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1.
We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.  相似文献   

2.
Rat plasma fibronectin has been isolated and characterized and monospecific antibodies were prepared to it. Two components of fresh rat plasma (in the presence of proteinase inhibitors) bound to a gelatin-Sepharose affinity column. One protein was eluted with 4.0 M-urea and was identified as fibronectin. Another protein was eluted from the gelatin-Sepharose column with 8.0 M-urea and was identified as a 70 000-Mr collagen-binding molecule. This 70 000-Mr fragment was found to be a normal constituent of blood plasma, and its presence did not represent a proteolytic degradation product formed during isolation. The antibodies prepared against rat fibronectin only weakly cross-reacted with plasma fibronectins of chicken, horse and human. These studies shed light on the metabolic interrelationships between fibronectin and other collagen-binding molecules.  相似文献   

3.
A monoclonal antibody specific for the chicken transferrin receptor was used to study receptor expression on circulating red cells from chick embryos of different ages. The use of indirect immunofluorescence with this antibody showed that all circulating immature reticulocytes and primitive series erythrocytes--but not erythrocytes from the definitive series--expressed the receptor. In all cells, the protein was synthesized as a 90-95-kD form. The retention of the transferrin receptor (and another proliferation-dependent cell surface protein) contrasted with the behaviour of a series of other developmentally regulated antigens which are lost during maturation of both primitive and definitive series erythroid cells.  相似文献   

4.
A 68 000-Mr protein is a major component of a Nonidet P-40-insoluble fraction of lymphocyte plasma membrane prepared from human B lymphoblastoid cells ( BRI 8) and pig mesenteric lymph nodes. The association of the protein with the detergent-insoluble complex depends on free Ca2+ concentrations of greater than 10 microM. The human and pig 68 000-Mr proteins were purified and appear to be homologous on the basis of amino acid composition and peptide mapping. The protein is monomeric, has pI 5.8 and a single high-affinity Ca2+-binding site (KD 1.2 microM). The results are discussed in terms of the possible role of the 68 000-Mr protein as an intracellular Ca2+ receptor in lymphocytes.  相似文献   

5.
Serum stimulation of quiescent chicken embryo fibroblasts resulted in a time-dependent, biphasic activation of S6 kinase activity. Chromatographic fractionation of serum-stimulated cell lysates resolved two distinct S6 kinase activities. Anti-Xenopus S6 kinase II antiserum immunoprecipitated a 90,000-Mr S6 kinase but did not cross-react with a smaller, 65,000-Mr S6 kinase. Phosphopeptide analysis confirmed that the 90,000- and 65,000-Mr proteins were structurally unrelated and established that the 65,000-Mr protein isolated by the current protocol was the same serum-stimulated chicken embryo fibroblast S6 kinase as that previously characterized (J. Blenis, C. J. Kuo, and R. L. Erikson, J. Biol. Chem. 262:14373-14376, 1987). These results demonstrate the contribution of two distinct S6 kinases to total serum-stimulated ribosomal protein S6 phosphorylation.  相似文献   

6.
Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.  相似文献   

7.
Summary The mechanism of iron uptake by avian erythroid cells was investigated using cells from 7 and 15-day chicken embryos, and chicken serum transferrin and conalbumin (ovotransferrin) labelled with125I and59Fe. Endocytosis of the protein was determined by incubation of the cells with Pronase at 4°C to distinguish internalized from surface-bound protein.Iron was taken up by the cells by receptor-mediated endocytosis of transferrin or conalbumin. The receptors had the same affinity for serum transferrin and conalbumin. Endocytosis of diferric transferrin and conalbumin and exocytosis of apo-protein occurred at the same rates, indicating that iron donation to the cells occurred during the process of intracellular cycling of the protein. The recycling time was approximately 4 min. The rate of endocytosis of diferric protein varied with incubation temperature and at each temperature the rate of endocytosis was sufficient to account for the iron accumulated by the cells. These results and experiments with a variety of inhibitors confirmed the role of endocytosis in iron uptake.The mean cell volumes, receptor numbers and iron uptake rates of 7-day embryo cells were approximately twice those of 15-day embryo cells but the protein recycling times were approximately the same. Hence, the level of transferrin receptors is probably the main determinant of the rate of iron uptake during development of chicken erythroid cells.Transferrins from a variety of mammalian species were unable to donate iron to the chicken cells, but toad (Bufo marinus) transferrin could do so at a slow rate. The mechanism of iron uptake by developing chicken erythroid cells appears to be similar to that described for mammalian cells, although receptor numbers and iron uptake rates are lower than those reported for mammalian cells at a similar stage of development.Abbreviations BSS Hanks balanced salt solution - PBS phosphate buffered saline - MCV mean corpuscular volume - CCCP carbonyl cyanide-M-chlorophenyl hydrazone  相似文献   

8.
A transferrin binding protein was isolated from normal rat placenta and from iron-deficient rat plasma using a human transferrin affinity column. The yield of the isolated pure protein from iron-deficient rat plasma was about 0.5 micrograms/ml plasma. The major protein had a molecular mass of 85 kDa and contained carbohydrate. Reduction with mercaptoethanol did not change the molecular mass of the plasma transferrin binding protein whereas the native placental transferrin receptor of 180 kDa was reduced to 90 kDa. The transferrin binding protein reacted with both monoclonal and polyclonal antibodies raised against rat transferrin receptor. Immunoblotting of both normal and iron deficient rat plasma showed that the transferrin binding protein had a molecular mass of 85 kDa. In vitro digestion of purified rat placental transferrin receptor and red blood cells with trypsin provided an identical peptide profile, suggesting that the transferrin binding protein in rat plasma is derived from proteolysis of the extracellular portion of the transferrin receptor of the erythroid tissues.  相似文献   

9.
Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.  相似文献   

10.
cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.  相似文献   

11.
Alterations in tubulin immunoreactivity; relation to secondary structure   总被引:2,自引:0,他引:2  
Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants. The phosphorylation reaction was studied at 33 degrees C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [gamma-32P]ATP or [gamma-32P]GTP essentially into one 170 000 Mr protein. Some incorporation was observed in a minor 120 000-Mr component which appears to be a degradation product of the 170 000-Mr component. No EGF-dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro. The dephosphorylation of the 170 000-Mr component was observed after 4 min of incubation at 33 degrees C. This dephosphorylation reaction was inhibited by addition of 5 mM p-nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate. The 170 000-Mr protein specifically bound 125I-labeled EGF and thus appeared to be the hepatic EGF receptor. The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170 000-Mr EGF receptor at 33 degrees C. Tryptic peptide maps of the 32P-labeled 170 000-Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P-labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170 000-Mr protein after it was covalently linked to 125I-labeled EGF showed only one 125I-labeled peptide, the migration of which appeared different from that of 32P-labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170 000-Mr protein, labeled with 32P or 125I-EGF.  相似文献   

12.
Physical characterization of the transferrin receptor in human placentae   总被引:4,自引:0,他引:4  
The physical properties and binding characteristics of the solubilized transferrin receptor isolated from the placental brush-border membrane of a human trophoblast cell were investigated. The receptor protein was isolated from solubilized 125I-labeled membranes by immunoprecipitation with anti-human transferrin in the presence of saturating amounts of human transferrin. Gel filtration on acrylamide agarose (AcA-22) at 23 degrees C in the absence of transferrin indicates the transferrin receptor has a Stokes radius of 4.6 nm. In the presence of transferrin, the Stokes radius of the receptor shifts to 6.3 nm. Sucrose density centrifugation studies indicate that it has a sedimentation coefficient of 9.8 S in the absence of transferrin and 11.2 S in the presence of transferrin. The molecular weight for the transferrin free receptor is calculated to be 213,000. Upon incubation with transferrin, it increases to 364,000. This is consistent with the idea that the active form of the solubilized receptor is a dimer and the dimer is in turn capable of binding two transferrin molecules.  相似文献   

13.
We have identified the product of the human c-myb proto-oncogene as a 80,000-Mr protein, p80c-myb, by using polyclonal and monoclonal antibodies raised against a bacterially synthesized polypeptide from the amino terminus of the viral myb protein. p80c-myb shares at least two distinct antigenic sites with the amino terminal region of the v-myb protein. p80c-myb is found only in hematopoietic cells or in cells that contain amplified c-myb genes. Like the chicken myb proteins, p80c-myb is a nuclear DNA-binding protein that is predominantly associated with chromatin and exhibits a short half-life of approximately 1 hour.  相似文献   

14.
Our earlier binding studies of the 22000- and 20000-Mr variants of human growth hormone (somatotropin) to pregnant-rabbit liver and mammary receptors [Closset, Smal, Gomez & Hennen (1983) Biochem. J. 214, 885-892] suggested that the 20000-Mr variant was a lower-affinity analogue of the 22000-Mr molecule. Since the receptor population in these tissues is not fully characterized, we have now investigated the binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line. The maximum bindability of radioiodinated 22000- and 22000-Mr to IM-9 cells was 60 and 45% respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10h at 30 degrees C), poor reversibility ('tight binding'), linear Scatchard plot, same specificity as shown by lack of competition by bovine, porcine or equine growth hormones or human growth hormone-(32-46)-(missing in the 20000-Mr variant),-(1-134)- and -(141-191)-peptides. Both unlabelled hormones inhibit binding of both tracers completely, with the 20000-Mr variant being only half as potent as the 22000-Mr one. The apparent affinity is 2.8 X 10(9)M-1 for the 22000-Mr variant and 1.6 X 10(9)M-1 for the 20000-Mr variant. This decreased affinity of the 20000-Mr variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the two variants that occupy about 15% of the total sites induce a marked down-regulation of the receptors after 18h incubation, but the 20000-Mr variant (50% decrease) has a smaller effect than the 22000-Mr variant (75% decrease). Thus the only consequence of the residues-32-46 deletion in the 20000-Mr variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the two forms suggest that the known differences in their insulin-like effects cannot be explained by differences in the nature of their interaction with the human-growth-hormone receptor.  相似文献   

15.
T Finkel  G M Cooper 《Cell》1984,36(4):1115-1121
Immunoprecipitation of extracts of human carcinoma cell lines with three different monoclonal antibodies generated against ras proteins revealed the coprecipitation of a 90,000 dalton protein. The coprecipitated protein was identified as the transferrin receptor by comigration in both reducing and nonreducing SDS-polyacrylamide gels, by absorption with a monoclonal antibody directed against transferrin receptor, and by analysis of partial proteolysis products. Coprecipitation of the transferrin receptor with three monoclonal antibodies with differing specificities to ras proteins, as well as the inability to coprecipitate the transferrin receptor from cell extracts from which ras proteins were depleted by preabsorption, indicates that ras proteins and the transferrin receptor form a molecular complex. This complex is disrupted by addition of transferrin to cell extracts. These findings suggest that ras proteins function in regulation of cell growth via interaction with the cell surface receptor for transferrin.  相似文献   

16.
The transferrin receptor of the human reticulocyte was isolated by two different immunoaffinity procedures. These included indirect immunoprecipitation with a transferrin/anti-transferrin complex and direct immunoprecipitation with antiserum to purified transferrin receptor from placentae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor isolated from reticulocytes reveals a polypeptide at Mr = 94,000 identical in molecular weight with that of the placenta. A radioimmunoassay using purified 125I-labeled transferrin receptor from placentae and antiserum to transferrin receptor fails to distinguish any immunological differences between the reticulocyte and placental forms of the protein. In addition, proteolytic digests of both of these polypeptides with Staphylococcus aureus protease show identical proteolytic patterns, indicating similar sequences.  相似文献   

17.
The function of the transferrin receptor is to transport iron-bound transferrin into the cell. In order to function properly, this dimeric glycoprotein must be expressed on the cell surface and be able to bind transferrin. Site-directed mutagenesis was performed to abolish the three asparagine-linked glycosylation consensus sequences of the human transferrin receptor. The DNA encoding the mutated transferrin receptor was stably transfected into mouse fibroblasts. This form of the human transferrin receptor shows reduced transferrin binding, reduced intersubunit bond formation, and reduced cell surface expression, indicating that the transferrin receptor which lacks asparagine-linked glycosylation is not fully functional. In addition, the mutated form of the receptor is not processed as quickly. It shows an association with an endoplasmic reticular chaperone protein, binding immunoglobulin protein, leading to the hypothesis that the mutated transferrin receptor experiences increased retention in the endoplasmic reticulum.  相似文献   

18.
We recently described an estrogen-inducible transferrin receptor from the chicken oviduct. We now report on the comparison of the oviduct transferrin receptor with the transferrin receptor obtained from chick embryo red blood cells. Western blot analysis reveals that rabbit polyclonal antibodies raised against one receptor do not cross react with the heterologous receptor. Furthermore, peptide map analyses of either affinity purified, native [125I]-labelled transferrin receptors (dimers) or dissociated, and repurified monomers obtained from oviducts and embryonic red blood cells yield distinct patterns. Therefore, the estrogen-modulated oviduct transferrin receptor appears to be structurally distinct from the iron-modulated red cell transferrin receptor.  相似文献   

19.
A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.  相似文献   

20.
1. Three of five monoclonal antibodies produced to chicken ovotransferrin bound quail ovotransferrin but none of the antibodies bound human, bovine or equine serum transferrin. 2. Equilibrium binding experiments indicate that both quail and chicken ovotransferrin bind to transferrin receptors on chick reticulocytes although the quail protein binds to 40% fewer sites with an affinity which is three times lower than chicken ovotransferrin. 3. The antibodies that recognize quail ovotransferrin block binding of both radiolabelled chicken and quail ovotransferrin to chick reticulocytes. 4. Quail NH2-terminal half-molecule domain appears to be unable to form a functional hybrid holo-ovotransferrin with chicken C-terminal half-molecule domain.  相似文献   

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