Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

3,5,3'-triiodo-L-thyronine binding sites in nuclei of human trophoblastic cells

Nuclear binding sites of T3 in human trophoblastic cells were biochemically characterized. Nuclei were isolated by a combination procedure with mild homogenization of the freshly obtained trophoblastic tissue aged term gestation, centrifugations and Triton X-100 treatment. The isolated nuclei were incubated with various concentrations of 125I-T3 at 20 degrees C for 3 h. The total number of T3 binding sites per nucleus was approximately 650. The apparent association constant (Ka) was 6.0 X 10(9)M-1. Nuclear proteins extracted from purified nuclei with 0.4M KCl were able to bind T3 giving rise to nuclear thyroid hormone binding protein-T3 complexes and they were precipitated with bovine IgG, as a carrier protein, by 12.5% polyethylene glycol. Binding was maximum in 3 h incubation at 20 degrees C or in 18 h at 0 degrees C, while it dropped quickly at 37 degrees C. The binding characteristics were analyzed by Scatchard plots. In nuclear proteins obtained from 8 term placentae there was a single set of high affinity-low capacity T3 binding sites with Ka of 7.0 X 10(9)M-1. The capacity is about 62.7 fmol T3/mg DNA. The binding sites were found to be specific for L-T3, while L-T4 was about 100-fold less effective, rT3 ineffective, and D-T3 and D-T4 were roughly 1/8 and 1/5 as active as L-T3 and L-T4, respectively in displacing 125I-T3 from the binding sites. These data confirmed that human placenta is a target organ of thyroid hormones; trophoblastic cells contain T3 nuclear receptors which are biochemically similar to those isolated from liver, although the capacity is low.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Cloning of the gene for myxobacterial hemagglutinin and isolation and analysis of structural gene mutations.

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation in the bacterium Myxococcus xanthus. It has been shown that this lectin is targeted to the cell surface and periplasmic space of developmental cells, suggesting that it may play a role in cell-cell recognition or agglutination. We have cloned the structural gene for MBHA by using synthetic deoxyoligonucleotides containing sequences deduced from the amino acid sequence of MBHA and have used the cloned gene to construct strains of M. xanthus that cannot synthesize MBHA. We found that although the MBHA-deficient strains are delayed in their developmental time course, they are otherwise able to aggregate and sporulate normally. Our results suggest that MBHA may function to increase the efficiency of fruiting-body formation but is not a critical component of cellular aggregation.     

Phaseolus vulgaris isolectin binding to human erythrocytes.

The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Increase of transferrin receptors in regenerating rat liver cells after partial hepatectomy

The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor.     

Interaction between squash inhibitors and bovine trypsinogen

Squash seeds proteinase inhibitors form stoichiometric complexes with bovine trypsinogen. In terms of association constants (Ka), the interaction is weak. The inhibitors bind to the zymogen with Ka values of approx. 10(4)M-1 i.e. 2 X 10(7) times weaker than to bovine beta-trypsin. Squash inhibitor with Lys at the P1 position binds to trypsinogen with a Ka value 2.1-fold higher than the inhibitor with Arg at P1. The Ile-Val binding cleft and the Ca2+ binding site of trypsinogen are cooperatively linked to the inhibitor binding site. Although these three sites are spatially separated, either binding of calcium ion or Ile-Val dipeptide to trypsinogen increase the Ka values 3-fold and more than 100-fold, respectively. In the presence of Ile-Val trypsinogen resynthetizes extremely slowly (about 10(4) times slower than beta-trypsin) the reactive site peptide bond in squash inhibitors.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Alterations in monoclonal antibody affinity and antigenic receptor site expression on mycoplasma-infected human colorectal cancer cells

The affinity of MoAb CO 17-1A and expression of its antigenic target were studied on uninfected and mycoplasma-infected colorectal cancer cell lines SW 1116 and SW 948. Binding of 125I-labeled CO 17-1A to SW 1116 cells was quantified at 37 degrees C by determination of the affinity constant (Ka) and the number of antigenic receptor sites (r) per cell using Scatchard plots. When mycoplasma-free SW 1116 cells were used as targets, Ka was 0.92 +/- 0.06 x 10(8) M-1 and r = 1.32 +/- 0.14 x 10(6) at 37 degrees C. One batch of unspeciated, mycoplasma-infected SW 116 cells had reduced affinity and a decreased number of antigenic receptor sites per cell for 125I-labeled 17-1A, while another batch of infected SW 1116 cells had a 4- to 5-fold increase in r and diminished Ka for the antibody compared with uninfected cells. When unspeciated, mycoplasma-infected SW 948 cells were exposed to 125I-labeled 17-1A and the data subjected to Scatchard analysis, the affinity of the antibody deviated markedly from linearity and rendered analysis for Ka and r meaningless. These data indicate that mycoplasma infection can produce variable effects on the cellular expression of antigenic receptor sites and the affinity of antibody for its target, and emphasize the importance of using mycoplasma-free cell lines in studies of these parameters.     

Insulin binding on cultured chick muscle cells: decrease in binding associated with cell fusion

Binding of 125I-bovine and chicken insulin to cultured embryonic chick skeletal muscle cells was studied. Bovine and chicken insulin bound cultured cells with high affinities of 2.4 X 10(9)M-1 and 4.8 X 10(9)M-1 and low affinities of 2.4 X 10(7)M-1 and 3.7 X 10(7)M-1, respectively. Maximum insulin binding was achieved after 90 min of incubation at 20 degrees C and the maximum value was maintained for an additional 3 hr. Insulin binding increased in a linear manner with increasing nuclei number over a 5-fold range. Maximum insulin binding per nuclei decreased as cell fusion increased between 24 and 72 hr in culture, primarily due to a decrease in the number of low affinity insulin receptors.     

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.     

Characterization and regulation of alpha-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells

Interferon-sensitive (IFN-S) and IFN-resistant (IFN-R) Daudi lymphoblastoid cells were studied for IFN-alpha receptor expression and regulation by steady state and kinetic procedures, utilizing a homogeneous 125I-IFN-alpha 2 probe. Heterogeneity in the binding of this probe to IFN-S cells was determined to result from negatively cooperative interactions between an initially homogeneous class of IFN receptor. No such heterogeneity was noted in the IFN-R cells, indicating an apparent difference in the interaction of IFN-alpha 2 with these cells. The apparent dissociation constants (Kd) for IFN-S cell receptors were calculated to be 1 X 10(-10) M and 1 X 10(-8) M, for the high and low affinity sites, respectively. The Kd for sites on the IFN-R cells was estimated to be 4 X 10(-9) M. IFN-R and IFN-S cells expressed 2.4 X 10(4) and 3.5 X 10(4) binding sites per cell, respectively, representing an increase of at least 6-fold over previous reports of IFN-S Daudi IFN receptor density. Both IFN-S and IFN-R cells were capable of down-regulating expression of the IFN-alpha receptor in response to low concentrations of IFN-alpha 2. Furthermore, both cell lines were shown to be capable of internalizing specifically bound 125I-IFN-alpha 2 to an equivalent degree. Accordingly, we propose that the relative insensitivity of the Daudi IFN-R phenotype involves the loss of a high affinity interaction between cellular receptors and IFN-alpha 2, in addition to the reduced level of expressed low affinity binding sites.     

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

Role of glycans in the binding of human serotransferrin and lactotransferrin to human alveolar macrophages

The optimal conditions of the binding of human lactotransferrin to human alveolar macrophages have been determined and the necessity to measure the binding in absence of bovine serum albumin was demonstrated. In these conditions, diferric lactotransferrin and iron-free lactotransferrin are reversibly bound with the following parameters: association constant Ka = 2 and 5 X 10(6) M-1, respectively, and the number of binding sites N = 1.2 and 1 X 10(7), respectively. The binding of the two forms of lactotransferrins was inhibited by various neoglycoproteins, the highest inhibition being obtained with L-fucosyl, then, in the following decreasing order: D-mannosyl greater than N-acetyl-D-glucosaminyl greater than D-galactosyl. In the same conditions, the binding of serotransferrin (Ka = 2 X 10(7) M-1 and 1.6 X 10(7) M-1; N = 5 X 10(4) and 8 X 10(4) for diferric and iron-free protein, respectively) was not inhibited. These results suggest that the recognition of lactotransferrin is mediated by one or several membrane lectins, the fucose being one of the sugar playing an important role in the association. On the contrary, the binding of serotransferrin does not depend on a membrane lectin system.     

An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4).

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.     

Role of galaptin in ovarian carcinoma adhesion to extracellular matrix in vitro

Immunohistochemical studies indicated that galaptin is a major protein of ovarian carcinoma cells present in patient effusions and it is distributed throughout the cytoplasm. Enzyme-linked immunoadsorbent assay (ELISA) and immunoprecipitation experiments demonstrated that galaptin is also a major protein of the A121 ovarian carcinoma cell line, constituting less than or equal to 1% of extractable protein bound by DEAE Sephacel. Western blot analyses revealed that the galaptin present in ovarian carcinoma consists of a 14.5 KD subunit. Ovarian carcinoma and mesothelial cells isolated from patient effusions display surface receptors for galaptin with an apparently greater density of receptors present on the carcinoma cells. A121 cells also display surface receptors for galaptin: binding sites/cell = 3 X 10(8) and Ka = 1.2 X 10(9) M-1. The presence of galaptin in bovine corneal endothelial cells (BCEC) and BCEC-derived extracellular matrix (ECM) was demonstrated by ELISA. Of the total ECM-bound galaptin, about 75% appears to be insoluble in phosphate-buffered saline (PBS) lactose. ECM was also found to contain abudnant receptors for galaptin. Treatment of ECM with lactose increased the apparent galaptin receptor density:binding sites/cm2 = 7 X 10(13) and Ka = 2.6 X 10(9) M-1. Pretreatment of A121 cells with galaptin inhibited adhesion to ECM. The addition of exogenous galaptin to ECM had variable effect on cell adhesion. The data presented here suggest that early adhesion events may be carbohydrate-specific involving interaction between ECM-bound galaptin and cell surface galaptin receptors.