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1.
C Bensimon H Wakasugi N Wakasugi S Jitsukawa T Tursz 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(7):2290-2298
We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R. 相似文献
2.
抗鳗弧菌独特型单克隆抗体的制备及鉴定 总被引:5,自引:0,他引:5
利用具有中和活性的抗鳗弧菌单克隆抗体 4A6作为免疫原 ,通过单克隆抗体技术制备出 7株分泌抗独特型单抗的杂交瘤细胞。以ELISA竞争抑制实验及诱导Ab3的功能实验证实 ,其中 4株属于Ab2 β,有可能用于疫苗生产。 相似文献
3.
The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated. 相似文献
4.
B Savage U M Marzec B H Chao L A Harker J M Maraganore Z M Ruggeri 《The Journal of biological chemistry》1990,265(20):11766-11772
In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction. 相似文献
5.
Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes 总被引:22,自引:0,他引:22
V M Dixit N J Galvin K M O'Rourke W A Frazier 《The Journal of biological chemistry》1986,261(4):1962-1968
Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP. 相似文献
6.
The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein 总被引:4,自引:0,他引:4
R J Andy C L Finstad L J Old K O Lloyd R Kornfeld 《The Journal of biological chemistry》1984,259(20):12844-12849
The antigen recognized by a mouse monoclonal antibody (mAb S27) raised against a human renal cancer cell line has been identified as the adenosine deaminase binding protein. mAb S27 immunoprecipitates binding protein purified from a soluble fraction of human kidney. It also recognizes the mature 120,000-dalton membrane form of binding protein from [35S]methionine-labeled human fibroblasts, HepG2 cells, and the renal cancer cell line against which the antibody was raised. A rabbit polyclonal antibody raised against purified kidney binding protein completely precipitates mAb S27-reactive material from labeled membrane extracts. mAb S27 does not precipitate the initially synthesized 110,000 molecular weight precursor of binding protein in fibroblasts and recognizes only a small portion of binding protein precursor in labeled HepG2 cells suggesting that the antigenic determinant recognized by mAb S27 may be a post-translational modification present on the mature form of binding protein or that mAb S27 recognizes molecules in a certain conformation. Glycopeptides derived from purified soluble kidney binding protein or exogenously added adenosine deaminase do not inhibit the immunoprecipitation of binding protein by mAb S27, indicating that the mature oligosaccharide chains of binding protein are not the determinant recognized by mAb S27 and that bound adenosine deaminase does not mask the antigenic sites on binding protein. The fact that monoclonal antibody S27, previously shown (Ueda, R., Ogata, S., Morissey, D. M., Finstad, C. L., Szkudlavek, J., Whitmore, W. F., Oettgen, H. F., Lloyd, K. O., and Old, L. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5122-5126) to detect a cell surface antigen on cultured renal cancer cells, is directed against the adenosine deaminase binding protein confirms and extends the earlier observation (Andy, R.J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925) that binding protein is located on the cell surface. 相似文献
7.
DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation 总被引:3,自引:0,他引:3
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Okamoto K Kashima K Pereg Y Ishida M Yamazaki S Nota A Teunisse A Migliorini D Kitabayashi I Marine JC Prives C Shiloh Y Jochemsen AG Taya Y 《Molecular and cellular biology》2005,25(21):9608-9620
8.
E Kornecki B Walkowiak U P Naik Y H Ehrlich 《The Journal of biological chemistry》1990,265(17):10042-10048
The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion. 相似文献
9.
Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY 总被引:1,自引:0,他引:1
Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells. 相似文献
10.
Cytochalasin inhibits the rate of elongation of actin filament fragments 总被引:41,自引:22,他引:19
Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends. 相似文献
11.
R U Chukwuocha E T Hsiao P Shaw J L Witztum P P Chen 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(8):4604-4611
We have isolated five monoclonal IgG anti-beta 2-glycoprotein-1 (anti-beta 2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three beta 2GP-1-enriched mAbs (B14, B22, B27) reacted with beta 2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with beta 2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 x 10-6 M for beta 2GP-1 vs 3.2 x 10-6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between beta 2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to beta 2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to beta 2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-beta 2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients. 相似文献
12.
D Bayle J C Robert K Bamberg F Benkouka A M Cheret M J Lewin G Sachs A Soumarmon 《The Journal of biological chemistry》1992,267(27):19060-19065
The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity. To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cys529 and Glu561. This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M. The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M. The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface. The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited. The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites. 相似文献
13.
Cytosol prepared in 0.3 M KCl from pituitary GH3 cells, but not from AtT-20 cells contains a receptor-like macromolecule that binds 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with specificity and high affinity (Kd = 2.9 x 10(-10) M). The GH3 cytosolic binding component sediments at 3.3 S in high-salt sucrose gradients and adsorbs to DNA-cellulose; its elution profile from DNA-cellulose and other biochemical properties are indistinguishable from those of classical 1,25(OH)2D3 hormone receptors. The presence of the 1,25(OH)2D3 receptor in pituitary cells which secrete primarily growth hormone and prolactin (GH3), but not in a line which secretes the 31,000-dalton ACTH precursor and its derived peptides (AtT-20), suggests that 1,25(OH)2D3 may play a regulatory role in specific pituitary cells. 相似文献
14.
Xiaoying Wu Xiaojun Li Qingshan Zhang Shaozhou Wulin Xiaofei Bai Tingting Zhang Yue Wang Ming Liu Yun Zhang 《PloS one》2015,10(2)
BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.
Methods and Results
To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.Conclusions and Significance
We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1. 相似文献15.
Usami M Mitsunaga K Ohno Y 《The Journal of steroid biochemistry and molecular biology》2002,81(1):47-55
We have developed a simple assay method for the evaluation of estrogen receptor (ER) binding capacity of chemicals without the use of radio- or fluorescence-labeled compounds. We used the solution competition assay by the BIACORE biosensor, a surface plasmon resonance biosensor, with estradiol as a ligand, human recombinant ER(alpha) (hrER(alpha)) as a high molecular weight (hmw) interactant and test chemicals as analytes. For the ligand, aminated estradiol with a spacer molecule (E2-17PeNH) was synthesized and immobilized on a carboxymethyl dextran-coated sensor chip by the amine coupling method. The injection of the hmw interactant hrER(alpha) to the biosensor raised the sensorgram, indicating its binding to the ligand E2-17PeNH. The binding of test chemicals to hrERalpha was determined as a reduction in the hrER(alpha) binding to E2-17PeNH. The dissociation constant for the binding to hrER(alpha) was calculated for estrone (4.29 x 10(-9)M), estradiol (4.04 x 10(-10)M), estriol (8.35 x 10(-10)M), tamoxifen (2.16 x 10(-8)M), diethylstilbestrol (1.46 x 10(-10)M), bisphenol A (1.35 x 10(-6)M) and 4-nonylphenol (7.49 x 10(-6)M), by plotting the data according to an equation based on mass action law. This method can also be used as a high throughput screening method. 相似文献
16.
Identification and synthesis of the epitope for a human monoclonal antibody which can neutralize human T-cell leukemia/lymphotropic virus type I 总被引:3,自引:0,他引:3
A monoclonal antibody (mAb), designated 0.5 alpha, derived from a patient with adult T-cell leukemia was found previously to neutralize the human T-cell leukemia/lymphotropic type I (HTLV-I) virus in in vitro assays and bind to the major envelope glycoprotein (gp46) of HTLV-I (Matsushita, S., Guroff, M.R., Trepel, J., Crossman, J., Mitsuya, H., and Broder, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2671-2676). We have designed experiments to determine the epitope for this mAb. Using simultaneous multiple peptide synthesis, we synthesized 481 overlapping octapeptides which corresponded to the sequence of gp46. We mapped the epitope for mAb 0.5 alpha to lie between residues 186 and 195 of gp46. This result was confirmed by independently synthesizing a peptide containing this epitope which bound specifically to mAb 0.5 alpha with an approximate Ka = 4 x 10(7) M-1. In addition, the peptide inhibited mAb 0.5 alpha binding to gp46 derived from T-cells infected with HTLV-I. This epitope containing peptide may facilitate understanding HTLV-1 infection of T-cells. 相似文献
17.
Localization of binding sites within human von Willebrand factor for monomeric type III collagen 总被引:19,自引:0,他引:19
Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Xiao X Wang F Saito A Majka J Schlösser A Schrempf H 《Molecular genetics and genomics : MGG》2002,267(4):429-439
During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo. 相似文献
19.
M Nakata S Tsuboi M Takenaka Y Konishi R Kawashima M Tomita T Nakagawa N Iwamura T Fujita 《Endocrinologia japonica》1981,28(6):741-746
Binding proteins for 1.25 (OH) 2D3 were investigated in thirty breast cancers. Human breast cancer was shown to contain specific, high affinity cytosol binding proteins for 1.25 (OH) 2D3 and 25 (OH) D3. The binding protein for 1.25 (OH) 2D3 sedimented at 3.7 S and the binding protein for 25 (OH) D3 at about 6.0 S on sucrose density gradient analysis containing 0.3 M KCl and 1 mM dithiothreitol in buffer. Kd for 1.25 (OH) 2D3 were from 0.1 x 10(-11) M to 7.1 x 10(-11) M measured by Scatchard plots. Competition binding studies indicated that the relative specificity of the binding protein for 1.25 (OH) 2D3 much greater than 25 (OH) D3 greater than 1 alpha (OH) D3, 24,25 (OH)2D3 greater than D3 much greater than Estradiol-17 beta. 1.25 (OH) 2D3 receptor-positive was detected in twenty-eight out of thirty breast cancers. 相似文献
20.
In model peptide systems, Ca2+ affinity is maximized in EF-hand motifs containing four carboxylates positioned on the +x and -x and +z and -z axes; introduction of a fifth carboxylate ligand reduces the affinity. However, in rat beta-parvalbumin, replacement of Ser-55 with aspartate heightens divalent ion affinity [Henzl, M. T., et al. (1996) Biochemistry 35, 5856-5869]. The corresponding alpha-parvalbumin variant (S55D/E59D) likewise exhibits elevated affinity [Henzl, M. T., et al. (2003) Anal. Biochem. 319, 216-233]. To determine whether these mutations produce a variation on the archetypal EF-hand coordination scheme, we have obtained high-resolution X-ray crystallographic data for alpha S55D/E59D. As anticipated, the aspartyl carboxylate replaces the serine hydroxyl at the +z coordination position. Interestingly, the Asp-59 carboxylate abandons the role it plays as an outer sphere ligand in wild-type rat beta, rotating away from the Ca2+ and, instead, forming a hydrogen bond with the amide of Glu-62. Superficially, the coordination sphere in the CD site of alpha S55D/E59D resembles that in the EF site. However, the orientation of the Asp-59 side chain is predicted to stabilize the D-helix, which may contribute to the heightened divalent ion affinity. DSC data indicate that the alpha S55D/E59D variant retains the capacity to bind 1 equiv of Na+. Consistent with this finding, when binding measurements are conducted in K(+)-containing buffer, divalent ion affinity is markedly higher. In 0.15 M KCl and 0.025 M Hepes-KOH (pH 7.4) at 5 degrees C, the macroscopic Ca2+ binding constants are 1.8 x 10(10) and 2.0 x 10(9) M(-1). The corresponding Mg2+ binding constants are 2.7 x 10(6) and 1.2 x 10(5) M(-1). 相似文献