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1.
Prostate cancer metastasizes predominantly to bone, where it induces osteoblastic lesions. Paracrine factors secreted by the metastatic cancer cells are thought to mediate these events. We previously isolated a novel bone metastasis-related factor (MDA-BF-1) from bone marrow aspirate samples from patients with prostate cancer and bone metastasis, and found that this factor stimulated osteoblast differentiation, possibly by interacting with a receptor on the osteoblasts. Identifying this putative MDA-BF-1 receptor biochemically requires the expression of MDA-BF-1 for receptor binding assays and for the preparation of a ligand-affinity column. We tagged MDA-BF-1 with a peptide containing a protein kinase A phosphorylation site plus a 7-histidine sequence to facilitate the labeling of MDA-BF-1 for receptor binding assay and the binding of MDA-BF-1 to an immobilized metal affinity column. The recombinant MDA-BF-1 protein (MDA-BF1-kinase-his) was expressed in Sf9 cells using a baculovirus expression system. About 0.8 mg of purified MDA-BF1-kinase-his protein was obtained from 4 x 10(8) Sf9 cells. MDA-BF1-kinase-his can be phosphorylated by PKA with a specific activity around 10(5)cpm/mug protein. Receptor binding assays using this (32)P-labeled MDA-BF-1 showed that MDA-BF-1 bound to membranes prepared from Saos-2, an osteosarcoma cell line, and C2C12, a mouse pluripotent mesenchymal precursor cell line that can be induced to become osteoblast by BMP-2. In contrast, MDA-BF-1 did not bind to membranes from PC-3 human prostate cancer cells or HEK293 human embryonic kidney cells. These observations suggest that the MDA-BF-1 receptor is expressed in cells of osteoblastic lineage. In addition to its use as a ligand for receptor binding assays, a ligand affinity column can be prepared by binding MDA-BF1-kinase-his to an IMAC for the purification of MDA-BF-1 receptor. 相似文献
2.
Further studies on the binding of vitamin B 12 to the cell wall of a B 12 -requiring Lactobacillus 总被引:1,自引:0,他引:1
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T Sasaki 《Journal of bacteriology》1972,109(1):169-178
The vitamin B(12)-binding property of Lactobacillus leichmannii ATCC 7830 has been studied. The organism could bind 0.52 mug of B(12) per mg of cells. With regard to the cellular site for B(12) accumulation, three-quarters of the B(12) bound to the cell was found in the crude cell wall fraction, and the remaining one-quarter was found in the particulate (ribosome) fraction. After receiving enzymatic treatments with ribonuclease, lipase, and trypsin, the wall fraction retained three-fifths of the initial B(12). The possibility of cross-contamination of the wall and particulate fractions was excluded by measuring the contents of ribonucleic acid and hexosamines in each fraction. The B(12)-binding activity of the wall was destroyed by pretreatment of the wall with pepsin, Pronase, or trypsin. However, once bound to the wall, the B(12) was not released by the same treatments. These facts suggest that B(12) is bound to a polypeptide in the wall on which these enzymes act and that, once bound, B(12) somehow inhibits the enzymatic actions as described earlier with L. delbrueckii no. 1. A B(12)-polypeptide complex was isolated by treatment with 0.2 n HCl from walls to which B(12) had been bound. The complex was then purified. The complex moves as a single band on polyacrylamide gel electrophoresis. Its molecular weight was estimated around 21,500 with microheterogeneity on a Sephadex G-75 column. The mode of B(12) binding was found to be similar to that of L. delbrueckii. 相似文献
3.
Bacteriophage T4D Receptor and the Escherichia coli Cell Wall Structure: Binding of Endotoxin-Like Particles to the Cell Wall 总被引:1,自引:1,他引:0
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Jorge Zorzopulos Sara DeLong Virginia Chapman Lloyd M. Kozloff 《Journal of bacteriology》1980,142(3):982-991
A variety of degradative treatments have been used to investigate the nature of the structure and components of the cell walls of Escherichia coli B. The binding and localization of the endotoxin-like particles found on the cell walls were of special interest because some of them are associated with the site where the inner tail tube of bacteriophage T4D penetrates the cell wall. Modified cell walls were obtained by heating a suspension of bacterial cells originally in 0.1 M phosphate, pH 7.0, after the addition of 12.5 M NaOH to a final concentration of 0.25 M. With regard to the endotoxin-like particles, it was found that: (i) at least part of them still remained bound to the modified cell wall after the alkali treatment; (ii) the subsequent incubation of alkali-treated cell walls with lysozyme destroyed the bacterial form and released a complex of endotoxin-like particles together with a fibrous material; (iii) on the other hand, treatment with 45% phenol at 70°C removed the endotoxin-like particles from the surface of the alkali-treated cell walls, but most of the fibrous material was left on the cell wall; and (iv) incubation of alkali-treated cell walls with 5 mM ethylenediaminetetraacetic acid at 20°C also removed the endotoxin-like particles, but did not disrupt the rodlike bacterial form. However, if the ethylenediaminetetraacetic acid treatment was performed at 55°C, the bacterium-like form was destroyed. These differential sensitivities to ethylenediaminetetraacetic acid suggested that loosely bound divalent metal ions normally hold these endotoxin-like particles on the cell wall surface, but that probably more tightly bound metal ions are involved in the determination of cell shape. Analysis of the protein components of the alkalitreated cell walls showed that only one protein was present in significant amounts, and this protein had an electrophoretic mobility similar to that of the Braun lipoprotein. This protein was released from the alkali-treated cell walls upon heating with 2% sodium dodecyl sulfate at 100°C. Phospholipids were also absent from this structure. The distribution of the remaining cell wall components on the alkali-treated cell walls is discussed. 相似文献
4.
Paul Y. Sze Patricia Sullivan Ralph F. Alderson Andrew C. Towle 《Neurochemistry international》1983,5(1):51-56
The binding of ATP to brain l-glutamate decarboxylase (GAD) was studied by means of ATP-agarose chromatography, utilizing partially purified GAD from mouse brain after DEAE-cellulose chromatography and ammonium sulfate fractional precipitation. GAD was found to bind with a high affinity to the ATP-agarose with the ATP molecule linked to the beaded agarose through the N6-amino group. Agarose with ATP attached through the ribosyl hydroxyls was totally ineffective to bind the enzyme. GAD bound to the immobilized ATP could be dissociated by free ATP (10–50 mM), but not by ADP at a concentration as high as 100 mM. Mg2+ was not a required factor for the binding. The enzyme binding to the ATP-agarose occurred under a saturating concentration (50 μM) of pyridoxal 5′-phosphate (PLP). Moreover, GAD bound to the ATP-agarose was not dissociated by PLP even at 1.0 mM, indicating no competition of PLP with ATP for the same binding site on the enzyme. Kinetic characterization showed that binding of ATP raised the Km of the enzyme for PLP. Our approach provides direct evidence that there is a specific binding site on GAD for ATP, which is distinct from the binding site for PLP. 相似文献
5.
Purification and properties of colony-stimulating factor from mouse lung-conditioned medium. 总被引:57,自引:0,他引:57
Colony-stimulating factor, which specifically stimulates mouse bone marrow cells to proliferate in vitro and generate colonies of granulocytes, or macrophages, or both, was purified 3500-fold from mouse lung-conditioned medium. Analysis by discontinuous polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate indicated that there was a single protein component. All of the colony-stimulating activity was coincident with the protein band. The molecular weight of colony-stimulating factor estimated by gel filtration was approximately 29,000 and by electrophoresis approximately 23,000. The specific activity of purified colony-stimulating factor from mouse lung-conditioned medium bound to concanavalin A-Sapharose, indicating that it is a glycoprotein. The small percentage of colony-stimulating factor in mouse lung-conditioned medium which did not bind to concanavalin A-Sepharose appeared to represent molecules which lacked the carbohydrate moieties required for binding to this lectin. It was necessary to include low concentrations (less than 0.01%, v/v) of polymers such as gelatin and polyethylene glycol, or nonionic detergents such as Triton X-100, in all of the buffers used throughout the purification scheme, otherwise colony-stimulating factor was lost from solution. At high concentrations (greater than 20 mug/ml) the factor stimulated the formation of granulocytic, macrophage, and mixed colonies from C57BL mouse bone marrow cells. As the concentration of purified colony-stimulating factor was decreased, the frequency of colonies containing granulocytes also decreased. At low concentrations of colony-stimulating factor (less than 70 pg/ml) only macrophage colonies were stimulated. 相似文献
6.
alpha-Amylase inhibitor from fungus Cladosporium herbarum F-828 总被引:1,自引:0,他引:1
N Saito 《The Journal of biological chemistry》1982,257(6):3120-3125
A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site. 相似文献
7.
Flood C Gustafsson M Richardson PE Harvey SC Segrest JP Borén J 《The Journal of biological chemistry》2002,277(35):32228-32233
An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic. 相似文献
8.
The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression. 相似文献
9.
A fraction with the ability to bind the terminal fragment of equine adenovirus (EAd) DNA was prepared from MDBK cell nuclei. The fraction (MDBK nuclear factor) bound to the terminal fragment of all human and animal adenovirus DNAs examined except avian adenovirus EDS-76. However, the terminal fragments of two animal adenoviruses, EAd and bovine adenovirus type 3 (BAd3), showed higher affinity for the nuclear factor than the others. The MDBK nuclear factor-binding site determined by footprinting analysis was the sequence located between nucleotides 22 and 46 in EAd, between 36 and 53 in canine adenovirus type 2, and between 20 and 46 in BAd3, counting from the terminus. The respective binding site contained a sequence resembling the consensus sequence. The binding site of Ad4 DNA was not within the inverted terminal repetition, but was located at least 550 base pairs apart from the terminus. 相似文献
10.
P G McCaffrey J Jain C Jamieson R Sen A Rao 《The Journal of biological chemistry》1992,267(3):1864-1871
Nuclear extracts from a nontransformed murine T lymphocyte clone contained two inducible factors that bound to a nuclear factor kappa B (NF-kappa B) site. One factor was NF-kappa B, and the other was differentiated from NF-kappa B by its mobility in the electrophoretic mobility shift assay and its lack of sensitivity to protein kinase C depletion. Competition and methylation interference assays showed that the binding site for the novel factor was limited to nucleotides in the 3' half of the kappa B site. This part of the kappa B site resembled sequences in the binding site for a second inducible nuclear factor of T cells, NF-AT, as well as a conserved sequence found in several lymphokine genes, termed "cytokine-1" (CK-1). Competition and methylation interference analysis showed that both NF-AT and CK-1 sequences bound a factor similar to the novel kappa B-binding factor and that binding involved a four-nucleotide sequence (TTCC) that the kappa B, CK-1, and NF-AT sites have in common. The complexes that form with each site have characteristics of NF-AT: they are induced upon T cell receptor stimulation, are sensitive to protein synthesis inhibitors and cyclosporin A, and are not sensitive to protein kinase C depletion. Thus, a factor or factors similar to NF-AT can bind to three distinct promoter sequences which occur commonly in several T cell activation genes. These results raise the possibility that related factors binding to kappa B, CK-1, and NF-AT sequences could play a role in the coordinate induction of T cell activation genes. In addition, our results suggest that kappa B and CK-1 sites represent potential cyclosporin-sensitive promoter elements by virtue of their ability to bind an NF-AT-like factor. 相似文献
11.
Detection of two tissue-specific DNA-binding proteins with affinity for sites in the mouse beta-globin intervening sequence 2. 总被引:20,自引:6,他引:14
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To identify proteins from uninduced murine erythroleukemia nuclear extracts which specifically bind to sequences from the DNase I-hypersensitive region within the mouse beta-globin intervening sequence 2 (IVS2), a gel electrophoretic mobility shift assay was used. Two distinct sequence-specific binding proteins were detected. The specific binding sites for these factors were delineated by both DNase I protection footprinting and methylation interference. Factor B1 bound specifically to two homologous sites, B1-A and B1-B, approximately 100 base pairs apart within the IVS2 and on opposite strands. These two regions could interact with factor B1 independently. Factor B1 was limited to cells of hematopoietic lineages. Factor B2 bound to a site approximately 5 base pairs away from the B1-A site and was limited to cells of the erythroid lineage. The limited tissue distribution of these factors and the locations of their binding sites suggest that one or both of these factors may be involved in the formation of the tissue-specific DNase I-hypersensitive site in the IVS2 of the mouse beta-globin gene. 相似文献
12.
Bozonnet S Jensen MT Nielsen MM Aghajari N Jensen MH Kramhøft B Willemoës M Tranier S Haser R Svensson B 《The FEBS journal》2007,274(19):5055-5067
Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates. 相似文献
13.
I Przylas Y Terada K Fujii T Takaha W Saenger N Str?ter 《European journal of biochemistry》2000,267(23):6903-6913
As a member of the alpha-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one alpha-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite -3 to +1 and a second one approximately 14 A away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites -3 to +1. Unlike the situation in other enzymes of the alpha-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite -1. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond. 相似文献
14.
A beta-d-glucanase highly specific for glucans containing a linkage sequence ... Glc 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc ... has been isolated from several commercial preparations of Bacillus subtilis alpha-amylase including one purified by repeated crystallization. The beta-d-glucanase will not hydrolyze cellulose or laminarin. Gel filtration on a Bio-Gel P-200 column results in separation of the glucanase from the alpha-amylase. The enzyme is of the endo type as changes in the substrate viscosity appear long before the appearance of detectable reducing sugars. No evidence of product inhibition was revealed and appropriate substrates were converted to oligosaccharides, the quantity of which approaches theoretical yields. The products of the reaction were separated according to molecular size by use of Bio-Gel P-2 gel filtration and found to be consistent with the action pattern of the enzyme. Kinetic studies show that the enzyme has an optimum activity at pH 6.5, a V(max) of 13.9 mug glucose equivalent released/mug protein.hour, and an apparent Km of 3.4 mg of lichenan per ml. Potential application of this enzyme for the structural characterization of plant cell wall glucans is discussed. 相似文献
15.
Y Li H Kobayashi Y Hashimoto S Iwasaki 《Biochemical and biophysical research communications》1992,187(2):722-729
The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated. Mycelial extracts to which RZX could bind contained beta-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained beta-tubulins with either Ile-100 or Val-100. Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX. PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined. The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins. 相似文献
16.
Molluscum contagiosum virus interleukin-18 (IL-18) binding protein is secreted as a full-length form that binds cell surface glycosaminoglycans through the C-terminal tail and a furin-cleaved form with only the IL-18 binding domain
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Some poxviruses and their mammalian hosts encode homologous proteins that bind interleukin-18 (IL-18) with high affinity and inhibit IL-18-mediated immune responses. MC54L, the IL-18 binding protein of the human poxvirus that causes molluscum contagiosum, is unique in having a C-terminal tail of nearly 100 amino acids that is dispensable for IL-18 binding. When recombinant MC54L was expressed and purified via a C-terminal six-histidine tag, a shorter fragment was detected in addition to the full-length protein. This C-terminal fragment resulted from the cleavage of MC54L by cellular furin, as it was greatly diminished when furin was specifically inhibited or when a furin-deficient cell line was used for expression. Furthermore, the N- and C-terminal fragments of MC54L were generated by cleavage of the recombinant protein with furin in vitro. The furin cleavage site was mapped within a 32-amino-acid segment that is C terminal to the IL-18 binding domain. Full-length MC54L, but not the N-terminal IL-18 binding fragment, bound to cells and to purified heparin and other glycosaminoglycans that are commonly found on the cell surface and in the extracellular matrix. MC54L bound to heparin with a nanomolar K(d) and could simultaneously bind to IL-18. Their different glycosaminoglycan and cell binding properties may allow the long and short forms of MC54L to inactivate IL-18 near the site of infection and at more distal locations, respectively. 相似文献
17.
Nogami K Shima M Hosokawa K Nagata M Koide T Saenko EL Tanaka I Shibata M Yoshioka A 《The Journal of biological chemistry》2000,275(33):25774-25780
Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg(1689) by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K(d) values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and the K(d) values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC(50), 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)'(2) of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg(1689). Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg(1689). 相似文献
18.
Beck CG Studer C Zuber JF Demange BJ Manning U Urfer R 《The Journal of biological chemistry》2001,276(46):43270-43276
Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine mainly acting on monocytes and T cells that elicits its biological effects by interacting with the seven-transmembrane helix receptor CCR2B. The vaccinia virus strain Lister and many other poxviruses express soluble proteins (vCCI) that bind MCP-1 and other CC chemokines and inhibit their function. In order to define the interaction site of MCP-1 with vCCI from vaccinia, surface exposed residues of MCP-1 were identified and mutated to alanine. The MCP-1 variants were expressed, purified, and their interaction with vCCI was characterized. The site on MCP-1 for vCCI binding is dominated by arginine 18 with important additional contributions from tyrosine 13 and arginine 24. These residues define a binding site that largely overlaps with the CCR2B receptor interaction site. The viral chemokine-binding protein vCCI thus inhibits the biological function of MCP-1 by directly masking its CCR2B receptor-binding site. 相似文献
19.
Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin 总被引:2,自引:0,他引:2
Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly. 相似文献
20.
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