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1.
Adult rat liver parenchymal cells in primary culture exhibit specific saturable binding of 125I-labeled murine epidermal growth factor (EGF). The Scatchard plot of the binding data obtained at 36 °C was curvilinear yielding two apparent dissociation constants of 1.5 × 10?10m and 1.2 × 10?9m with 27,000 and 57,000 sites per cell, respectively. The binding data obtained at 2 °C yielded a linear Scatchard plot with an apparent dissociation constant of 4.4 × 10?9m and 78,000 sites per cell. Exposure of the hepatocytes to EGF at 36 °C resulted in a loss of EGF binding capacity due to down regulation of receptors. The cells recovered the capacity to bind EGF upon incubation in medium which did not contain EGF; this recovery was inhibited by cycloheximide. The cultures appeared to internalize and degrade bound EGF at 36 °C but not at 2 °C. The degradation of EGF was inhibited by chloroquine, an inhibitor of lysosomal enzymes. These data indicate that liver specifically binds and further processes EGF, and therefore, may be a physiological target tissue for this growth factor.  相似文献   

2.
Although D.discoideum amoebae do not bind AMP at their surface if they are not disrupted, total cell lysates display high levels of AMP binding activity specifically associated with the plasma membrane. The binding of AMP is not competed by adenosine and only poorly by ADP and ATP. The AMP binding sites have a single affinity of 0.6 μM for AMP; the association and dissociation rate constants are respectively 8×103 sec?1M?1 and 4.8 ×10?3sec?1. The AMP binding occurs at a site distinct from the cAMP binding site and from the catalytic site of a membrane bound enzyme.  相似文献   

3.
Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with Kd (app) values of 3.5 × 10?9 M and 2.4 × 10?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 105 M?1 sec?1 and 2.6 × 105 M?1 sec?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with T12 approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.  相似文献   

4.
Previous studies have shown that thrombin action at the cell surface is sufficient to bring about division of cultured fibroblast-like cells (Carney and Cunningham, 1978). This prompted the present binding experiments with 125I-thrombin which led to the Identification of a thrombin receptor on the surface of mouse embryo cells. Scatchard plots of binding data at 4, 22 and 37°C were linear over a broad range of thrombin concentrations, indicating a single affinity class of receptors. The association constant was about 1 × 109 M?1 and there were approximately 2 × 105 receptors per cell. Neither insulin, epidermal growth factor nor prothrombin competed for thrombin binding to its receptor, indicating that It was unique for thrombin. Comparisons of thrombin binding and the amount of cell division produced by various concentrations of thrombin indicated that there was a relationship between receptor occupancy and increase in cell number. Low concentrations of serum (0.1%) inhibited both the mitogenic action of thrombin and the specific binding of thrombin to its receptor. It did not, however, inhibit nonspecific association of 125I-thrombin with the cells. Experiments showed that this inhibition by serum resulted from a masking of thrombin receptors on the cells and not from binding of thrombin by serum factors. Together these studies suggest that thrombin must bind to Its surface receptor to stimulate cell division.  相似文献   

5.
The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [125I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10?9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [125I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.  相似文献   

6.
Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k on) of 1.41?×?104 M?1 sec?1, dissociation rate constant (k off) of 2.03?×?10?4 sec?1 and K d of 1.44?×?10?8 M to sulfatide immobilized on a sensor chip. The k off values of sHA were similar for sulfatide and GD1a, whereas the k on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.  相似文献   

7.
Epidermal growth factor (EG factor) and insulin stimulate the incorporation of thymidine into contact-inhibited rabbit lens epithelial cells in culture. The maximal stimulation observed with EG factor is greater than with insulin. Half-maximal stimulation by EG factor is observed at 6 × 10?10m and for insulin at 1 × 10?9m. [125I]-labeled EG factor (2000 Ci/mmol, about 1 g atom of iodine per mol) is equipotent with native EG factor in stimulating DNA synthesis. Both insulin and EG factor bind to distinct high-affinity sites in intact lens cell monolayers; half-maximal binding is observed at about 10?9m for both polypeptides. A maximum of approximately 8 × 104 insulin molecules and 4 × 104 EG factor molecules are bound per cell. These observations indicate that cultured rabbit lens cells possess receptors for insulin and EG factor by biological and physicochemical criteria and raise the possibility that both peptides may play a role in lens growth and development.  相似文献   

8.
Effect of prolactin on the testicular luteinizing hormone binding was studied in a serum-free culture system. By the collagenase digestion of decapsulated testes taken out from 25-day-old rats, Leydig cells were isolated and cultured for 7 days in DME/F12 (1:1) medium supplemented with insulin, transferrin, epidermal growth factor, and gentamicin. The cultured cells exhibited the 3β-hydroxysteroid dehydrogenase activity. Hill plots constructed from the data of competition experiment showed that the dissociation constant (Kd) was 0.33 × 10–10M. The Kd value was approximately the same as the known value for the rat testicular homogenates. When the Leydig cells were cultured with ovine prolactin for the last 3 days of 7-day culture period, the binding of luteinizing hormone increased to 1.7-fold ofthat in the control group. From these results it is concluded that prolactin acts to up-regulate the binding of luteinizing hormone to rat testicular Leydig cells in serum-free culture  相似文献   

9.
Hydrocortisone modulates the binding capacity of HeLa cells for 125I-labeled epidermal growth factor (EGF). A twofold increase in 125I-labeled EGF binding is observed within 24 hours after the addition of pharmacological concentration of hydrocortisone (5 × 10?8?1 × 10?6 M). This enhancement of binding is reversible, and occurs when the cells are cultured in either serum-supplemented or completely defined, serum-free, hormone-supplemented medium. Scatchard analysis of the binding data indicates that the number of 125I-EGF binding sites is increased, and that no appreciable change in the affinity of the EGF receptor for labeled EGF occurs. In the serum-free condition hydrocortisone stimulates the growth of HeLa cells, but we have observed no connection between this growth stimulation and the enhancement of EGF binding. The growth response to hydrocortisone is independent of EGF, and the concentration dependency of the growth response to EGF is unaltered by the addition of hydrocortisone to the medium. Hydrocortisone elicits the growth response at a concentration as low as 5 × 10?9 M, while a concentration higher than 5 × 10?8 M is required to affect the binding capacity for 125I-EGF. These effects are specific for glucocorticoid steroids. Similar concentrations of progesterone, testosterone, or estradiol produce no measurable response. Although the elevation of EGF receptor levels in the serum-supplemented medium is similar to that observed in the serum-free cultures, hydrocortisone is growth-inhibitory under these conditions. This growth inhibition occurs at pharmacological concentrations of hydrocortisone with a concentration dependency that is similar to that of the EGF receptor modulation.  相似文献   

10.
Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 106 epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.  相似文献   

11.
Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca2+ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of α-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth factor occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecular weight molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.  相似文献   

12.
The reaction between the mouse (BALB/c) anti-idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 103 M?1 sec?1) and a resulting low equilibrium constant (Ka = 2 × 105M ?1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.  相似文献   

13.
The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (KCN) of the peroxidase-cyanide complex and both forward (k+) and reverse (k?) rate constants are independent of the H+ concentration over the pH range 2.7 to 7.1. The values obtained are kcn = (9.5 ± 1.0) × 10-5 M, k+. = (5.2 ± 0.5) × 104 M?1 sec?1 and k- = (5.0± 1.4) sec-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pKa values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pKa of the heme-linked acid group.  相似文献   

14.
The specific binding of [3H] Prostaglandin (PG) F2α to bovine corpus luteum cell membranes prepared in homogenizing buffer containing either 1 mM EDTA (H-EDTA) or 1 mM Ca2+ (HCa2+) was examined. The membranes prepared in H-EDTA buffer bound less [3H] PGF2α and had a single class of PGF2α receptors with an apparent dissociation constant (Kd) of 2.7 × 10?8M. The addition of Ca2+ to these membranes resulted in increased binding with the appearance of new PGF2α receptors of Kd = 4.3 × 10?9M. The membranes prepared in HCa2+ buffer contained two classes of receptors with Kds = 2.9 × 10?9M and 2.9 × 10?8M. The removal of Ca2+ from these membranes resulted in lower binding as well as a complete disappearance of receptors of Kd = 2.9 × 10?9M. These results suggest the dependency of high affinity PGF2α receptors, in bovine corpus luteum cell membranes, on cations.  相似文献   

15.
The binding of [G-3H]nitrobenzylthioinosine to intact Chinese hamster ovary cells has been studied kinetically and thermodynamically. The association of nitrobenzylthioinosine with cells is a second-order process which proceeds at 24°C with a rate constant of 2·107 M?1·s?1. Dissociation of the complex was characterized as a simple first-order process with rate constant on the order of 7·10?3 s?1. The quotient of these is comparable to the dissociation constant as measured in equilibrium binding studies, 2.2·10?10 M. The temperature dependence of the rate of association indicated an Arrhenius activation energy of 8.4 kcal·mol?1, while that of the equilibrium constant for dissociation indicated a standard enthalpy change of 8.8 kcal·mol?1. The large increase in affinity of nitrobenzylthioinosine as compared to natural nucleosides is attributable to an entropy-driven interaction with the binding site. Thymidine, dipyridamole and papaverine each decrease the apparent dissociation constant for the nitrobenzylthioinosine-cell complex; the latter, inhibitors of nucleoside transport, decrease the rate of dissociation of the complex.  相似文献   

16.
The epidermal growth factor can be isolated from the male mouse submaxillary gland as part of a high molecular weight complex. The complex is composed of two molecules of epidermal growth factor and two molecules of epidermal growth-factor binding protein (J.M. Taylor, W.M. Mitchell, and S. Cohen, 1974, J. Biol. Chem.249, 3198–3203). The proteolytic activity of epidermal growth-factor binding protein was demonstrated by its self-proteolysis in moderate (3–7 m) concentrations of urea, and, its inhibition by formation of a complex with pancreatic trypsin inhibitor. This complex was characterized by its pI and by its ability to yield pancreatic trypsin inhibitor and epidermal growth factor-binding protein in sodium dodecyl sulfate-urea gel electrophoresis. The association equilibrium constant was determined to be 3.6 × 107m?1 by inhibition studies of the esteropeptidase. These results, which indicate that epidermal growth factor-binding protein is capable of autodigestion and of forming a stable complex with a macromolecular inhibitor of trypsin, lend strong support to the hypothesis that epidermal growth factor-binding protein is capable of cleaving a larger precursor by its proteolytic action.  相似文献   

17.
The kinetics of the reaction of Helix pomatia haemocyanin with oxygen have been studied under conditions where ligand binding is co-operative (n = 4.5). The dissociation of oxygen from oxyhaemocyanin in the presence of sodium dithionite and the combination of deoxyhaemocyanin with oxygen were studied by the stopped-flow technique. The combination with oxygen, as well as the dissociation of oxyhaemocyanin, are clearly autocatalytic. The initial rate constant for oxygen combination to the fully deoxygenated state is 0.2 to 0.3 × 106m?1 s?1; during the course of the reaction the rate constant increases to a value higher than 106m?1s?1.The initial rate of oxygen dissociation from fully saturated haemocyanin is 10 s?1, increasing to about 30 s?1 as the reaction proceeds. Thus, both the combination and the dissociation rate constants contribute to the co-operativity of oxygen binding.Temperature-jump relaxation experiments were carried out at fractional oxygen saturations larger than 0.7. The dependence of the relaxation rate upon the concentration of the reactants indicates the presence of one principal bimolecular process. The calculated combination and dissociation rate constants for this process are: 3.8 × 106m?1 s?1 and 10 s?1, respectively. Evidence is presented which shows that the transition from the T-state to the R-state of the protein is relatively slow. Both the T and R-state seem to be largely stabilized at the expense of intermediate states.Under other conditions, where oxygen binding is non-co-operative, temperature-jump and stopped-flow experiments reveal considerable kinetic heterogeneity.  相似文献   

18.
Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law
where Ka is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k1 = 3.94 × 102M?1 sec?1, k2Ka = 1.18 × 10?1 sec?1, k3 = 3.6 × 10?1 sec?1. Activation parameters for k1 are ΔH = 11.7 kcal mole?1 and ΔS = ?8 cal K?1 mole?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step  相似文献   

19.
The binding of the fluorescent analog of adenosine diphosphate (ADP)1, 1,N6-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (± .5) × 10?5 M?1. This is identical to the values found by Young (J. Biol. Chem., 242, 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of Vmax and Km were 25 μM phosphate sec?1 (gm protein)?1 and 5 × 10?5 M?1 for ATP, and 80 μN phosphate sec?1 (gm protein)?1 and 45 × 10?5 M?1 for εATP. The results indicate that the increased Vmax that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in Vmax may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex.  相似文献   

20.
Insulin-receptor interactions in liver cell membranes   总被引:17,自引:0,他引:17  
The specific binding of 125I-insulin to liver cell membranes is a saturable process with respect to insulin. Binding is displaced by low concentrations of native insulin but not by biologically inactive insulin derivatives or by other peptide hormones. The rate constants of association (3.5 × 106 mole−1 sec−1) and of dissociation (2.7 × 10−4 sec−1) of the insulin-membrane complex can be determined independently. The dissociation constant of the complex, determined from the rate constants and from equilibrium data, is about 7 × 10−11M. Complex formation does not result in degradation of the insulin molecule. The binding interaction is a dissociable process involving a homogeneous membrane structure which is almost certainly the biologically significant receptor. The kinetic properties, and the effects of enzymic perturbations of the membrane, suggest that the insulin receptors of liver and of adipose tissue cells may be very similar structures.  相似文献   

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