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PubMed1997

Novel mechanisms of RTK signal generation.

Recent findings shed new light on the process of receptor tyrosine kinase (RTK) activation and signal definition. In extension to the established mechanism of ligand-induced homodimeric receptor complex formation, recent findings highlight heterodimeric receptor aggregation as a powerful means of signal diversification. Promiscuous receptor interactions involve different ligand binding kinetics and generate divergent receptor phosphorylation sites that could allow enhanced or modified signal generation. Besides activation by a specific ligand, a newly defined RTK function involves signal integration of a variety of stimuli, including calcium-dependent responses in neuronal cells, activation of G-protein-coupled receptors or cellular stress such as UV irradiation. On the basis of existing evidence for such crossactivation pathways, RTKs must be considered as representing critical foci and switch points for multiple environmental and internal stimuli.

Animals↗
PubMed2005

Castration reduces platelet thromboxane A2 receptor density and aggregability.

BACKGROUND: Exogenously administered testosterone upregulates platelet thromboxane A2 (TXA2) receptors and increases aggregation response to thromboxane mimetics in healthy male volunteers. However, the biological impact of endogenous testosterone on platelet TXA2 receptor expression, especially in older men at risk of coronary artery disease, is unclear. AIM: To investigate the impact of reduction in circulating testosterone on platelet TXA2 receptor expression in older men. DESIGN: Cross-sectional case-control study. METHODS: We studied surgically and/or medically castrated men with prostate cancer (group A, n = 8, aged 71 +/- 8 years) and age-matched, uncastrated urology patients (group B, n = 7, aged 67 +/- 9 years). Plasma testosterone was measured by radioimmunoassay. Platelet TXA2 receptor expression was assessed by radioligand binding studies using radioactive 125I-BOP. Platelet aggregation responses to TXA2-mimetic I-BOP, and to thrombin, were also studied. RESULTS: Group A had significantly lower plasma testosterone than group B (16 +/- 5 ng/dl vs. 308 +/- 47 ng/dl, p<0.001). Platelet TXA2 receptor density (B(max)) but not affinity (K(d)) was lower in group A (0.50 +/- 0.12 vs. 1.01 +/- 0.17 pmol/mg protein, p = 0.03). Maximum platelet aggregation response to I-BOP (E(max)), but not sensitivity (EC50) was lower in group A (53 +/- 2% vs. 63 +/- 2%, p = 0.003 ANOVA). In vitro, high concentrations of hydroxyflutamide (100 microM) competitively inhibited U46619-induced platelet aggregation in washed platelets, without affecting the binding of 125I-BOP to platelet TXA2 receptors. DISCUSSION: Endogenous testosterone regulates platelet TXA2 receptor B(max) and the E(max) aggregation response to thromboxane mimetic I-BOP. Blockade of androgen receptors or inhibition of testosterone production may reduce platelet aggregation responses. Preliminary evidence suggests the presence of functional androgen receptors on human platelets, which may regulate TXA2 receptor expression.

Aged↗
PubMed2004

Beta-1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering.

The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. Here we attempted to assess quantitatively the functional importance of receptor clustering. We studied beta-1 integrin-mediated attachment of THP-1 cells to fibronectin-coated surfaces under low shear flow. Cells displayed multiple binding events with a half-life of the order of 1 s. The duration of binding events after the first second after arrest was quantitatively accounted for by a model assuming the existence of a short-time intermediate binding state with 3.6 s(-1) dissociation rate and 1.3 s(-1) transition frequency toward a more stable state. Cell binding to surfaces coated with lower fibronectin densities was concluded to be mediated by single molecular interactions, whereas multiple bonds were formed <1 s after contact with higher fibronectin surface densities. Cell treatment with microfilament inhibitors or a neutral antiintegrin antibody decreased bond number without changing aforementioned kinetic parameters whereas a function enhancing antibody increased the rate of bond formation and/or the lifetime of intermediate state. Receptor aggregation was induced by treating cells with neutral antiintegrin antibody and antiimmunoglobulin antibodies. A semiquantitative confocal microscopy study suggested that this treatment increased between 40% and 100% the average number of integrin receptors located in a volume of approximately 0.045 microm(3) surrounding each integrin. This aggregation induced up to 2.7-fold increase of the average number of bonds. Flow cytometric analysis of fluorescent ligand binding showed that THP-1 cells displayed low-affinity beta-1 integrins with a dissociation constant in the micromolar range. It is concluded that the initial step of cell adhesion was mediated by multiple incomplete bonds rather than a single equilibrium-state ligand receptor association. This interpretation accounts for the functional importance of integrin clustering.

Actin Cytoskeleton↗
PubMed1978

Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose.

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.

Animals↗
PubMed1986

Quantitative relationships between aggregation of IgE receptors, generation of intracellular signals, and histamine secretion in rat basophilic leukemia (2H3) cells. Enhanced responses with heavy water.

RBL-2H3 cells (a tumor analog of rat mast cells) have plasma-membrane receptors that bind immunoglobulin E, which when aggregated, initiate degranulation. As in other systems, secretion is preceeded by enhanced hydrolysis of inositol phospholipids and by a rise in intracellular Ca2+. Unlike the responses of many other cells, however, both of these earlier events require extracellular Ca2+. The relationship of these events to each other and to the subsequent secretory process is thus unclear. By exposing cells to covalent oligomers of IgE one can demonstrate substantial increases in secretion of histamine by increasing the concentration and size of the oligomers or by using heavy water (D2O) in the medium. We have used such maneuvers to examine the quantitative relationships between aggregation of the receptors and the breakdown of inositol phospholipids, the increase in cytosolic Ca2+ and secretion. Our principal findings were: all treatments that increased secretion, correspondingly increased the changes that precede degranulation. These early events correlated with the degree of aggregation of the receptors even when the stimulatory conditions resulted in maximal secretion. Although the results were insufficient to prove that the hydrolysis of inositol phospholipids is required for the rise in cytosolic Ca2+, the studies with D2O and other observations supported this view. Since a plasma-membrane ion channel for Ca2+ has been implicated in the IgE-mediated rise in cytosolic Ca2+ in RBL 2H3 cells, this in turn suggests a heretofore undescribed role for hydrolysis of inositol phospholipids.

Animals↗
PubMed2005

Platelet aggregation induces platelet aggregate stability via SLAM family receptor signaling.

Platelet aggregation is a dynamic entity, capable of directing its own growth and stability via the activation of signaling cascades that lead to the expression and secretion of various secondary agonists. Here we show that the signaling pathways triggered during platelet aggregation include an intrinsic pro-thrombotic activity mediated by 2 homophilic adhesion molecules, CD84 and CD150 (SLAM [signaling lymphocyte activation molecule]), which are tyrosine phosphorylated in a platelet aggregation-dependent fashion. The 2 CD84/SLAM adapter proteins, SAP (SLAM-associated protein) and EAT-2 (EWS-activated transcript-2), were found in platelets; only SAP, however, was found to immunoprecipitate with tyrosine-phosphorylated SLAM. The immobilized extracellular domain of CD84 promoted microaggregate formation, while SAP-deficient platelets demonstrated defective spreading on immobilized CD84, demonstrating a functional role in platelets for SLAM family interactions. Finally, analysis of SLAM-deficient mice revealed an overall defect in platelet aggregation in vitro and a delayed arterial thrombotic process in vivo. The data indicate that signaling of the adhesion molecules in the SLAM family, activated by proximity during aggregation, further stabilize platelet-platelet interactions in thrombosis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Opioid receptor homo- and heterodimerization in living cells by quantitative bioluminescence resonance energy transfer.

Opioid receptors have been shown to dimerize or oligomerize among themselves and each other, affecting their functional properties. This study used bioluminescence resonance energy transfer (BRET) between the mu, delta, and kappa opioid receptors to study opioid receptor aggregation in transfected human embryonic kidney 293 cells. Titration of receptor levels indicated that all three opioid receptors have a similar affinity to form homo- or hetero-oligomers in combination with any other opioid receptor type. In contrast, none of the opioid receptors formed detectable oligomers with the muscarinic M2 receptor, indicating that interactions among opioid receptors are selective. The formation of opioid receptor dimers, rather than higher order oligomers, is supported by binding kinetics in competition experiments between labeled and unlabeled receptors. Opioid receptor dimerization occurred at physiological temperatures upon receptor biosynthesis, before trafficking to the plasma membrane. Moreover, using BRET, coimmunoprecipitation, receptor binding, and G protein coupling, we demonstrate for the first time functional mu opioid receptor-kappa opioid receptor heterodimerization. These combined results demonstrate that opioid receptors can undergo homo- and heterodimerization, a process with potential implications for opioid physiology and pharmacology.

Animals↗
PubMed1993

Biochemical evidence for a homophilic interaction of the alpha 3 beta 1 integrin.

The purpose of this study was to determine if the alpha 3 beta 1 integrin could interact in a homophilic manner. Several earlier reports have shown that certain integrin adhesion receptors, namely alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 localize to intercellular adhesion structures and, therefore, may participate in cell-cell interactions (Carter, W. G., Wayner, E. A., Bouchard, T. S., and Kaur, P. (1990) J. Cell Biol. 110, 1387-1404; Kaufmann, R., Frosch, D., Westphal, C., Weber, L., and Klein, C. E. (1989) J. Cell Biol. 109, 1807-1815; Hynes, R. O. (1987) Cell 48, 549-554; Symington, B. E., Takada, Y., and Carter, W. G. (1993) J. Cell Biol. 120, 523-535). We present data herein suggesting that the integrin alpha 3 beta 1 may interact homophilically in such cell-cell adhesion structures which contain this specific receptor or, alternatively, in receptor aggregates found in focal adhesions. The alpha 3 beta 1 receptor was purified by affinity chromatography on either human laminin or peptide GD-6-Sepharose and subsequently used as a substrate in cell adhesion assays. The immobilized alpha 3 beta 1 supported the adhesion of cells containing alpha 3 beta 1, and this attachment was specifically inhibited by monoclonal antibodies to both beta 1 and alpha 3 subunits. In addition, an affinity matrix containing purified alpha 3 beta 1 showed specific binding of only alpha 3 beta 1 from detergent extracts of cell surface proteins and such binding was cation-dependent. Finally, using biosensor technology involving the principle of surface plasmon resonance (BIAcore, Pharmacia Biosensor), alpha 3 beta 1, when bound to a carboxymethyl dextran-modified gold surface, was found to bind only other soluble alpha 3 beta 1 receptors and did not bind other purified integrins, including alpha 5 beta 1 and alpha v beta 3. These data strongly suggest that alpha 3 beta 1 likely interacts in a homophilic manner under our experimental conditions.

Biosensing Techniques↗
PubMed1994

Membrane clustering and bungarotoxin binding by the nicotinic acetylcholine receptor: role of the beta subunit.

Nicotinic acetylcholine receptors (nAChRs) are localised at morphologically distinct regions of the post-synaptic membrane by interactions between the receptor subunits and cytoskeletal proteins, such as the 43-kDa protein. We have used Xenopus oocytes to examine the localisation and pharmacological properties of muscle nAChRs associated with 43-kDa protein and to compare them with hybrid muscle nAChRs containing a beta subunit derived from a neuronal source. Receptors expressed on the oocyte outer membrane were visualised using confocal scanning laser microscopy. Coexpression of mouse muscle subunit alpha 1 beta 1 gamma delta and 43-kDa protein transcripts produced discrete receptor aggregates with a diameter of 1-5 microns whose function was partially blocked by application of neuronal bungarotoxin (NBT) at 100 nM. Substitution of the beta 1 subunit by the neuronal beta 2 protein produced a functioning receptor that did not aggregate in the presence of 43-kDa protein and was substantially blocked by the same concentration of NBT. Hybrid alpha 1 beta 4 gamma delta receptors exhibited a combination of characteristics in that they clustered like normal muscle subunits in the presence of 43-kDa protein, but showed a sensitivity to NBT intermediate between that of muscle receptors and that of hybrids containing beta 2. These results suggest that the beta subunit is an important determinant in receptor localisation and sensitivity to NBT.

Animals↗
PubMed1997

Surface-secreted von Willebrand factor mediates aggregation of ADP-activated platelets at moderate shear stress: facilitated by GPIb but controlled by GPIIb-IIIa.

We previously showed that ADP activation of washed human platelets in plasma-free suspensions supports aggregation at moderate shear stress (0.4-1.6 Nm-2) in Poiseuille flow. Although most activated platelets expressed maximal fibrinogen-occupied GPIIb-IIIa receptors, aggregation appeared to be independent of bound fibrinogen, but blocked by the hexapeptide GRGDSP. Here, we tested the hypothesis that von Willebrand factor (vWF) secreted and expressed on activated platelets mediates aggregation at moderate shear rates from 300 to 1000 s-1 corresponding to shear stresses from 0.3 to 1.1 Nm-2. Relatively unactivated platelets (< 15% expressing prebound fibrinogen) were prepared from acidified citrated platelet rich plasma (cPRP) by single centrifugation with 50 nM stable prostacyclin derivative ZK 36374 and resuspended in Tyrodes-albumin at 5 x 10(4) cells microliter-1. Flow cytometric measurements with monoclonal antibody (mAb) 2.2.9 reporting on surface-bound vWF, and with mAb S12 reporting on alpha-granule secreted P-selectin, showed that 65% and 80%, respectively, of all platelets were maximally activated with respect to maximal secretion and surface expression of these proteins. "Resting" washed platelets exhibited both surface-bound vWF and significant P-selectin secretion. We showed that mAbs 6D1 and NMC4, respectively blocking the adhesive domains on the GPIb receptor recognizing vWF, and on the vWF molecule recognizing the GPIb receptor, partially inhibited ADP-induced aggregation under shear in Couette flow, the degree of inhibition increasing with increasing shear stress. In contrast, mAb 10E5, blocking the vWF binding domain on GPIIb-IIIa, essentially blocked all aggregation at the shear rates tested. We conclude that vWF, expressed on ADP-activated platelets, is at least the predominant cross-bridging molecule mediating aggregation at moderate shear stress. There is an absolute requirement for free activated GPIIb-IIIa receptors, postulated to interact with platelet-secreted, surface bound vWF. The GPIb-vWF cross-bridging reaction plays a facilitative role becoming increasingly important with increasing shear stress. Since aurin tricarboxylic acid, which blocks the GPIb binding domain on vWF, was also found to completely block aggregation in Poiseuille flow, we conclude that it too affects the GPIIb-IIIa interaction.

Adenosine Diphosphate↗
PubMed1972

The mechanism of adenosine diphosphate induced platelet aggregation: binding to platelet receptors and inhibition of binding and aggregation by prostaglandin E 1 .

1. Normal human blood platelets in stirred plasma were incubated with [(14)C]ADP for 10-360 sec and the aggregation responses were correlated with platelet bound radioactivity, the platelets being separated from the plasma within 25 sec of the end of the experiment.2. The platelet aggregation response, measured as a change in light transmittance through platelet-rich plasma, was related to the plasma [(14)C]ADP concentration and linearly related to the log platelet bound [(14)C]ADP 60-120 sec after addition of nucleotide to plasma.3. Thin layer chromatography of the platelet bound radioactivity showed that 78-90% was unmetabolized ADP, the remainder being AMP. Plasma radioactivity consisted of ADP, AMP and adenosine. There was no detectable radioactive cyclic AMP in either platelets or plasma.4. Further accumulation of radioactivity occurred after 180 sec but was not related to the aggregation response.5. Prostaglandin E(1) inhibited aggregation and platelet [(14)C]ADP accumulation when added to platelet-rich plasma 60 sec before [(14)C]ADP. There was a significant correlation between inhibition of aggregation and inhibition of [(14)C]ADP accumulation.6. Prostaglandin E(1) also reversed ADP aggregation when added to platelet-rich plasma after the nucleotide, with an accompanying decrease in platelet bound [(14)C]ADP.7. It is concluded that ADP induces platelet aggregation by binding to specific receptors probably located on the plasma membrane, and that prostaglandin E(1) inhibits this effect by interfering with the ADP binding.

Adenine Nucleotides↗
PubMed1984

Transformation by Rous sarcoma virus prevents acetylcholine receptor clustering on cultured chicken muscle fibers.

Acetylcholine receptors aggregate in the membrane of cultured chicken myotubes; the process of receptor clustering can be stimulated by exogenous factors that we, among others, have begun to characterize. Chicken myoblasts transformed by temperature-sensitive mutants of Rous sarcoma virus, such as tsNY68, fuse to form multinucleated myotubes at 42 degrees C, the nonpermissive temperature for transformation. However, tsNY68-infected myotubes do not cluster acetylcholine receptors at 42 degrees C, even in the presence of active clustering agents. This phenomenon is not merely a result of viral infection, since myotubes infected with a transformation-deficient viral mutant, td107A, behave like noninfected myotubes with respect to receptor clustering; thus, the effects of tsNY68 on the clustering process must be mediated by the src gene product. These experiments may provide a method of identifying essential elements of acetylcholine receptor clusters.

Animals↗
PubMed1992

Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity.

Most neurotransmitter receptors examined to date have been shown either to be regulated by protein phosphorylation or to contain consensus sequences for phosphorylation by protein kinases. Neurotransmitter receptors that mediate rapid synaptic transmission in the nervous system are the ligand-gated ion channels and include the nicotinic acetylcholine receptors of muscle and nerve and the excitatory and inhibitory amino acid receptors: the glutamate, GABAA, and glycine receptors. These receptors are multimeric proteins composed of homologous subunits which each span the membrane several times and contain a large intracellular loop that is a mosaic of consensus sites for protein phosphorylation. Recent evidence has suggested that extracellular signals released from the presynaptic neuron, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein, regulate the phosphorylation of ligand-gated ion channels. The functional effects of phosphorylation are varied and include the regulation of receptor desensitization rate, subunit assembly, and receptor aggregation at the synapse. These results suggest that phosphorylation of neurotransmitter receptors represents a major mechanism in the regulation of their function and may play an important role in synaptic plasticity.

Amino Acid Sequence↗
PubMed1976

Subpopulations of human lymphoblastoid cell lines. Correlation with the expression of surface receptors and content of Epstein-Barr virus genome.

Forty lymphoblast cell lines derived from normal subjects, patients with infectious mononucleosis, leukemia, and Burkitt's lymphoma have been studied for surface receptors including surface Ig, complement receptors by the EAC rosette and fluorescent (Raji cell) techniques, and Fc (aggregate) receptor by direct and indirect immunofluorescence. Because of the B-cell tropism of the Epstein-Barr virus (EBV), an effort was made to correlate the expresion of various surface properties of lymphoblastoid cell lines with the content of EBV viral DNA as determined by complementary RNA-DNA (cNRA-DNA) hybridization on membrane filters or by DNA-DNA renaturation kinetic analysis. The only correlation established was with the Fc receptor determined by direct immunofluorescence. No correlation of EBV genome equivalents per cell with complement receptor or surface Ig was noted, suggesting that the expression of these receptors is not influenced by EBV viral DNA content. Subgroups of lymphoblastoid cell lines were on the basis of variable expression of surface receptors, designated B1, B2, B3, B4, and T. The distribution of lymphoblastoid cell lines into these subgroups were in the ratio of 14:4:1:4:1. The B1, B2, and B4 cell lines (except Molt 4F) were found to contain EBV. The B3 subgroup, for wich cell line 698 was the sole example, expressed surface immunoglobulins but no other B-cell characteristics, and H.S.B., a T-cell line, lacked detectable EBV.

Burkitt Lymphoma↗
PubMed1999

One lyn molecule is sufficient to initiate phosphorylation of aggregated high-affinity IgE receptors.

In response to antigenic stimuli, the multisubunit immune recognition receptors become aggregated and then phosphorylated on their cytoplasmic tyrosines. For the clonotypic receptors of B and T cells and for Fc receptors such as the high-affinity receptor for IgE (FcepsilonRI), a Src family kinase initiates this phosphorylation. We ask whether aggregation of the initiating kinase itself is required for signal transduction or whether, alternatively, a single associated kinase molecule can phosphorylate the receptors in an aggregate. We formulate the alternative molecular mechanisms mathematically and compare predictions with experimental findings on FcepsilonRI-bearing cells expressing varying amounts of the transfected Src family kinase Lyn. The data are consistent with the requirement of only a single Lyn molecule per FcepsilonRI aggregate to initiate signaling and are inconsistent with a mechanism requiring more than one Lyn molecule.

Animals↗
PubMed1993

Rapid non-genomic actions of progesterone stimulate Ca2+ influx and the acrosome reaction in human sperm.

This review summarizes some recent findings in human sperm which show that progesterone and 17 alpha hydroxyprogesterone are able rapidly (within seconds) to elevate [Ca2+]i and elicit the acrosome reaction (AR) via a non-genomic cell surface receptor. Progesterone promotes a transient elevation in [Ca2+]i which is blocked by extracellular La3+ and Ni2+ and removal of extracellular Ca2+ following chelation with EGTA. Some studies suggest that polyamines, trypsin-like proteases, and progesterone receptor aggregation are involved in progesterone-induced Ca2+ influx and AR. The receptor is not stimulated by the potent synthetic progestigins (e.g. promegestone, norethynodrel, megestrol acetate, cyproterone acetate) and is weakly antagonized by the genomic anti-progestins RU 486 and ZK 98.299. The sedative-hypnotic 3 alpha hydroxyl A-ring reduced pregnane steroids, which are powerful activators of the GABAA Cl- channel, are weak activators of Ca2+ influx and the AR. These data suggest that human sperm have a cell surface steroid receptor which is unlike the genomic progesterone receptor and the GABAA Cl- channel steroid receptor.

Acrosome↗
PubMed1979

Electrophoresis and diffusion in the plane of the cell membrane.

Electrophoretic and diffusional movements of concanavalin A (Con A) receptors and acetylcholine (ACh) receptors in the plane of the plasma membrane of mononucleate, spherical Xenopus myoblasts were studied by microfluorimetry and iontophoresis. We found that (a) a uniform electric field of 10 V/cm applied along the cell surface produces a partial accumulation of both types of receptors toward the cathodal pole of the cell within 30 min: (b) post-field relaxation of the culture results in the complete recovery of the uniform distribution of the Con A receptors within 10 min; and (c) in contrast to the Con A receptor in general, accumulation of ACh receptors by the electric field results in the formation of stable, localized receptor aggregates. Theoretical analyses were carried out for the distribution of charged membrane receptors at equilibrium between electrophoresis and diffusion, and for the rate of back diffusion after the removal of the field. These analyses indicated that, at 22 degrees C, the average electrophoretic mobility of the electrophoretically mobile population of the Con A receptors is about 1.9 X 10(-3) micron/s per V/cm, while their average diffusion coefficient is 5.1 X 10(-9) cm2/s.

Animals↗
PubMed1979

Oestrogen receptor of calf mammary gland. Purification by use of sodium bromide and heparin-sepharose.

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In 'low-salt' buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.

Animals↗