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1.
Ben D.-M. Chen Shin Hsu Hsiu-San Lin 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(4):366-373
Iodinated colony-stimulating factor produced by L-cells (125I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37°C, the cell-bound 125I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0°C, the cell-bound 125I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37°C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH4Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added 125I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37°C but not at 0°C in culture medium containing fetal calf serum. 相似文献
2.
Distribution of cells bearing receptors for a colony-stimulating factor (CSF-1) in murine tissues
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《The Journal of cell biology》1981,91(3):848-853
CSF-1 is a subclass of the colony-stimulating factors that specifically stimulates the growth of mononuclear phagocytes. We used the binding of 125I-CSF-1 at 0 degrees C by single cell suspensions from various murine tissues, in conjunction with radioautography, to determine the frequency of binding cells, their identity, and the number of binding sites per binding cell. For all tissues examined, saturation of binding sites was achieved within 2 h at 2--3 x 10(-10) M 125I-CSF-1. The binding was irreversible and almost completely blocked by a 2 h preincubation with 5 x 10(-10) M CSF-1. 125I-CSF-1 binding was exhibited by 4.3% of bone marrow cells, 7.5% of blood mononuclear cells, 2.4% of spleen cells, 20.5% of peritoneal cells, 11.8% of pulmonary alveolar cells and 0.4% of lymph node cells. Four morphologically distinguishable cell types bound 125I-CSF-1: blast cells; mononuclear cells with a ratio of nuclear to cytoplasmic area (N/C) greater than 1; cells with indented nuclei; and mononuclear cells with N/C less than or equal to 1. No CSF-1 binding cells were detected among blood granulocytes or thymus cells. Bone marrow promyelocytes, myelocytes, neutrophilic granulocytes, eosinophilic granulocytes, nucleated erythroid cells, enucleated erythrocytes, and megakaryocytes also failed to bind. The frequency distribution of grain counts per cell for blood mononuclear cells was homogenous. In contrast, those for bone marrow, spleen, alveolar, and peritoneal cells were heterogeneous. The monocytes in blood or bone marrow (small cells, with either indented nuclei or with N/C greater than 1) were relatively uniformly labeled, possessing approximately 3,000 binding sites per cell. Larger binding cells (e.g., alveolar cells) may possess higher numbers of receptors. It is concluded that CSF-1 binding is restricted to mononuclear phagocytic cells and their precursors and that it can be used to identify both mature and immature cells of this series. 相似文献
3.
Both granulocyte-macrophage CSF and macrophage CSF control the proliferation and survival of the same subset of alveolar macrophages 总被引:4,自引:0,他引:4
The effect of granulocyte-macrophage (GM)-CSF on the proliferation of murine pulmonary alveolar macrophages in vitro was investigated. About 20% of freshly isolated alveolar macrophages formed colonies in both liquid and soft agar cultures in the presence of GM-CSF. GM-CSF was also found to be capable of maintaining the survival of these colony-forming cells in vitro. Moreover, GM-CSF could substitute for CSF-1 in maintaining the survival of CSF-1-responding pulmonary alveolar macrophage colony-forming cells in the absence of CSF-1. The concentration of GM-CSF required for maintaining the survival of colony-forming cells without proliferation was much lower than that required for the proliferation of these cells in vitro. It also enhanced the CSF-1-dependent clonal growth of alveolar macrophages. These data suggest that the colony-forming cells that respond to GM-CSF are the same subset of macrophages that form colonies in the presence of CSF-1. GM-CSF did not inhibit the binding of 125I-CSF-1 to alveolar macrophages at 0 degrees C. However, the preincubation of macrophages with GM-CSF at 37 degrees C resulted in a transient down-regulation of CSF-1 binding activity. 相似文献
4.
G A Gao D X Zhu X Tao J Zheng A Scharfman G Lamblin Y W Shing K K Han 《The International journal of biochemistry》1989,21(4):391-399
1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase. 相似文献
5.
《Bone and mineral》1994,24(2):151-164
It has been shown that both calcitonin gene-related peptide (CGRP) and amylin bind weakly to calcitonin (CT) receptors in osteoclast-like cells formed in vitro and inhibit bone resorption by a cAMP-dependent mechanism. Osteoclasts are thought to be derived from cells of the monocyte macrophage lineage, in which CGRP, but not CT, induces cAMP production. In this study, we determined the presence of functional receptors for CGRP in mouse alveolar macrophages and the effects of this peptide on proliferation and osteoclastic differentiation in mouse alveolar and bone marrow-derived macrophages. Human CT did not stimulate cAMP production in macrophages. Human CGRP stimulated cAMP production in mouse alveolar macrophages and bone marrow-derived macrophages dose-dependently. Human amylin, which has 43% homology with human CGRP, also stimulated these macrophages to produce cAMP, but only at a 100-fold higher concentration. The increment in cAMP production induced by human CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. Specific binding of [125I]human CGRP to alveolar macrophages was detected (dissociation constant, 2.5 × 10−8 M; binding sites, 1.4 × 104/cell). Amylin, but not CT, displaced the bound [125I]human CGRP from alveolar macrophages, but at a 100-fold higher concentration. No specific binding of [125I]human CT and [125I]human amylin to alveolar macrophages could be detected. Pretreatment with human CGRP for 24 h dose-dependently suppressed DNA synthesis in alveolar macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). CGRP also suppressed the number of macrophage colonies formed from bone marrow cells induced by macrophage colony-stimulating factor (M-CSF). Pre-treatment of alveolar macrophages with CGRP inhibited differentiation into osteoclast-like cells in co-cultures with primary osteoblastic cells in the presence of 1α,25-dihydroxy vitamin D3. These results indicate that specific receptors for CGRP are present in macrophages and that CGRP modulates proliferation and differentiation of macrophages into osteoclast-like cells by a receptor-mediated mechanism involving cAMP. 相似文献
6.
Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages 总被引:1,自引:0,他引:1
Iodinated colony-stimulating factor produced by L-cells (125I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37 degrees C, the cell-bound 125I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0 degree C, the cell-bound 125I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37 degrees C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH4Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added 125I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37 degrees C but not at 0 degrees C in culture medium containing fetal calf serum. 相似文献
7.
Sren Kragh Moestrup Erik Ils Christensen Lars Sottrup-Jensen Jrgen Gliemann 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,930(3)
125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3–4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4°C was 2–3-times higher than binding of the homologous α2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2–3 min at 37°C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the α2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes. 相似文献
8.
Isolated, intact rat liver nuclei have high-affiity (Kd=10−9 M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd=10−9 M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (Kd=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (Kd=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites. 相似文献
9.
Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages 总被引:6,自引:0,他引:6
L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity. 相似文献
10.
Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor 总被引:4,自引:0,他引:4
J H Shieh R H Peterson D J Warren M A Moore 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(8):2534-2539
The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis. 相似文献
11.
Using 3H-leukotriene D4, a specific receptor assay has been developed for human alveolar macrophages, obtained by broncho-alveolar lavage of patients undergoing fiberoptic bronchoscopy because of suspected bronchial carcinomaa. Lavage was performed in a carcinoma-free lobe of the lung and alveolar macrophages were subsequently isolated and incubated for binding studies. 3H-Leukotriene D4 was found to bind specifically with high affinity (Kd = 3.8 nM), in a saturable manner (Bmax = 90 fmol/106 cells), reversible and selective. Specific binding was linear with protein concentration and equilibrium binding at 4°C was reached at 50 min. Scatchard and Hill analysis revealed a single class of binding sites with no cooperativity among the sites. displacement studies with LTD4, the selective SRS-A antagonist FPL 55712 and with leukotriene C4 revealed respective Ki values of 3.4; 16; and 110 nM. The data suggest that human alveolar macrophages may contain a specific receptor type for LTD4, which has a relatively low affinity for LTC4, and are discussed in relation to modulatory processes in the lung, apart from direct actions of LTD4 on smooth muscle receptors. From the data here acquired, it may be apparent that the study of characteristics of receptors specific for a broncho-active substance like LTD4 on huma alveolar macrophages, which play an important role in immuno-inflammatory processes seen in many chronic lung diseases, may yield major insights into the pathogenesis and therapy decisions involved in these diseases. 相似文献
12.
Juan-Carlos Prieto Maria-Jose Carmena 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,763(4):408-413
Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of 125I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15°C. At apparent equilibrium, the binding of 125I-labelled peptide was competitively inhibited by native VIP in the 1·10−10−10−7 M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a Kd = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium. 相似文献
13.
Junji Tsuzuki Henry C. Wu 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(4):390-399
The nature of binding of abrin to Chinese hamster ovary cells was examined in relation to the ensuing intoxication of the treated cells. Approx. 20% of [125I]abrin bound to CHO cells at 37°C was found to be resistant to the addition or presence of 0.1 M lactose. The extent of lactose-resistant binding depended inversely upon the temperature of incubation. Among various proteins, lectins and sugars, only non-labeled abrin could strongly inhibit the lactose-resistant binding of [125I]abrin. Lactose-resistant binding could lead to an inhibition of cellular protein synthesis and to a loss of cell viability. Abrin molecules bound at the lactose-sensitive and lactose-resistant binding sites apparently have an equal probability of being internalized by CHO cells. Binding of approx. 3·103 abrin molecules per CHO cell was required to elicit 50% loss of cell viability regardless of whether the binding occurs in the presence or absence of lactose. The result of a cross-linking experiment suggested that a membrane protein with an Mr of about 45 000 may be responsible for the lactose-resistant binding of abrin. 相似文献
14.
Robert J. Mason Thomas P. Stossel Martha Vaughan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,304(3):864-870
The initial rate of phagocytosis by rabbit alveolar macrophages of paraffin oil emulsions stabilized with albumin was markedly increased by prior incubation of the emulsion with serum. The active component(s) of serum was non-dialyzable and heat-labile and was absent from zymosan-treated serum. Magnesium and calcium were both required for the maximal rate of phagocytosis. At 4 °C or in the presence of 1 mM N-ethylmaleimide, the rate of phagocytosis was less than 2% of the control (37° C) rate. The initial rates of phagocytosis of this emulsion by alveolar macrophages from rabbits injected with Freund's adjuvant were not demonstrably different from those observed with macrophages from normal rabbits. Per of mg of cell protein, polymorphonuclear leukocytes ingested serum-treated emulsion more rapidly than did macrophages, but per cell the rates were not different. 相似文献
15.
Gerhard Fleischmann Miguel Beato 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,540(3):500-517
The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [3H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 108 M−1 at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [3H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [3H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [3H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [3H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C
. Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [3H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K−1 · mol−1.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight. 相似文献
16.
Receptors for α2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled α2-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α2-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat α1-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-α1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor. 相似文献
17.
Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages 总被引:15,自引:0,他引:15
Rabbit alveolar macrophages internalize α-macroglobulin 125I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · 125I-T at 37°C than at 0°C. At 0°C cell-bound αM · 125I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · 125I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · 125I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · 125I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM125I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization. 相似文献
18.
Solubilization and assay of a colony-stimulating factor receptor from murine macrophages 总被引:3,自引:0,他引:3
The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl). 相似文献
19.
S.U. Jadhav S.D. Kalme S.P. Govindwar 《International biodeterioration & biodegradation》2008,62(2):135-142
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl−1) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl−1 molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l−1 concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l−1 concentration.
The scientific relevance of the paper
The azo dye Methyl red (100 mg l−1) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants. 相似文献20.
Intact Sendai virus particles were radiolabeled by the use of chloramine-T and Na 125I. The method described is reproducible, efficient and appropriate for the preparation of large quantities of biologically active virus with relatively high specific activity. Gel electrophoresis analysis of the radiolabeled virus revealed that approx. 50% of the total 125I incorporated in the virus are associated with the two viral envelope glycoproteins, while the remaining 50% are evenly distributed throughout the other viral polypeptides. The 125I-virus particles were used to study some of the kinetic parameters of the interaction between Sendai virus particles and human erythrocytes. Binding of virus particles at 4 °C is irreversible, non-cooperative and exhibits a characteristic saturation curve. A maximum of 1–2 × 103 virus particles bound per cell was derived from the saturation curve. Non-radioactive native virus particles as well as isolated glycophorin molecules competitively inhibit binding of the 125I-virus particles to human erythrocytes. Incubation at 37 °C of the virus-erythrocyte complex resulted in the release of about 33% of the bound virus to the surrounding medium. 相似文献