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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

The polymeric immunoglobulin receptor (pIgR) on mucosal epithelial cells binds dimeric IgA (dIgA) on the basolateral surface and mediates transport of dIgA to the apical surface. Using Madin-Darby canine kidney epithelial cells stably transfected with pIgR cDNA, we found that soluble immune complexes (ICs) of 125I-labeled rat monoclonal antidinitrophenyl (DNP) dIgA (125I-dIgA) and DNP/biotin-bovine serum albumin were transported from the basolateral to the apical surface and then released. Monomeric IgA ICs were not transported, consistent with the specificity of pIgR for polymeric immunoglobulins. Essentially all the 125I-dIgA in apical culture supernatants was streptavidin precipitable, indicating that dIgA remained bound to antigen during transcytosis. While both dIgA and dIgA ICs bound pIgR with equal affinity (Kd approximately 8 nM), the number of high-affinity binding sites per cell was 2- to 3-fold greater for dIgA than for dIgA ICs. The extent of endocytosis of dIgA and dIgA ICs was correlated with the number of high-affinity binding sites. SDS/PAGE analysis of intracellular dIgA and dIgA ICs demonstrated that in both cases IgA remained undegraded during transport. The results suggest that the pathways of epithelial transcytosis of free dIgA and dIgA ICs are the same. Given the high population density of mucosal IgA plasma cells and the enormous surface area of pIgR-expressing mucosal epithelium, it is likely that significant local transcytosis of IgA ICs occurs in vivo. Such a process would allow direct elimination of IgA ICs at the mucosal sites where they are likely to form, thus providing an important defense function for IgA.
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PMID:The polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across epithelial cells: a local defense function for IgA. 192 41

We have previously reported the isolation of a 66 kDa melanoma-associated antigen, identified by autologous antibody, in serum and unfractionated spent tissue culture media by Western blot analysis. The antigen, detected by autologous serum S150, was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma and head and neck carcinoma cell lines. S150 did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, autologous cultured lymphocytes or fetal calf serum. To further characterize the antigen, spent tissue culture media, obtained from autologous melanoma cell line, Y-Mel 84:420, was separated by an isoelectric focusing column. Unabsorbed control serum S150 was noted to have a maximum titer of 1:2040 against autologous melanoma cells as measured by protein A hemadsorption. Following isoelectric focusing the greatest decrease in autologous antibody titer (30-fold) occurred with fractions having a pI between 2 and 3. Further resolution of the antigen was accomplished with high-pressure ion-exchange chromatography. One of these fractions showed a significantly higher concentration of antigen and was distinctly resolved from bulk serum albumin. Subsequent Western blot analysis, with autologous antibody, of the isolated antigen-containing fraction, confirmed the presence of a single 66 kDa band. Exposure of the antigen, purified by high-pressure ion-exchange chromatography, to neuraminidase ablated recognition by autologous antibody and suggests that sialic acid is present on the protein and may be part of the antigenic epitope. Binding of antigen, obtained following DEAE anion exchange chromatography, was noted to lectins derived from Triticum vulgaris, Dolichos biflorus and Lycopersicon esculentum. Preparative purification of the antigen was accomplished by anion exchange followed by lectin affinity chromatography with a Dolichos biflorus column. Antigen obtained following lectin affinity chromatography subjected to SDS-PAGE and silver stain revealed a single band at 66 kDa. We conclude that a melanoma-associated antigen detected by autologous antibody in spent tissue culture media is an unusually acidic glycoprotein (pI 2-3).
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PMID:Purification and partial characterization of a shed 66 kDa melanoma-associated antigen identified by autologous antibody. 193 77

The production of koala immunoglobulins (Ig) was elicited by the immunisation of a koala (Phascolarctos cinereus) with bovine serum albumin. This Ig was then purified using the highly specific techniques of affinity chromatography. The purified protein was compared with a "potential" koala Ig protein which was subsequently purified by Protein G chromatography and both proteins were further characterised by agarose/SDS-PAGE and immunoelectrophoresis. Results indicate that koalas do produce Ig in response to antigen challenge, koala Ig has a higher net negative charge than that seen in most other mammals and possibly two subclasses of IgG and IgM are present in normal koala serum.
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PMID:Purification and initial characterisation of koala immunoglobulins. 194 80

Protocols are described for the isolation from human placental extracts of a preparation which is active in the rosette inhibition assay by inducing an increased rosette inhibition titre. In this respect the preparation mimics the effects of pregnancy sera, an ability ascribed to the presence in these sera of a so-called 'early pregnancy factor' (EPF). In addition to this activity in the rosette inhibition assay, the preparation was also shown to modulate the expression of cell surface immunoglobulin on peripheral blood mononuclear cells. The polypeptide composition of the preparation was relatively simple, as revealed by SDS-PAGE. There was a major 12 kd polypeptide previously isolated in a rosette inhibition titre-active preparation from ovine placental extracts, and a small amount of 68 kd polypeptide. N-Terminal amino acid sequence analyses obtained after blotting onto polyvinylidene difluoride membranes identified the latter polypeptide as serum albumin and the major 12 kd polypeptide as human thioredoxin. Gel permeation analysis partially resolved activity expression away from the 12 kd polypeptide as activity expression was also found to be associated with low mol. wt material. It is concluded that EPF activity expression in pregnancy sera involves molecules related to and associated with thioredoxin.
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PMID:Isolation from human placental extracts of a preparation possessing 'early pregnancy factor' activity and identification of the polypeptide components. 195 57

An automated Minigel electrophoresis system (PhastSystem, Pharmacia, Uppsala, Sweden) was tested for human tear protein analysis. Tear samples were treated under nonreducing or reducing conditions before sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE). Micro-amounts of tears (2 microliters) were sufficient for analysis and separation and visualization of proteins were completed within 2 hr. Tear proteins were identified using purified control proteins and immunoblotting techniques, using antisera against immunoglobulin (Ig) A (alpha) heavy chains, Ig heavy and light chains, secretory components and lactoferrin. In nonreduced tears, lactoferrin (seen as a double band), serum albumin, tear-specific prealbumin (TSPA), and lysozyme were clearly separated. Secretory IgA (sIgA) was seen as a smear on top of the gel. Immunostaining also showed a major Ig light chain containing protein. After reduction, the protein profiles showed marked changes. In reduced tears, immunoglobulin heavy and light chains (molecular weight [MW]: 64 and 28 kD, respectively) were detected on the SDS-PAGE profile after immunostaining, and represented disulfide cleavage fragments, which originated from sIgA. Reduction resulted in the liberation of the secretory component piece (MW: 85 kD), which was found to co-migrate with the tear lactoferrin bands. Both lactoferrin and serum albumin acted as larger proteins on SDS-PAGE after reduction. The authors found that the two methods of sample treatment, before electrophoresis, resulted in marked differences on the electropherograms.
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PMID:SDS-Minigel electrophoresis of human tears. Effect of sample treatment on protein patterns. 199 90

Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain.
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PMID:Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum. 201 7

Pepstatin A, a pentapeptide aspartyl protease inhibitor, can interact with intermediate filament (IF) subunit proteins and induce their polymerization in the absence of salt into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appears to be driven primarily by non-ionic interactions between pepstatin A and polymerization-competent forms of IF proteins, resulting in a composite filament. Proteolytic fragments of vimentin, lacking portions of only the head domain or of both the head and tail domains, failed to copolymerize with pepstatin A into long filaments under these conditions. Rather, these peptides, as well as control proteins like bovine serum albumin, were found to decorate pepstatin A polymers (filaments, ribbons, and sheets) by sticking to their surfaces. In addition to the electron microscopy experiments, UV difference spectra, ultracentrifugation, and SDS-PAGE analysis of in vitro cleavage products of vimentin obtained with HIV-1 protease all provided independent evidence for a direct association of pepstatin A with IF subunit proteins, with subsequent alterations in the IF subunit protein conformation. These data show that non-ionic interactions can substitute for the effect of salt and effectively drive the higher-order polymerization of IF subunit proteins.
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PMID:Effect of pepstatin A on structure and polymerization of intermediate filament subunit proteins in vitro. 205 52

Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. (We refer to this interaction as a "crosslink," without intending to imply knowledge as to its chemical nature.) Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. [14C]Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H2O2 is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.
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PMID:Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein. 206 86

The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.
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PMID:Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin. 215 67


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