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1.
M Gonzalez-Gronow G E Siefring F J Castellino 《The Journal of biological chemistry》1978,253(4):1090-1094
The object of this investigation was to distinguish between two potential mechanisms of activation of human plasminogen (HPg) to plasmin (HPm) by catalytic levels of the activator complex, streptokinase.plasmin (SK.HPm). One mechanism, which is widely supported, postulates an enzymatic role for SK.HPm in the conversion of molar excesses of plasminogen to plasmin. A more recently described kinetic mechanism involves a direct conversion of HPg to HPm by streptokinase (SK). Here, it is believed that displacement of HPm from SK.HPm by excess HPg is the major source of free HPm in the activation process. The present paper shows that SK is not capable of undergoing rapid exchange from SK.HPm to other HPg or HPm molecules, thus precluding the possibility of direct activation of HPg by SK. Our evidence supports a mechanism involving an enzymatic role for SK.HPm as the major means of converting free HPg to HPm. 相似文献
2.
Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids 总被引:3,自引:0,他引:3
An Escherichia coli expression vector, containing the alkaline phosphatase promoter and the stII heat-stable enterotoxin signal sequence, along with the cDNA of the kringle 1 (K1) region of human plasminogen (HPg), has been employed to express into the periplasmic space amino acid residues 82-163 (E163----D) of HPg. This region of the molecule contains the entire K1 domain (residues C84-C162) of HPg, as well as two non-kringle amino-terminal amino acids (S82-E83) that are present in their normal locations in HPg and a carboxyl-terminal amino acid, D163, that results from mutation of the E163, normally present at this location in the HPg amino acid sequence. After purification of r-K1 by chromatographic techniques, we have investigated its omega-amino acid binding properties by titration calorimetry, intrinsic fluorescence, and differential scanning microcalorimetry (DSC). The antifibrinolytic agent, epsilon-aminocaproic acid (EACA), possesses a single binding site for r-K1. The thermodynamic properties of this interaction, studied by calorimetric titrations of the heats of binding with this ligand, reveal a Kd of 12 +/- 2 microM at 25 degrees C and pH 7.4, a corresponding delta G of -6.7 +/- 0.1 kcal/mol, a delta H of -3.6 +/- 0.1 kcal/mol, and a delta S of 10.5 +/- 0.8 eu. The intrinsic fluorescence of r-K1 decreases by approximately 44% when its binding site is saturated with EACA, and titrations of this perturbation with EACA lead to calculation of a Kd of approximately 13 microM, a value in good agreement with that obtained from titration calorimetric analysis. EACA represents the strongest binding ligand of a variety of simple aliphatic omega-amino acids examined. A cyclic analogue of EACA, trans-4-(aminomethyl)cyclohexanecarboxylic acid, interacts with r-K1 with an approximate 12-fold tighter Kd (1.0 +/- 0.2 microM). Investigations by DSC, at pH 7.4, demonstrate that a significant stabilization of the r-K1 structure occurs when EACA binds to this domain. The temperature of maximum heat capacity change (Tm) in the thermal denaturation of r-K1 increases from approximately 340.8 to 359.1 K as a consequence of EACA binding. These studies demonstrate that a fully functional EACA-binding kringle from HPg can be expressed and secreted in E. coli, purified by techniques that do not require refolding, and investigated as an independent structural unit. 相似文献
3.
Malembic S Saillard C Bové JM Garnier M 《Applied and environmental microbiology》2002,68(5):2113-2119
Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed. 相似文献
4.
Edward M. Conway Barbara Nowakowski Marta Steiner-Mosonyi 《Journal of cellular physiology》1994,158(2):285-298
The plasminogen activation (PA) system of human Co 115 colon carcinoma cells was investigated. Analysis at the levels of protein and mRNA of cultured cells and of histozymography of tumor xenografts in nude mice showed that Co 115 cells produce only tissue type PA (t-PA) and no urokinase (u-PA). Also, mRNA for the u-PA receptor and for PA inhibitorr type 2 (PAI-2), but not for PAI-I, were detected. We developed a quantitative degradation assay using glutaraldehyde-immobilized125I-Iaminin to investigate the capacity of Co 115 cells to degrade laminin. Laminin degradation by Co 115 cells was completely inhibited by 100 μg/ml of polyclonal anti-t-PA IgG, by the plasmin inhibitors aprotinin (100μg/ml) or ε-aminocaproic acid (EACA; at 0.3 M), but not by antibodies against u-PA or u-PAR nor by nonimmune IgG. Cycloheximide-treated Co 115 cells were unable to degrade laminin but increased laminin degradation induced by conditioned medium of Co 115 cells or recombinant t-PA. No potentiation was observed when Co 115 cells and laminin were kept separated by Transwell™ inserts. Our results suggest that Co 115 human colon carcinoma cells degrade laminin by potentiating t-PA-mediated plasminogen activaton at the cells surface which requires close contact between tumor cells and laminin substrate. © 1994 Wiley-Liss, Inc. 相似文献
5.
Mitogen-like monoclonal anti-actin antibodies 总被引:1,自引:0,他引:1
H M Rosenblatt N Parikh J E McClure I Meza S Y Hwo J Bryan W T Shearer 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(2):995-1000
Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism. 相似文献
6.
Recombinant Autographa california baculoviruses expressing genes for pseudorabies virus glycoprotein (gp50T), human plasminogen (HPg), and beta-galactosidase (beta-gal) were used to infect 23 cell lines or strains. The objectives were to compare amounts of recombinant proteins expressed in the cell lines, compare yields from clones and parent lines, investigate the effects of long-term culture in serum-free medium on production, and determine if some lines yield gp50T with different glycosylation patterns. For HPg, IZD-MB0503 had the highest yield and four other lines (IPLB-TN-R2, IPLB-SF-1254, IPLB-LdEIta, and CM-1) had levels above that of SF-9 cells. For gp50T, four lines (IPLB-HvT1, IPLB-SF21AE, IPLB-SF21AE-15, and IPLB-SF-1254) had higher amounts than SF-9 cells. Some lines yielded gp50T with molecular mass about 1000 daltons larger than that from SF-9 cells, which suggests increased oligosaccharide processing. Equally high levels of beta-gal were expressed in three lines (SF-9, IZD-MB0503, and BCIRL-PX2-HNV3). The major conclusion is that no single cell line produced highest yields for all three recombinant proteins. Four lines were cultured in serum-free medium for 31-34 passages and then infected with the three recombinant viruses. For most cell line-recombinant combinations, the yields in serum-free medium were equal to or better than those in serum-supplemented medium. Medium composition had a much stronger effect on foreign gene expression than on susceptibility of cells to wild-type virus. 相似文献
7.
Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri
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Sylvie Malembic Colette Saillard Joseph M. Bov Monique Garnier 《Applied microbiology》2002,68(5):2113-2119
Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed. 相似文献
8.
Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development 总被引:1,自引:0,他引:1
Summary The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats. 相似文献
9.
Monoclonal antibody DH12 reacts with a cell surface and a precursor form of the beta subunit of the human fibronectin receptor 总被引:1,自引:0,他引:1
B De Strooper M Saison M Jaspers M Spaepen F Van Leuven H Van den Berghe J J Cassiman 《Cell biology international reports》1988,12(1):9-16
Monoclonal and polyclonal antibodies were raised against a placenta plasma membrane protein preparation, which was obtained by fractionation on Blue B dye matrix and by HPLC-anionexchange, and which was shown to contain fibronectin receptors. Immunochemical and functional evidence showed that monoclonal antibody DH12 recognized the beta subunit of the human fibronectin receptor on fibroblasts. This monoclonal antibody reacted with two proteins in Western blots and in double immune precipitations of whole cell preparations. Only the higher Mr protein became labeled by surface iodination of intact fibroblasts. The lower Mr protein is thought to be an intracellular precursor of the beta subunit of the fibronectin receptor. 相似文献
10.
The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats. 相似文献
11.
A M Vandamme M Dewerchin H R Lijnen H Bernar F Bulens L Nelles D Collen 《European journal of biochemistry》1992,205(1):139-146
A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro. 相似文献
12.
Incubation of a Spodoptera frugiperda (IPLB-SF-21AE) cell extract with the oligosaccharide Man9GlcNAc2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant Asn residue in the nascent protein, results in its trimming to Man6GlcNAc2, an intermediate that is relatively stable to further alpha-D-mannosidase action in these cells. On the other hand, incubation of a similar extract from cells that had been infected for various times with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) or a recombinant baculovirus (r-BAC)/human plasminogen (HPg) construct employed for expression of HPg led to rapid trimming of Man6GlcNAc2 to Man5GlcNAc2 and Man3GlcNAc2. These latter reactions displayed temporal effects, in that an enhancement of this latter trimming process occurred as a function of the time of infection of the cells with the wild-type and recombinant viral constructs. We have previously demonstrated that the nature of the oligosaccharide assembled on Asn289 of HPg expressed in several lepidopteran insect cell lines was dependent on the time of infection of the cells with r-BAC/HPg and that the amount of complex glycan found on this recombinant protein increased with an increase in infection times [Davidson, D. J., & Castellino, F. J. (1991) Biochemistry 30, 6167-6174].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance 总被引:1,自引:0,他引:1
The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead. 相似文献
14.
J Wojta R Beckmann L Turcu O F Wagner A J van Zonneveld B R Binder 《The Journal of biological chemistry》1989,264(14):7957-7961
Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes. 相似文献
15.
The heavy (H) and light (L) chains of murine monoclonal autoantibody 62 reacting with thyroglobulin independently express idiotypic (Id) determinants that are very similar if not identical with the Id62 expressed on the intact protein. In this report, we describe the production and characterization of rabbit antibodies to isolated H62 and L62 chains to further prove that individual chains express Id62 in an immunogenic form. The results demonstrate that both chains are capable of eliciting antibodies that, after appropriate adsorption, behave like conventional anti-Id62 antibodies prepared against the intact antibody molecule. By direct radioimmunoassay binding, competition of Id binding and Western blot anti-H62 and anti-L62 antibodies identify as Id-positive the same group of IgG1, bind in a reciprocal fashion to H- and L-chains of parental monoclonal antibody 62, and detect Id62-positive polyclonal serum autoantibodies to thyroglobulin. We conclude that monoclonal antibody 62 expresses independently a similar Id on both polypeptide chains and the intact antibody molecule, or its isolated chains, induce qualitatively similar anti-Id responses. These results are discussed in light of the possible structure/function implication such autoantibodies may have within the Id network. 相似文献
16.
Recombinant polyclonal antibodies for cancer therapy 总被引:4,自引:0,他引:4
Although monoclonal antibodies are increasingly used for cancer therapy, remissions are only temporary due to emergence of tumor cell escape variants that are no longer affected by the antibody. The emergence of escape variants could be minimized by multi-targeting of tumor cells with polyclonal antibodies, which would also be more efficient than monoclonal antibodies at mediating effector functions for target destruction. A technology for generating recombinant polyclonal antibodies for cancer therapy has been developed based on the construction and selection of tumor-reactive Fab phage display libraries. The selected Fabs are mass-converted to full-length polyclonal antibody libraries (PCALs) of any isotype and any species. Prototypic PCALs generated against human colorectal cancer cell lines showed that libraries of diverse recombinant antibodies, enriched for reactivity to the cancer cells compared to normal human cells, can be obtained. The success of recombinant polyclonal antibodies as cancer therapeutics will depend on the ability to generate, characterize, and mass-produce PCALs with high ratios of cancer-to-normal reactivities that cross-react with many cancers of the same type. 相似文献
17.
Liu L Sazonova IY Turner RB Chowdhry SA Tsai J Houng AK Reed GL 《The Journal of biological chemistry》2000,275(48):37686-37691
The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing. 相似文献
18.
A mitogenic and plasminogen activator (PA)-inducing activity for endothelial cells has been identified in serum-free culture medium of normal AG 7680 and transformed tumorigenic GM 7373 fetal bovine aortic endothelial (FBAE) cells. The activity binds to heparin-Sepharose and it is quenched by polyclonal anti-human placental basic fibroblast growth factor (bFGF) antibodies. In the serum-free conditioned medium of FBAE cells, the anti-bFGF antiserum recognizes an immunorective Mr 20,000 molecule which co-purifies with the mitogenic and PA-inducing activity on a heparin-Sepharose column. The partially purified Mr 20,000 bFGF-like molecule competes with the typical Mr 18,000 125I-bFGF form for the binding to high-affinity bFGF receptors in intact GM 7373 cells. Immunoprecipitation of biosynthetically labeled GM 7373 cells with anti-bFGF antiserum confirms the presence of a Mr 20,000 bFGF-like molecule in the conditioned medium of these cells and identifies the typical Mr 16,000 and Mr 18,000 bFGF forms and two high-molecular-weight immunoreactive Mr 22,000 and Mr 25,000 bFGF forms in their cell extract. Immunoreactive Mr 20,000 bFGF is detectable also in the conditioned medium of transformed nontumorigenic FBAE GM 7372 cells and of adult bovine aortic endothelial cells, but not in the culture medium of nonendothelial cell types, including rat and mouse fibroblasts, human hepatoma, and human endometrial adenocarcinoma cells. The results indicate that bovine endothelial cells secrete a Mr 20,000 bFGF-like molecule which shares several biological, biochemical, and immunological characteristics with the typical cell-associated Mr 18,000 bFGF. 相似文献
19.
A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred. 相似文献
20.
We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C. 相似文献