibits the ribosomal activity but not the high-speed supernatant activity. The incubation of lysate above 34°C results from a dissociation of polysomes to monosomes. These results indicate that ribosomes are the most sensitive component to the increased temperature on protein synthesis in lysate cell free system by spermatids and that the inhibition of protein synthesis in spermatids above 34°C is at least partly explained by the breakdown of polysomes in these cells.     

Characterization of the Galactose-Specific Binding Activity of a Purified Soluble Entamoeba histolytica Adherence Lectin

ABSTRACT. We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein to glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffnity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4°C by radioimmunoassay; the dissociation coefficient (Kd was 2.39 × 10^?8 M^?1 with 5.97 × 10⁴ lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cell mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 × 10⁵/cell) rather than a significantly reduced dissociation constant (4.93 × 10^?8 M^?1). At 4°C, there was no measurable Gal-specific binding of the adherence protein to the Lec and IdlD.Lecl CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 μg/ml) to CHO cells was equivalent at 4°C and 37°C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 μg/ml). Gal-specific binding of the lectin at 4°C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells. In summary, these findings indicate that the purified E. histolytica adherence lectin demonstrates saturable Gal-specific binding to 1–6 branched-N-linked and not O-linked galactose terminal cell surface carbohydrates; however, apparently only a small percentage of purified amebic lectin molecules actually possess galactose binding activity.     

Radioreceptor Assay for α;-Msh Using Mouse B16 Melanoma Cells

Abstract

A radioreceptor assay for α;-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle⁴]-α;-MSH tracer. The assay was used (1) to study the binding characteristics of α;-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of α;-MSH to B16 cells reached a stable plateau after 3 h at 15°C. At 25° or 37°C, the binding was transient and at 0-1°C, the association was very slow. The hormone-receptor complex was relatively stable between 0° and 15°C whereas a 50% dissociation was reached after 90 min at 25°C and after 35 min at 37°C. The mean K_D for α;-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably     

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.     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.