Studies of fibronectin matrices in living cells with fluoresceinated gelatin

We have used fluorescein isosthiocyanate-conjugated gelatin (FITC- gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC- gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC- gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.     

Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb

Variations in sulfation of heparan sulfate (HS) affect interaction with FGF, FGFR, and FGF-HS-FGFR signaling complexes. Whether structurally distinct HS motifs are at play is unclear. Here we used stabilized recombinant FGF7 as a bioaffinity matrix to purify size-defined heparin oligosaccharides. We show that only 0.2%-4% of 6 to 14 unit oligosaccharides, respectively, have high affinity for FGF7 based on resistance to salt above 0.6M NaCl. The high affinity fractions exhibit highest specific activity for interaction with FGFR2IIIb and formation of complexes of FGF7-HS-FGFR2IIIb. The majority fractions with moderate (0.30-0.6M NaCl), low (0.14-0.30M NaCl) or no affinity at 0.14M NaCl for FGF7 supported no complex formation. The high affinity octasaccharide mixture exhibited predominantly 7- and 8-sulfated components (7,8-S-OctaF7) and formed FGF7-HS-FGFR2IIIb complexes with highest specific activity. Deduced disaccharide analysis indicated that 7,8-S-OctaF7 comprised of DeltaHexA2SGlcN6S in a 2:1 ratio to a trisulfated and a variable unsulfated or monosulfated disaccharide. The inactive octasaccharides with moderate affinity for FGF7 were much more heterogenous and highly sulfated with major components containing 11 or 12 sulfates comprised of predominantly trisulfated disaccharides. This suggests that a rare undersulfated motif in which sulfate groups are specifically distributed has highest affinity for FGF7. The same motif also exhibits structural requirements for high affinity binding to dimers of FGFR2IIIb prior to binding FGF7 to form FGF7-HS-FGFR2IIIb complexes. In contrast, the majority of more highly sulfated HS motifs likely play FGFR-independent roles in stability and control of access of FGF7 to FGFR2IIIb in the tissue matrix.     

Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography

This protocol describes a method for purification of fibronectin (Fn) from human plasma based on a combination of gel filtration and affinity chromatography steps. Clarified plasma is first loaded onto a Sepharose CL-4B column and unbound material is sequentially purified on columns containing covalently coupled gelatin and Arg. The elution conditions are optimized to obtain a homogeneous preparation of Fn on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the Fn yield is expected to be lower than that obtained using other methods, affinity adsorbents based on gelatin and Arg and gentle elution steps offer advantages including a high purity of the preparation and a correctly folded protein. The preparation can be useful for interaction studies and analysis of biological and immunological activities of Fn.     

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

A domain of the Leptospira LigB contributes to high affinity binding of fibronectin

Adhesion of pathogenic Leptospira spp. to mammalian cells is mediated by their adhesins interacting with host cell receptors. In a previous study, we have identified two potential fibronectin (Fn) binding sites in central variable region (LigBCen) and C-terminal variable region (LigBCtv) of LigB, an adhesin of pathogenic Leptospira spp. In this study, we have further localized the Fn-binding site on LigBCen and found a domain of LigB (LigBCen2) (amino acids 1014-1165) strongly bound to Fn. LigBCen2 bound to a 70kDa domain of Fn including N-terminal domain (NTD) and gelatin binding domain (GBD), but with a higher binding affinity to NTD (K(d)=272nM) than to GBD (K(d)=1200nM). Except Fn, LigBCen2 also bound laminin and fibrinogen. LigBCen2 could bind MDCK cells, and blocked the binding of Leptospira on MDCK cells by 45%. These results suggest that LigBCen2 contributed to high affinity binding on NTD or GBD of Fn, laminin, and fibrinogen and mediated Leptospira binding on host cells.     

Interaction of culture-derived macrophages with the fibroblast-binding domain of fibronectin is a necessary but inefficient signal for fibronectin enhancement of CR1-mediated phagocytosis

Human plasma fibronectin (Fn) enhances ingestion of opsonized particles through its interaction with phagocytic cells. To better characterize the site or sites on Fn responsible for this effect, we subjected Fn to limited proteolytic cleavage by chymotrypsin and used affinity and gel filtration chromatography to isolate a 110,000 dalton cell-binding fragment, a 60,000 dalton fragment that bound both heparin and gelatin, and 50,000 and 45,000 dalton fragments that bound to gelatin but not heparin. The cell-binding fragment mediated adhesion and spreading of fibroblasts on glass slides, whereas the heparin-gelatin and gelatin-binding fragments failed to cause fibroblast spreading. At high concentrations, the cell-binding fragment doubled phagocytosis of C4b-coated sheep erythrocytes by human monocyte-derived macrophages, whereas equal concentrations of the other fragments had minimal enhancing effect on phagocytosis. Interestingly, the effect of the cell-binding fragment on CR1-mediated phagocytosis was always less than the effect of intact Fn, even when a 40-fold higher molar concentration of the cell-binding fragment was used. Fab of a monoclonal anti-Fn, HFn 7.1, which recognizes the 110,000 dalton cell-binding fragment of Fn and inhibits fibroblast binding, blocked enhancement of CR1-mediated phagocytosis by intact Fn. Fab of Fn 8, a monoclonal anti-Fn that binds the heparin-gelatin-binding fragment, failed to inhibit the Fn effect. These data suggest that interaction of the macrophage with the cell-binding domain of Fn is critical for the Fn effect on CR1-mediated phagocytosis. In addition, there may be other domains of the Fn molecule that have a role in augmenting the Fn-phagocyte interaction.     

Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies

Binding of fluorescyl ligand by five IgG anti-fluorescyl hybridoma proteins (4-4-20, 6-10-6, 20-4-4, 20-19-=1, 20-20-3) was examined. Relative reduction in fluorescence of bound fluorescein, deuterium oxide (D2O)-induced enhancement of fluorescence, and the effects of pH on binding kinetics were measured for each clone. Individual hybridoma proteins (all of which bind fluorescein with relatively high affinity) exhibited significant differences in the relative contribution of various forces (hydrophobicity, hydrogen bonding, ionic interactions) to binding and hence, affinity. The extent of such variations in binding mechanisms among monoclonal antibodies binding the same hapten is indicative of the extreme functional diversity of active sites. In addition, ligand binding by clone 20-20-3 was examined in greater detail. ABsorption spectra of ligand bound by purified intact antibody, Fab fragments, and reassociated heavy and light chains indicated that protonation of the fluorescyl ligand by a residue within the active site contributed significantly to the binding free energy. Comparative dissociation rates of fluorescein and a structural analog, rhodamine 110, were used to quantitatively substantiate the contribution of this interaction. Association and dissociation rate studies with fluorescein and antibody indicated that: 1) the active site appeared to undergo a conformational change upon ligand binding, and 2) neither intact disulfides nor intersite cooperativity affected the dissociation rate of bound ligand. Observed mechanisms of ligand binding are discussed in terms of proposed mechanisms of antibody affinity maturation and diversity.     

Influence of steroids and retinoic acid on peanut-lectin binding of human breast cancer cells

Membrane binding sites for peanut lectin or peanut agglutinin (PNA) were investigated in the established mammary carcinoma cell lines MCF-7, 734-B, ZR-75.1 and BT-20. The determination of PNA binding sites was performed in a flow cytometer after staining with fluorescein(FITC)-labeled PNA. It appeared that only the estrogen-sensitive cell lines exhibited PNA binding sites, whereas the hormone-insensitive cell line BT-20 was clearly negative. Steroid hormones, when administered singly to the cells in physiological concentrations (10(-9)-10(-8) M) had no effect on PNA binding expression. Only the combination of estradiol and progesterone together increased PNA binding sites. Pharmacological doses (10(-6) M) of medroxyprogesteroneacetate (MPA) and dexamethasone increased the number of binding sites, whereas retinoic acid decreased them. A preliminary characterization of the binding sites revealed that they have high capacity and moderate affinity for PNA (KD greater than 10(-7) M). FITC-PNA binding could be inhibited selectively by fetuin (greater than 10(-5) M) and by galactose (greater than 10(-2) M). Cytosol from MCF-7 cells and from some primary breast cancer specimens were able to decrease PNA binding to the surface of 734-B cells.     

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as: 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.     

The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells

Leptospira spp. are pathogenic spirochetes that cause the zoonotic disease leptospirosis. Leptospiral immunoglobulin (Ig)-like protein B (LigB) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin, fibrinogen, laminin, elastin, tropoelastin and collagen. A high-affinity Fn-binding region of LigB has been localized to LigBCen2, which contains the partial 11th and full 12th Ig-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, the gelatin binding domain of fibronectin was shown to interact with LigBCen2R (K_D = 1.91±0.40 µM). Not only LigBCen2R but also other Ig-like domains of Lig proteins including LigAVar7''-8, LigAVar10, LigAVar11, LigAVar12, LigAVar13, LigBCen7''-8, and LigBCen9 bind to GBD. Interestingly, a large gain in affinity was achieved through an avidity effect, with the terminal domains, 13th (LigA) or 12th (LigB) Ig-like repeat of Lig protein (LigAVar7''-13 and LigBCen7''-12) enhancing binding affinity approximately 51 and 28 fold, respectively, compared to recombinant proteins without this terminal repeat. In addition, the inhibited effect on MDCKs cells can also be promoted by Lig proteins with terminal domains, but these two domains are not required for gelatin binding domain binding and cell adhesion. Interestingly, Lig proteins with the terminal domains could form compact structures with a round shape mediated by multidomain interaction. This is the first report about the interaction of gelatin binding domain of Fn and Lig proteins and provides an example of Lig-gelatin binding domain binding mediating bacterial-host interaction.     

Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin

Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.     

Biochemical characterization of the fibronectin binding sites for IgG

Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation.     

Elements in the C terminus of apolipoprotein [a] responsible for the binding to the tenth type III module of human fibronectin

In previous studies, we showed that the C-terminal domain, F2, but not the N-terminal domain, F1, is responsible for the binding of apolipoprotein [a] (apo[a]) to human fibronectin (Fn). To pursue those observations, we prepared, by both elastase digestion and recombinant technology, subsets of F2 of a different length containing either kringle (K) V or the protease domain (PD). We also studied rhesus monkey apo[a], which is known to contain PD but not KV. In the case of Fn, we used both an intact product and its tenth type III module (10FN-III) expressed in Escherichia coli. The binding studies carried out on microtiter plates showed that the affinity of F2 for immobilized 10FN-III was approximately 6-fold higher than that for Fn (dissociation constants = 1.75 +/- 0.31 nM and 10.25 +/- 1.62 nM, respectively). The binding was also exhibited by rhesus apo[a] and by an F2 subset containing the PD linked to an upstream microdomain comprising KIV-8 to KIV-10 and KV, inactive by itself. Competition experiments on microtiter plates showed that both Fn and 10FN-III, when in solution, are incompetent to bind F2. Together, our results indicate that F2 binds to immobilized 10FN-III more efficiently than whole Fn and that the binding can be sustained by truncated forms of F2 that contain the catalytically inactive PD linked to an upstream four K microdomain.     

Interactions of calf-thymus histone fractions in aqueous solution with 8-anilinonaphthalene-1-sulphonic acid

1. The interactions of histone fractions with 8-anilinonaphthalene-1-sulphonic acid were investigated by fluorimetry and spectrofluorimetry and the results were interpreted with the aid of equilibrium-dialysis techniques. 2. Characteristic differences were found between the various histone fractions, and with fractions F3 and F2a the binding was found to be salt-dependent. 3. Evidence was obtained indicating a slow change of the physical state of fractions F3 and F2a in the presence of salt, and the binding by these two fractions in the presence of salt was greater by an order of magnitude than by fractions F1 and F2b. 4. Conditions favouring binding were also those favouring histone aggregation; SO(4) (2-) ions activated binding at a lower concentration than Cl(-) ions; urea, guanidinium ions and high concentrations of I(-) ions were inhibitory to binding. 5. After histones had been kept in the presence of salt for a long time the reversal of interaction on decreasing the salt concentration was incomplete. 6. The inhibition of binding by fraction F2a in the presence of urea or fraction F2b depended on the time sequence of addition of the reagents. 7. Artificial nucleoproteins made by precipitating DNA with the histone fractions in neutral 0.14m-sodium chloride showed the same order of interaction as was found for the fractions in solution. 8. Comparison of the binding by fraction F2a with that by bovine plasma albumin showed that in both cases there were a large number of weakly binding sites but that fraction F2a lacked the small number of strongly binding sites found in albumin. No slow change of binding in the presence of salt was found for albumin. 9. Binding by fraction F2b increased the affinity of the protein for further molecules of the adsorbate. 10. The results are discussed in relation to the close relationship between binding and aggregation and the possible role of non-polar interactions as determined by the balance between polar and non-polar amino acids in the histone fractions.     

Lipase-colipase interactions during gel filtration. High and low affinity binding situations

The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.     

Binding analysis of 1alpha- and 17alpha-dihydrotestosterone derivatives to homodimeric sex hormone-binding globulin

Binding studies of the interaction of immobilized 1alpha- and 17alpha-aminoalkyl derivatives of 5alpha-dihydrotestosterone (DHT) with purified N-deglycosylated homodimeric human sex hormone-binding globulin (SHBG) were performed using a surface plasmon resonance biosensor. These 1alpha- and 17alpha-derivatives with spacers of appropriate lengths between the amine function and the steroid ring skeleton enabled privileged, sterically undisturbed, interactions of either the 17- or 3-characteristic functional groups of DHT with SHBG. The association constants (K(a)1) for the binding of these immobilized DHT derivatives to the first binding site of SHBG, determined by SPR measurements, were 0.16 x 10(7) M(-1) for 17alpha-aminopropyl-17beta-hydroxy-5alpha-androstan-3-one (1), 1.64 x 10(7) M(-1) for 17alpha-aminocaproyl-17beta-hydroxy-5alpha-androstan-3-one (2), and 1.2 x 10(8) M(-1) for 1alpha-aminohexyl-17beta-hydroxy-5alpha-androstan-3-one (3). These values were compared with global K(a) data for the corresponding nonimmobilized DHT derivatives from equilibrium measurements using competitions with a tritiated testosterone tracer: the K(a) values were 1.25 x 10(7) M(-1) for 1, 1.50 x 10(7) M(-1) for 2, and 140 x 10(7) M(-1) for 3, confirming a remarkably high binding affinity of this latter compound for SHBG. A global fitting analysis of the biosensor data revealed that the interaction of the three immobilized steroids with SHBG was best described by a kinetic model assuming two structurally independent binding sites. This hypothesis of a bivalent binding model was also directly suggested by a dual fluorescent signal observed by the flow cytometry analysis of SHBG immobilized as a hybrid complex binding simultaneously two 1alpha-aminohexyl DHT ligands, one formed by 3, covalently coupled to phycoerythrin-labeled latex microspheres, and the other by the same DHT derivative, coupled to a fluorescein derivative (4).     

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.