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D R Clemmons M A Cascieri C Camacho-Hubner R H McCusker M L Bayne 《The Journal of biological chemistry》1990,265(21):12210-12216
Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity. They are present in extracellular fluids and modulate the interactions of their ligands with the type 1 IGF cell surface receptor. These studies utilized IGF-I analogs that have reduced binding affinity for either the type 1 IGF receptor or binding proteins to study the ligand specificity of IGF-BP-1 and the role of IGF-BP-1 in modulating the biological activity of IGF-I. The data indicate that the regions of IGF-I which are responsible for binding to IGF-BP-1 and to human serum-binding proteins are distinct but overlapping and are clearly distinct from the type I receptor binding sites. In the absence of exogenously added IGF-BP-1, the analogs with reduced affinity for IGF-BP-1 are more potent than IGF-I in stimulating DNA synthesis by porcine aortic smooth muscle cells. In contrast, when cells are concomitantly exposed to IGF-BP-1, two of the analogs with reduced affinity for binding protein give only 40-65% of the maximal IGF-I response. [Leu24, 1-62]IGF-I, which has a 100-fold reduced affinity for the type 1 IGF receptor, gave a value that was 62% of the maximal IGF-BP-1 potentiated response. A second biological response, that of stimulating binding protein secretion by IGF-I, was also examined. [Leu24, 1-62]IGF-I is more potent than IGF-I whereas the activity of the analogs with lower affinity for IGF-BP-1 is significantly reduced. Thus, the ability to activate DNA synthesis and binding protein secretion maximally in the presence of IGF-BP-1 is dependent on the affinity of IGFs for both type 1 receptors and binding proteins. 相似文献
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C Domenicucci H A Goldberg T Hofmann D Isenman S Wasi J Sodek 《The Biochemical journal》1988,253(1):139-151
Osteonectin, extracted from foetal porcine calvariae with 0.5 M-EDTA, was purified to homogeneity by using gel filtration and polyanion anion-exchange fast protein liquid chromatography under dissociative conditions without the need of reducing agents. The purified protein migrated with an Mr of 40,300 on SDS/polyacrylamide gels and was similar to bovine osteonectin in both amino acid composition and in its ability to bind to hydroxyapatite in the presence of 4 M-guanidinium hydrochloride (GdmCl). However, unlike the bovine protein, porcine osteonectin did not bind selectively to hydroxyapatite when EDTA tissue extracts were used. In addition, purified porcine osteonectin did not show any apparent affinity for either native or denatured type I collagen, but did bind to serum albumin. Primary sequence analysis revealed an N-terminal alanine residue, with approximately one-half of the subsequent 35 residues identified as small hydrophobic amino acids and one-quarter as acidic amino acids. The only significant difference between the N-terminal sequences of the bovine and porcine proteins was the deletion of the tripeptide Val-Ala-Glu in porcine osteonectin. In contrast with bovine osteonectin, far-u.v.c.d. of porcine osteonectin revealed considerable secondary structure, of which 27% was alpha-helix and 39% was beta-sheet. Cleavage of the molecule with CNBr under non-reducing conditions generated five fragments, of which two major fragments (Mr 27,900 and 12,400) stained blue with Stains All, a reagent that stains sialic-acid-rich proteins/phosphate-containing proteins and/or Ca2+-binding proteins blue while staining other proteins pink. The 12,400-Mr fragment bound 45Ca2+ selectively, indicating a Ca2+-binding site in this part of the molecule. The 27,900-Mr fragment did not bind Ca2+, and since biosynthetic studies with 32PO4(3-) did not show phosphorylation of porcine osteonectin, this fragment is likely to be highly acidic. The incomplete cleavage of the molecule with CNBr and the ability of the molecule to regain its secondary structure after exposure to 7 M-urea are features consistent with the molecule having a compact structure that is stabilized by numerous disulphide bridges. The chemical and binding properties of porcine osteonectin are closely similar to the recently described 'culture shock', SPARC and BM-40 proteins, indicating that these are homologous proteins. 相似文献
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Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane 总被引:3,自引:0,他引:3
Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders. 相似文献
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Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases 总被引:13,自引:0,他引:13
Glycoproteins which bind concanavalin A (Con A) can be located on nitrocellulose sheets after electrophoretic transfer from slab gels, by sequential incubation of the sheets with Con A and peroxidase, and visualization of the peroxidase by an insoluble reaction product. We refer to this method as affinoblotting. Differential elution of Con A from the blots by washing the sheets with different concentrations of alpha-methylglycosides is used to demonstrate the affinity of Con A for the oligosaccharide side chains, and to differentiate between proteins with weak and those with high affinity for Con A. Concanavalin A has a high affinity for the four plant glycoproteins (phaseolin, phytohemagglutinin, jackbean alpha-mannosidase, and the glycosylated precursor of Con A) studied here. Incubation of the blots with alpha-mannosidase and endoglycosidase H (endo H) is used to demonstrate that the oligosaccharide chains can be degraded by glycosidases while the proteins are immobilized on the nitrocellulose. With this approach we show here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation. 相似文献
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Porcine uterine flushings obtained on day 15 of the estrous cycle were fractionated using gel filtration, and preparative poly-acrylamide gel electrophoresis was employed to separate one molecular weight fraction into two groups of small uterine-specific proteins designated pAP and pLAP. The two groups were assayed for immuno-suppressive ability using phytohemagglutinin (PHA) stimulated porcine lymphocyte cultures and incorporated 3H-thymidine. It was found that the pAP preparation which was composed of two proteins inhibited lymphocyte reactivity to PHA (p<.001) while the pLAP preparation failed to exhibit a similar activity at levels as high as 1000 μg/ml. The immunosuppressive effect was determined to be independent of cytotoxicity, PHA inactivation by binding and other non-specific phenomena. The results of this study indicate that the immunosuppressive activity of porcine uterine flushings is caused, at least in part, by one or both of these proteins present in the pAP preparation. 相似文献
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Porcine uterine flushings obtained on day 15 of the estrous cycle were fractionated using gel filtration, and preparative poly-acrylamide gel electrophoresis was employed to separate one molecular weight fraction into two groups of small uterine-specific proteins designated pAP and pLAP. The two groups were assayed for immuno-suppressive ability using phytohemagglutinin (PHA) stimulated porcine lymphocyte cultures and incorporated 3H-thymidine. It was found that the pAP preparation which was composed of two proteins inhibited lymphocyte reactivity to PHA (p<.001) while the pLAP preparation failed to exhibit a similar activity at levels as high as 1000 μg/ml. The immunosuppressive effect was determined to be independent of cytotoxicity, PHA inactivation by binding and other non-specific phenomena. The results of this study indicate that the immunosuppressive activity of porcine uterine flushings is caused, at least in part, by one or both of these proteins present in the pAP preparation. 相似文献
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P S Bachorik J N Livingston J Cooke P O Kwiterovich 《Biochemical and biophysical research communications》1976,69(4):927-935
Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of 125I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL. 相似文献
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A simple and rapid method for the purification of porcine ligandin with glutathione-S-transferase activity is presented. After ion-exchange chromatography on DEAE-Sephadex, ligandin is isolated from porcine liver cytosol by affinity chromatography on bromosulphophthalein-Sepharose and gel filtration of Sephadex G-100. Evidence is presented that the purified ligandin is homogeneous with respect to polyacrylamide-gel electrophoresis (7.5%) and sodium dodecylsulphate-gel electrophoresis. Physico-chemical investigations show that the purified ligandin has properties similar to those of ligandin isolated from other species with respect to molecular weight, amino-acid composition, secondary structure and catalytic activity. As is the case for human and rat ligandin, porcine ligandin binds bilirubin. Evidence is also presented that porcine liver cytosol contains several bromosulphophthalein-binding proteins with basic isoelectric points lacking catalytic activity. 相似文献
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E C Theil 《The Journal of biological chemistry》1975,250(10):3807-3813
A group of abundant (15% of the soluble protein) nonhemoglobin proteins was isolated from the primitive (embryonic) red cells found in tadpoles, using the cationic properties of the proteins at pH 8.6 to separate them from hemoglobin and other red cell proteins. The cathodal proteins (CP) were resolved into five components, and the two most predominant proteins were separated and characterized. Purified CP-1b and CP-2 had an amino acid composition similar to that of unfractionated cathodal proteins and to each other, except for small variations in the lysine and half-cystine content. The molecular weight of the purified CP-1b and CP-2 was 13 to 14,000, determined by gel filtration chromatography and electrophoresis in the presence of sodium dodecyl sulfate. Cathodal proteins were immunologically related although there were quantitative differences in reactivity. The concentration of cathodal proteins in primitive (embryonic) red cells was 100 times that in definitive (adult) red cells coincided with the replacement of primitive red cells. The synthesis of the cathodal proteins appeared to continue throughout the life of the primitive red cells; when hemoglobin synthesis declined in primitive red cells, approximately half of the protein synthesized by the cells was cathodal protein. Although the function of the cathodal proteins is as yet unknown, the data suggest that the cathodal proteins are a unique characteristic of erythroid differentiation in early development. 相似文献
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Substrate specificity of a human-specific esterase 总被引:1,自引:0,他引:1
A human species-specific esterase has been identified in tissues, cell cultures, and urine. It is the most slowly migrating (i.e., cathodal) of the esterase isoenzymes in agarose electrophoresis; it is not a choline estrase, a pseudocholine esterase, an acetyl phenylalanine-3-naphthyl esterase or N-benzoyl-arginine-3-naphthyl esterase. Hydrolysis of N-methyl indoxyl acetate caused by this esterase is not inhibited by eserine, eserine sulfate, or EDTA. Phenylmethylsulfonyl fluoride, however, did inhibit hydrolysis. Furthermore, this cathodal esterase does not show any chymotrypsin, trypsin, or leucine aminopeptidase enzyme activity. 相似文献
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Tubulin is detected among the DNA-binding proteins when an extract from fibroblasts is chromatographed on DNA-cellulose. Further purification of the colchicine-binding activity shows that purified tubulin from fibroblasts does not bind to DNA. Depolymerized brain microtubule proteins show a high affinity for DNA. The fraction bound is composed of tubulin and microtubule-associated proteins. Experiments with fractionated microtubule proteins indicate that tubulin-free microtubule associated proteins bind to DNA, while tubulin free of microtubule-associated proteins does not. Microtubule-associated proteins bind better to eukaryotic than to phage DNA suggesting a specificity of the interaction. 相似文献
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Partial agonist/antagonist mouse interleukin-2 proteins indicate that a third component of the receptor complex functions in signal transduction. 总被引:4,自引:9,他引:4
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Some mouse interleukin-2 (mIL-2) proteins with substitutions at residue Gln141 are unable to trigger a maximal biological response. The Asp141 protein induces the lowest maximal response. The Asp141 protein can weakly antagonize the biological activity of mIL-2 and strongly antagonizes the biological activity of active mIL-2 mutant proteins that have defects in interactions with the high affinity receptor. Residue 141 mutant proteins bind with reduced affinity to T cells expressing the high affinity IL-2 receptor, yet bind normally to transfected fibroblasts expressing only the alpha and beta chains of the receptor. These results suggest that a third receptor component is important for both binding and signal transduction. 相似文献
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Characterization of Annexins in Mammalian Brain 总被引:3,自引:2,他引:1
Three annexins--p68, endonexin, and p32--have been isolated from porcine brain using their calcium-dependent affinity for membranes. Large amounts (20-50 mg/kg of tissue) of p68 and p32 can be isolated from cerebrum and cerebellum. The p68 is present as up to 0.3% of total porcine brain protein. The p68 and p32 from porcine brain bind to phosphatidic acid (half-maximal binding at 6 and 34 microM free calcium, respectively) and to phosphatidylserine (8 and 34 microM, respectively). They do not bind to phosphatidylcholine at calcium concentrations up to 1 mM. Two other major proteins (Mr 180,000 and Mr 76,000) were isolated with the annexins in a calcium-dependent manner but do not bind to phospholipids. The 180-kilodalton protein is the heavy chain of clathrin. From immunohistochemical studies, p68 is strongly associated with the plasma membranes of Purkinje cell bodies and dendrites in porcine cerebellum. It is also an intracellular component of Purkinje cells localized to perinuclear structures. Staining of axons in the white matter and granule cell layer was also seen. In contrast, p32 is completely absent from Purkinje cells and their dendrites; it is predominantly located in the molecular layer and in white matter of the cerebellar folds. The distribution of p32 may be consistent with a predominantly glial localization. 相似文献
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Hiroko Matsushita Mio Takenaka Haruko Ogawa 《The Journal of biological chemistry》2002,277(7):4680-4686
Porcine pancreatic alpha-amylase was shown by interaction analyses using a resonance mirror detector and alpha-amylase-immobilized Sepharose to bind with glycoproteins possessing N-glycans but not O-linked mucin-type glycans. Direct binding of three types of N-glycans to the alpha-amylase was demonstrated by surface plasmon resonance. Binding with biotin-polymer sugar probes revealed that the alpha-amylase has affinity to alpha-mannose, alpha-N-acetylneuraminic acid, and beta-N-acetyllactosamine, which are components of N-glycans. The binding of glycoproteins or carbohydrates enhanced the enzyme activity, indicating that the recognition site for N-glycans is different from its catalytic site. The binding activity was unique to porcine pancreatic alpha-amylase and was not observed for alpha-amylase from saliva, wheat, and fungus. 相似文献
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Fc and C3bi receptors and the differentiation antigen BH2-Ag are randomly distributed in the plasma membrane of locomoting neutrophils 总被引:2,自引:1,他引:1
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Reports from several laboratories suggest that neutrophils arrested during locomotion preferentially bind immune complexes at the front of the cell. Such asymmetry of binding has been interpreted as indicating an active modulation of phagocytic receptors to the anterior of the cell. To investigate this further, we have used digital analysis of fluorescence images to determine the binding patterns of mAbs directed against the Fc receptors, the receptors for the C3bi fragment of C3, and a neutrophil-specific antigen. We found that all three proteins are distributed nearly identically along the length of migrating neutrophils, and their distribution very closely parallels the anterior to posterior distribution of the plasma membrane. The use of mAbs offered an important advantage in that the binding of antireceptor antibodies, unlike the binding of ligands, should be independent of potential changes in the affinity of the receptors. We conclude that the anterior distribution of the phagocytic receptors in the plasma membrane of locomoting neutrophils parallels the overall increase in membrane area at the front of a migrating cell and that specific translocation of phagocytic receptors does not occur. 相似文献
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The ability of several D-galactose and N-acetyl-D-galactosamine-binding lectins to bind to Sepharose was investigated. Lectins from soybean, Wistaria floribunda, Bauhinia purpurea alba, and Sophora japonica could be isolated by affinity chromatography on acid-treated Sepharose 6B. These lectins would not bind to untreated Sepharose 2B, 4B, and 6B. The binding of B. purpurea alba and S. japonica was temperature-dependent. The S. japonica lectin would bind only at high pH. Ricinus communis toxin also showed a temperature-dependence of binding; acid-treated Sepharose 6B was a better affinity support for the toxin than was untreated Sepharose 4B Lectins from lima bean, Dolichos biflorus, and kidney-bean phytohemagglutinin did not bind to Sepharose under any of the conditions studied. 相似文献
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G Gil T F Osborne J L Goldstein M S Brown 《The Journal of biological chemistry》1988,263(35):19009-19019
The gene for 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-controlling enzyme of cholesterol biosynthesis, is transcribed at a relatively high level when cellular sterols are depleted and is repressed when sterols accumulate. We have previously reported that the regulatory region of the hamster reductase gene contains eight different sequences that bind nuclear proteins as determined by DNase I footprinting assays. We here report the purification of a single activity that accounts for six of these footprints. This activity was found in a doublet of proteins (designated reductase promoter factor 1, RPF-1) that have apparent molecular weights of 33,000 and 35,000. They were isolated by DNA affinity chromatography using oligonucleotides corresponding to either of two footprinted sequences. The 33- and 35-kDa species were present as monomers, as indicated by gel filtration and gradient ultracentrifugation. Oligonucleotides corresponding to any one of the six footprinted sequences prevented the binding of RPF-1 to all of the other sequences, indicating that all six bind to a single site in RPF-1. The only sequence shared by all six footprinted sequences is the trinucleotide, TGG, both of whose guanosines made contact with RPF-1, as determined by methylation interference assays. The footprinted sequence that binds RPF-1 with highest affinity contains the palindrome, TGG(N7)CCA, which conforms to the consensus sequence for binding NF-1, a nuclear protein that stimulates replication of adeno-virus-2. Purified RPF-1 was shown to bind to the adenovirus NF-1 binding site with high affinity. Although the apparent molecular weight of the RPF-1 doublet was lower than the molecular weight range for NF-1 proteins (52,000-66,000), it is likely that the 33-35-kDa doublet is derived from a larger NF-1-like protein as a result of proteolysis. We conclude that RPF-1 belongs to a group of TGG-binding proteins that includes NF-1 and other proteins previously described as CCAAT binding proteins. This protein binds to six sites in the promoter region for hamster 3-hydroxy-3-methylglutaryl CoA reductase, where its function remains to be determined. 相似文献