共查询到20条相似文献,搜索用时 31 毫秒
1.
S Fong P P Chen T A Gilbertson R I Fox J H Vaughan D A Carson 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(3):1955-1960
The monoclonal antibody (MoAb) 17.109 recognizes a cross-reactive idiotype (CRI) associated with the light chains of Waldenstrom's macroglobulins with rheumatoid factor (RF) activity. The MoAb also reacts with a proportion of IgM-RF molecules from the sera of rheumatoid arthritis and primary Sjogren's syndrome patients, and from the sera of seropositive normal human subjects. In the present experiments, we used affinity chromatography to purify the 17.109 CRI-positive immunoglobulin from serum and have analyzed the isolated material by Western blotting. The purified 17.109 CRI-positive material from the sera of rheumatoid arthritis patients, Sjogren's syndrome patients, and normal subjects contained exclusively kappa light chains, and had demonstrated RF activity. In every case the 17.109 CRI-positive isolates reacted with antibodies against synthetic peptides corresponding to both the conserved second and third complementarity-determining regions (CDR) of the monoclonal kappa IgM-RF paraprotein Sie. The binding was inhibited specifically by the free peptides in solution. The antipeptide antibodies did not react appreciably with unfractionated human immunoglobulin. The data establish that the 17.109 CRI-positive immunoglobulin from diverse human sera have similar or identical second and third light chain CDR. These results suggest i) that the MoAb 17.109 identifies the protein product of a single or a very few V kappa genes, ii) that the ability to make kappa light chains with the 17.109-associated variable region is widespread in the human population, and iii) that the 17.109-defined kappa variable region segment is associated with IgM-RF autoantibodies. 相似文献
2.
I Bosc-Bierne L De la Fournière J Rathelot M Hirn L Sarda 《Biochimica et biophysica acta》1987,911(3):326-333
Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase. 相似文献
3.
The majority of human monoclonal IgM rheumatoid factors express a "primary structure-dependent" cross-reactive idiotype 总被引:16,自引:0,他引:16
P P Chen F Go?i S Fong F Jirik J H Vaughan B Frangione D A Carson 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(5):3281-3285
Genetic studies of human immunoglobulin variable regions have been hampered by the lack of anti-idiotypic antibodies that recognize specific heavy and light chain variable region sequences. Sixty percent of human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors [RF]) from unrelated individuals share a cross-reactive idiotype (CRI) termed Wa. In previous experiments in which we used an enzyme-linked immunosorbent assay, we reported that a synthetic peptide (PSL2), corresponding to the second hypervariable region in the kappa light chain of a monoclonal IgM-RF (Sie), induced rabbit antibodies reactive with several RF paraproteins. In the present experiments, to avoid interference due to the human IgM-RF binding toward rabbit IgG, the reactivity of the anti-PSL2 antibody to the separated heavy and light chains of multiple IgM proteins and Bence-Jones proteins was assessed by the Western blot technique. The PSL2-induced anti-CRI reacted well with the separated kappa chains from 10 out of 12 IgM-RF, zero out of four light chains from IgM proteins lacking anti-IgG activity, and one out of six kappa Bence-Jones proteins. The results show that the PSL2-CRI is associated with RF and is not a kappa subgroup marker. Furthermore, a comparison of the reported light chain sequences of the PSL2-CRI-positive IgM-RF suggests that the majority of human IgM-RF light chains derive from a single germ-line VK gene or from a family of closely related VK genes that is highly conserved in the human population. Synthetic peptide-induced anti-CRI provide a potent tool for analyzing the genetic basis of CRI and abnormal autoantibody production in humans. 相似文献
4.
A bispecific (Fab')2 molecule was constructed by linking the monovalent Fab' from an anti-fibrin monoclonal antibody to the Fab' from an anti tissue plasminogen activator (tPA, single chain) monoclonal antibody by means of inter-heavy-chain disulfide bonds. An immunochemical complex composed of the bispecific (Fab')2 molecule bound to tPA [tPA-bispecific (Fab')2 complex] was then generated and purified. Its molecular weight was 170 kDa [less than half the molecular weight of a previously described tPA-bispecific antibody complex containing the entire anti-fibrin and anti-tPA immunoglobulin molecules; Runge, M. S., et al. (1987) Trans. Assoc. Am. Phys. 100, 250-255]. The tPA-bispecific (Fab')2 complex was 8.6-fold more efficient in fibrinolysis than tPA alone and 94-fold more potent than urokinase. This enhancement in the fibrinolytic potency of tPA compares favorably with that observed for the bispecific whole-antibody complex. These results suggest that this smaller, less immunogenic molecule is capable of binding both fibrin and tPA with high affinity and of enhancing the thrombolytic efficiency of exogenous and, perhaps, endogenous tPA. 相似文献
5.
Christel Ostermann-Latif Michael Mäder Jürgen W. Unger Ilse Bartke† Kurt Naujoks† Johann H. Peters‡ Klaus Felgenhauer 《Journal of neurochemistry》1992,58(3):1060-1065
Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA. 相似文献
6.
7.
Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinity-resin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dose-dependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 30,000 polypeptide. The purified enzyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3 Nan and N-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, alpha 1 PI and alpha 2 M with a Ki of 10(-9)M for the NP4-alpha 1 PI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and NP4-alpha 1 PI complexes present in inflammatory exudates. 相似文献
8.
Tayebi M Enever P Sattar Z Collinge J Hawke S 《Molecular medicine (Cambridge, Mass.)》2004,10(7-12):104-111
Prion diseases such as Creutzfeldt-Jakob disease are believed to result from the misfolding of a widely expressed normal cellular prion protein, PrPc. The resulting disease-associated isoforms, PrP(Sc), have much higher beta-sheet content, are insoluble in detergents, and acquire relative resistance to proteases. Although known to be highly aggregated and to form amyloid fibrils, the molecular architecture of PrP9Sc) is poorly understood. To date, it has been impossible to elicit antibodies to native PrP(Sc) that are capable of recognizing PrP(Sc) without denaturation, even in Pm-P(o/o) mice that are intolerant of it. Here we demonstrate that antibodies for native PrPc and PrP(Sc) can be produced by immunization of Pm-P(o/o) mice with partially purified PrPc and PrP(Sc) adsorbed to immunomagnetic particles using high-affinity anti-PrP monoclonal antibodies (mAbs). Interestingly, the polyclonal response to PrP(Sc) was predominantly of the immunoglobulin M (IgM) isotype, unlike the immunoglobulin G (IgG) responses elicited by PrP(c) or by recombinant PrP adsorbed or not to immunomagnetic particles, presumably reflecting the polymeric structure of disease-associated prion protein. Although heat-denatured PrP(Sc) elicited more diverse antibodies with the revelation of C-terminal epitopes, remarkably, these were also predominantly IgM suggesting that the increasing immunogenicity, acquisition of protease sensitivity, and reduction in infectivity induced by heat are not associated with dissociation of the PrP molecules in the diseased-associated protein. Adsorbing native proteins to immunomagnetic particles may have general applicability for raising polyclonal or monoclonal antibodies to any native protein, without attempting laborious purification steps that might affect protein conformation. 相似文献
9.
Khosraviani M Blake RC Pavlov AR Lorbach SC Yu H Delehanty JB Brechbiel MW Blake DA 《Bioconjugate chemistry》2000,11(2):267-277
A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution. 相似文献
10.
Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells. 总被引:2,自引:0,他引:2
Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress. 相似文献
11.
Idiotypic interactions in type II mixed cryoglobulins 总被引:2,自引:0,他引:2
J C Renversez S Roussel M J Vallee G Brighouse P H Lambert 《Revue fran?aise de transfusion et immuno-hématologie》1984,27(6):737-755
An analysis of interactions between immunoglobulin molecules within cryoglobulins has been carried out in 18 patients with type II mixed cryoglobulinemia. In this series, there was a prevalence of VH I and VK I variable regions subgroups in the monoclonal IgM component. Our analysis of the IgG component indicated a particular selection of IgG molecules during cryoprecipitation. There was a prevalence of IgG3 and of the VH I subgroup and the isoelectrofocusing pattern revealed a very restricted spectrotype in two thirds of these IgG. These results which suggested a restricted reactivity between cryo-IgM and IgG fractions were confirmed by the analysis of the interaction between each IgG and each IgM from the cryoprecipitates. All IgM reacted with intact IgG or Fc fragments but another reaction was observed between cryo-IgM and Fab fragments from a limited number of cryo-IgG, with a pattern suggestive of idiotypic specificity. Results of the absorption of each cryo-IgM on Fc or on Fab fragments from the corresponding cryo-IgG also suggested the existence of a reaction between IgM and IgG Fab in addition to that involving IgM Fab and IgG Fc. The coexistence of the 2 reactions should confer a higher stability to the IgM-IgG complex. Therefore, it is possible that the proliferation of one clone of IgM-RF producing B cells would be followed in certain cases by a relatively restricted anti-idiotypic IgG response. The IgM-RF would preferentially react with these anti-idiotypic IgG. 相似文献
12.
Properties of monoclonal antibodies to nicotinic acetylcholine receptor from chick muscle 总被引:3,自引:0,他引:3
Four stable, hybrid-cell lines secreting monoclonal antibodies to distinct determinants on the nicotinic acetylcholine receptor from chick muscle have been established. These were characterised by the following criteria: immunoglobulin isotype, ability to produce experimental autoimmune myasthenia gravis in mice and reactivity towards homologous and heterologous acetylcholine receptor proteins. Two monoclonal antibodies were found to inhibit the reaction of alpha-bungarotoxin with homologous acetylcholine receptor; in addition one of these, on binding to receptor-toxin, induced a rapid dissociation of the complex (t1/2 = 0.5 h at 23 degrees C). Three of the antibody preparations recognised epitopes on this receptor from muscle of other species and two of these caused experimental autoimmune myasthenia gravis in BALB/c mice following passive transfer. The latter two recognised to significant extents the alpha-bungarotoxin binding component purified from chick optic lobe and brain cortex. Sedimentation analysis demonstrated that two of the monoclonal antibodies form a distinct size (s20, w = 12S) of complex with the receptor of chick muscle which most probably corresponds to a 1:1 attachment of antibody and receptor; this may involve cross-linking of two determinants within the same oligomer. A similar observation was made with the alpha-bungarotoxin binding component from optic lobe using one of the cross-reacting antibodies. Another monoclonal antibody was found to be capable of forming much heavier complexes with the receptor from chick muscle, these are thought to involve inter-molecular cross-linking of oligomers. The observed properties of these antibodies are discussed in relation to their myasthenogenicity and with reference to the extent of structural similarities between the peripheral nicotinic acetylcholine receptor and the alpha-bungarotoxin binding protein from brain. 相似文献
13.
In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis
Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor. 相似文献
14.
The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers. 相似文献
15.
A complex of a fragment of gamma-chain of IgG and beta2-microglobulin (beta2M) was isolated from the urine of a patient with plasma cell leukemia. The approximate m.w. of the gamma-fragment was 19,000 and this gamma-fragment was found to be associated with beta2M noncovalently. The complex could be dissociated in 7.5% (v/v) n-propanol which suggests an important role of hydrophobic bonds in the association of the gamma-fragment and beta2M. The beta2M did not bind monoclonal IgG from this patient. Several anti-beta2M antisera tested which had been prepared with free beta2M did not react with the beta2M associated with the gamma-fragment, but reacted with the free beta2M obtained by the dissociation of the complex. 相似文献
16.
S Ben-Or 《Journal of steroid biochemistry》1989,33(5):899-906
Previous studies have shown that the exposure of molybdate-stabilized nontransformed glucocorticoid receptor (GR) of the chick embryonic neural retina to 0.4 M KCl dissociated the 9.5 S complex to a 5 S GR complex, which is an intermediate state in GR transformation. The present study was designed to characterize the 5 S GR complex. It shows that molybdate-stabilized nontransformed 9.5 S GR complex and 5 S GR interact with monoclonal antibodies (MAb) directed against 90 kDa heat shock protein (hsp90), as evidenced by the increase in the sedimentation velocity of these GR-complexes. Electrofocusing of the partially purified molybdate-stabilized nontransformed GR, prepared from [32P]-labeled neural retinas, and of the 5 S GR (derived from molybdate-stabilized preparation) showed that nontransformed GR complex, which has an apparent pI (pI') value of 5.0 +/- 0.2, and 5 S GR, which was resolved in a major peak with a pI' value of 5.8, are phosphorylated. Partially purified 5 S GR, cleared of molybdate and exposed to 25 degrees C, was resolved by electrofocusing into two phosphorylated fractions, one with a pI' value of 6.5, representing the monomeric GR form and the other with a pI' value of 5.1, apparently representing the acidic hsp90. The dissociation of hsp90 from the molybdate-cleared 5 S heterodimer seems to account for the decrease in the negative net charge of 5 S GR from pI' 6.5. Monomeric GR, derived from a molybdate-cleared, partially purified GR preparation, by the exposure to 25 degrees C, did not retain glucocorticoid-binding activity. Molybdate-stabilized 5 S GR was apparently re-assembled into the oligomeric nontransformed state when the salt concentration was reduced. This phenomenon was evident under the low-salt conditions of electrofocusing, by the shift in pI' value of GR from 5.8 to 5.0; and in glycerol density gradients containing 0.15 M KCl, by the shift in the sedimentation of the GR complex from 5 S to 9.5 S. 相似文献
17.
Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma 总被引:9,自引:0,他引:9
Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor. 相似文献
18.
Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions. 相似文献
19.
R M Ulloa C P Rubinstein L Molina y Vedia H N Torres M T Téllez-I?ón 《FEBS letters》1988,241(1-2):219-222
Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS-polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin. 相似文献
20.
A monoclonal antibody capable of inhibiting opioid binding to rat neural membranes has been produced. Spleen cells from a BALB/c mouse, immunized with a partially purified opioid receptor complex, were fused with P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM that was capable of partially inhibiting opioid binding to rat neural membranes under equilibrium binding conditions, while not affecting the binding of nonopioid ligands. Control mouse immunoglobulins and heat-denatured OR-689.2.4 did not inhibit opioid binding to membranes. The purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a titrable, saturable, and reversible manner, as well as the binding of the delta-ligand [3H][D-Ala2,D-Leu5]enkephalin, the kappa-ligand [3H] ethylketocyclazocine, and 3H-labeled antagonists. In addition to blocking the binding of opioids to membranes, the immunoglobulin could also displace bound [3H]dihydromorphine from neural membranes. The 125I-labeled immunoglobulin specifically bound to neural membranes with a Kd of 1.3 nM and a maximal number of binding sites of 41.8 fmol/0.25 mg of membrane protein. In a titrable manner, the immunoglobulin precipitated opioid binding sites from a solubilized preparation of neural membranes. When OR-689.2.4 conjugated to Sepharose was incubated with the partially purified opioid receptor complex, labeled with 125I, a 35,000-dalton protein was specifically bound by the immunoglobulin. This antibody provides a tool for probing the multiple opioid binding sites. 相似文献