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PubMed1992

Polymeric porogens used in the preparation of novel monodispersed macroporous polymeric separation media for high-performance liquid chromatography.

A novel approach to monosized macroporous polymeric separation media with vastly enhanced pore size distributions and chromatographic properties has been developed. Key to this approach is the combined use of monodispersed polymeric particles and suitable solvents as porogens in the copolymerization of styrene and divinylbenzene. Following polymerization, the polymeric porogen is dissolved, leaving behind the monosized beads with a controlled pore structure. The exact pore size and pore size distribution of the final beads are largely controlled by the amount of soluble polymer in the polymerizing mixture: the larger the proportion of soluble polymer in the system, the larger the pores. The uniformly sized macroporous beads prepared with an optimized ratio of polymeric and low molecular weight porogens proved to be very efficient even in short columns for the separation of polystyrene standards in the SEC mode and the separation of proteins in the reversed-phase mode. The relationship between pore size and specific surface area, on one hand, and chromatographic properties of the stationary phase, on the other, have been clearly documented.

Chromatography, High Pressure Liquid↗
PubMed2003

Molecular cloning of a chondroitin polymerizing factor that cooperates with chondroitin synthase for chondroitin polymerization.

We recently cloned human chondroitin synthase (ChSy) exhibiting the glucuronyltransferase-II (GlcATII) and N-acetylgalactosaminyltransferase-II (GalNAcTII) activities responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate, but chondroitin polymerization was not demonstrated in vitro using the recombinant ChSy. We report here that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor (ChPF) with ChSy. The human ChPF consists of 775 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 23% identity to that of human ChSy. The expression of a soluble recombinant form of the protein in COS-1 cells produced a protein with little GlcAT-II or GalNAcT-II activity. In contrast, coexpression of the ChPF and ChSy yielded markedly augmented glycosyltransferase activities, whereas simple mixing of the two separately expressed proteins did not. Moreover, using both UDP-glucuronic acid (GlcUA) and UDP-N-acetylgalactosamine (GalNAc) as sugar donors, chondroitin polymerization was demonstrated on the so-called glycosaminoglycan-protein linkage region tetrasaccharide sequence of alpha-thrombomodulin. These results suggested that the ChPF acts as a specific activating factor for ChSy in chondroitin polymerization. The coding region of the ChPF was divided into four discrete exons and localized to chromosome 2q35-q36. Northern blot analysis revealed that the ChPF gene exhibited a markedly different expression pattern among various human tissues, which was similar to that of ChSy. Thus, the ChPF is required for chondroitin polymerizing activity of mammalian ChSy.

Amino Acid Sequence↗
PubMed1976

Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin.

The kinetics and thermodynamics for the polymerization of purified Acanthamoeba actin were studied and compared to muscle actin. Polymerization was qualitatively similar for the two actins with a rate-limiting nucleation step followed by rapid polymer extension. Polymerization occurred only above a threshold critical concentration which varied with polymerization conditions for each actin. In the presence of 2 mM MgCl2, nucleation of both actins was rapid and their critical concentrations were similarly low and not detectably dependent on temperature. In 0.1 M KCl, the rates of nucleation of both actins were much slower than when Mg2+ was present and were significantly different from each other. Also, under these conditions, the critical concentrations of Acanthamoeba and muscle actin were significantly different and both varied markedly with temperature. These quantitative differences between the two actins could be attributed to differences in both their enthalpies and entropies of polymerization, Acanthamoeba actin having the more positive deltaH and delta S. Co-polymerization of the two actins was also demonstrated. Overall, however, there were no qualitative differences between Acanthamoeba and muscle actin that would suggest a unique role for the monomer-polymer equilibrium of cytoplasmic actin in cell motility.

Actins↗
PubMed1984

Polymerization of AS hybrid hemoglobin. Potent inhibitory effect of hemoglobin A on the polymerization of AS hybrid hemoglobin.

Asymmetrically cross-linked AS hybrid hemoglobin (Hb AS-fumarate) was prepared by reacting a mixture of oxy-Hb A and oxy-Hb S with a cross-linking agent, bis(3,5-dibromosalicyl)fumarate. Kinetics of the polymerization of Hb AS-fumarate in 1.8 M phosphate buffer showed that Hb AS-fumarate polymerized similarly to Hb S-fumarate despite the presence of a beta A chain. The kinetics of the polymerization of a mixture of Hb AS-fumarate and Hb S-fumarate was similar to that of Hb S-fumarate or Hb AS-fumarate alone. However, when Hb AS-fumarate was mixed with Hb A, the delay time prior to polymerization of the mixture was markedly prolonged. Although the solubility of Hb AS-fumarate was similar to that of Hb S-fumarate, it nearly doubled upon the addition of Hb A. Analysis of Hb A in the polymer phase showed that a significant amount (35-40%) of Hb A copolymerized with Hb AS-fumarate. No such effect was observed when Hb A was mixed with Hb S-fumarate, where the delay time prior to polymerization and the solubility of the mixture were regulated by the concentration of Hb S-fumarate in the mixture. The strong inhibitory effect of Hb A on the polymerization of Hb AS hybrid hemoglobin may help explain the benign clinical course in sickle cell trait.

Chromatography, High Pressure Liquid↗
PubMed1998

Radical Nature of Cu-Catalyzed Controlled Radical Polymerizations (Atom Transfer Radical Polymerization).

Copper-catalyzed atom transfer radical polymerization (ATRP) is one of the most robust and precise techniques for controlling radical polymerization. The very good control of molecular weights, polydispersities, functionalities, chain composition, and topologies unusual for radical systems combined with the application of transition metals as catalysts requires more detailed mechanistic studies and proof of the radical nature of active species. The following results are in agreement with the radical nature of ATRP: reverse ATRP, chemoselectivities similar to those for conventional radical polymerization (effect of additives and inhibitors/scavengers, reactivity ratios, transfer coefficients), regioselectivities similar to those for conventional radical polymerization (low proportion of head-to-head units and expected structure of both tail and head end groups), stereoselectivities (tacticities) similar to that in conventional radical polymerization, EPR detection of X-Mtn+1 species resulting from the persistent radical effect, and confirmation of the termination by doubling molecular weights as well as cross-linking with multifunctional initiators and inimers. In addition, it seems that, in most ATRP systems, the contribution of degenerative transfer and reversible formation of organometallic intermediates is small and a halogen atom is transferred in the concerted process rather than in a two-step process with the involvement of radical anions.

Journal Article↗
PubMed1986

An intermediate state of G-actin between native and denatured: polymerization rate decreases but extent of polymerization remains unchanged.

The rate of actin polymerization gradually decreased without changing the final level of polymerization, when incubated in the presence of 0.2 mM ATP at pH 8.0 and 25 degrees C. This change was much faster in Mg2+-actin than Ca2+-actin, and Mg2+-actin became denatured and unpolymerizable on prolonged incubation. The drop in the polymerization rate was due both to weakened nucleation and a slowed elongation rate in the incubated actin. The change in the polymerization rate was partially reversible by storing the sample at 0 degrees C. When the rate of polymerization dropped markedly on prolonged incubation, a gel filtration profile showed that Ca2+-actin existed as monomer not as oligomer. On the other hand, Mg2+-actin formed dimers, and other oligomers, as revealed by crosslinking analysis. There were changes in fluorescence intensities due to tyrosine and/or tryptophan residues of the actin molecule, and in difference absorption spectra, suggesting that conformational changes intermediate between native and denatured states occurred during incubation.

Actins↗
PubMed2001

Fibrinogen Matsumoto V: a variant with Aalpha19 Arg-->Gly (AGG-->GGG). Comparison between fibrin polymerization stimulated by thrombin or reptilase and fibrin monomer polymerization.

Fibrinogen Matsumoto V (M-V) is a dysfibrinogen identified in a 52-year-old woman with systemic lupus erythematous. The triplet AGG encoding the amino acid residue Aalpha19 was replaced by GGG, resulting in the substitution of Arg-->Gly. Residue Aalpha19 has been shown to be one of the most important amino acids in the so-called 'A' site or alpha-chain knob. The thrombin-catalyzed release of fibrinopeptide A from M-V fibrinogen was only slightly delayed yet release of fibrinopeptide B was significantly delayed. Both thrombin-catalyzed fibrin polymerization and fibrin monomer polymerization were markedly impaired compared to normal fibrinogen. In addition, reptilase-catalyzed fibrin polymerization of M-V was much more impaired than thrombin-catalyzed fibrin polymerization. These results indicate 'B' and/or 'b' site of M-V fibrinogen play a more important role in thrombin-catalyzed fibrin polymerization than that of normal control fibrinogen.

Batroxobin↗
PubMed2002

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor.

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.

Animals↗
PubMed1986

Mechanical properties of polymeric membranes obtained by radiation cast-polymerization of hydroxyalkyl and hydroxypolyethyleneglycol methacrylate monomers.

Polymeric membranes have been prepared by radiation cast-polymerization of hydroxyalkyl and hydroxypolyethyleneglycol methacrylate monomers, and the mechanical properties of the membranes before and after swelling have been studied as a function of the molecular structure of the monomers. The degree of hydration of the polymers from hydroxypolyethyleneglycol methacrylate monomers increased with increasing the number of ethyleneglycol units in the monomers, and that from hydroxyalkyl methacrylate monomers decreased with an increase in the number of methylene units. The mechanical properties such as tensile strength, tear strength, Young's modulus, and elongation at break varied with the length of the methylene and ethyleneglycol units in the monomers. It was found that flexible polymeric membranes having various properties for medical applications could be obtained by radiation cast-polymerization of hydroxyalkyl and hydroxypolyethyleneglycol methacrylate monomers.

Acrylates↗
PubMed1991

Osmotic effects of protein polymerization: analysis of volume changes in sickle cell anemia red cells following deoxy-hemoglobin S polymerization.

Polymerization-depolymerization of proteins within cells and subcellular organelles may have powerful osmotic effects. As a model to study these we analyzed the predicted volume changes following hemoglobin (Hb) S polymerization in sickle cell anemia (SS) red cells with different initial volumes. The theoretical analysis predicted that dehydrated SS red cells may sustain large polymerization-induced volume shifts whose direction would depend on whether or not small solutes were excluded from polymer-associated water. Experiments with SS cells from promptly fractionated venous blood showed oxygenation-induced swelling, maximal in the densest cells, in support of nonexclusion models. The predicted extent of cell dehydration on polymerization was strongly influenced by factors such as the dilution of residual soluble Hb and the increased osmotic contribution of Hb in cells dehydrated by salt loss, largely overlooked in the past. The osmotic effects of polymer formation may thus play an important part in microcirculatory infarction by dense SS cells, as they become even denser and stiffer during deoxygenation in the capillaries.

Anemia, Sickle Cell↗
PubMed1985

Polymerization properties of two normally circulating fibrinogens, HMW and LMW. Evidence that the COOH-terminal end of the a-chain is of importance for fibrin polymerization.

The plasma fibrinogen fractions HMW (mw 340,000) and LMW (mw 305,000) were prepared from purified (beta-alanine precipitated) fibrinogen by step-wise precipitation with ammonium sulfate. The thrombin clotting times were 14" and 20" respectively. The enzymatic phase of coagulation, measured as release of fibrinopeptide-A during incubation with thrombin, was found to be identical for HMW and LMW. Polymerization was studied by light scattering (at 605 nm) using preformed monomers (des-AA and des-AABB) prepared from HMW and LMW in the presence of 3.3 M urea by incubation with thrombin (100 NIH U/ml final conc.) and reptilase (1 U/ml final conc.). The HMW-monomers polymerised at a substantially higher rate than the corresponding LMW-monomers. Thus, the prolonged clotting time of LMW was explained by retarded polymerization. It is suggested that the -COOH terminal end of the a-chain, containing the molecular difference between HMW and LMW, is of importance for polymerization. Furthermore, the release of fibrinopeptide B (des-AABB-monomers) improved polymerization properties in HMW as well as in LMW, and all types of monomers polymerised more rapidly in the presence of Ca++.

Fibrin↗
PubMed1997

Free radical induced polymerization of synthetic peptides into polymeric immunogens.

Free radical induced polymerization of vinyl monomers such as the acryloyl peptides described here is a facile and rapid reaction used routinely, for example, in the polymerization of acrylamide and bisacrylamide for the assembly of polyacrylamide gels. The technology allows the incorporation of many of the same or different peptide determinants into a single polymer chain. In this study large polymers containing multiple copies of peptides representing T- and B-cell determinants of influenza haemagglutinin were constructed. The determinants retained antigenicity after the polymerization procedure and the polymers were highly immunogenic; the levels of antibody obtained after a single dose of polymeric immunogen were at least as great as those achieved only after repeated doses of the equivalent monomeric peptide. The technology has a wide range of potential applications, not the least significant of which is the construction of designer immunogens for third generation vaccine candidates.

Amino Acid Sequence↗
PubMed1997

Localization of an effective fibrin beta-chain polymerization site: implications for the polymerization mechanism.

To examine whether fibrin N-terminal Aalpha 17-23 and Bbeta 15-25 may contain high-affinity polymerization sites, GPRVVER and GHRPLDKKREE analogs were prepared, and their abilities to inhibit fibrin monomers from repolymerizing were compared in turbidity and clottability assays. Within Aalpha 17-23, GPR is the most active site (IC30 of 0.95-1.36 mM). Its extension into GPRVVER (IC30 of 1.75-2.3 mM) reduced activity. Within Bbeta 15-25, acyl-DKKREE (IC30 of 0.30-0.53 mM) can account for GHRPLDKKREE activity (IC30 of 0. 33-0.44 mM). Comparison of the assays showed that calcium, whose presence induces thick fibrin fibers, elicited a higher turbidity than clottability inhibition. Similarly, the lateral-association-promoting GHRP (IC30 of 1.25-1.43 mM) gave a high turbidity vs clottability inhibition ratio (137%). In contrast, low ratios were found for the linear-association-initiating GPR (73%) and for acyl-DKKREE (34%). Structure-activity correlation showed that fibrinogen-like acyl-GPRP and acyl-GHRP could inhibit D. E association at the millimolar range, but in a manner different from fibrin-related GPR peptides did, which required the NH2 as well as Arg presence. To explain Bbeta 20-25 masking, it is proposed that DKKREE in fibrinogen may engage in ionic and hydrogen bonds with KDSDW, the Aalpha 29-33 sequence implicated in thrombin binding. To explain acyl-GPRP and acyl-GHRP inhibition of D.E association, it is proposed that fibrinogen packing may be mediated by E domain association with alphaC (Aalpha 220-609) fragments of adjacent molecules, and by alphaC-alphaC association. A modified polymerization mechanism is deduced by taking into account fibrinogen N-terminal conformation as well as E domain binding to thrombin vs alphaC fragments. This model proposes the following. (1) Upon thrombin binding to fibrinogen KDSDW, DKKREE may become exposed. (2) Fibrinopeptide A cleavage further unmasks the NH2 and Arg group of GPR, leading to DKKREE and GPR initiation of polymerization. (3) The micromolar-effective thrombin-fibrin(ogen) binding may initiate a partial alphaC repulsion. Subsequent DKKREE and GPR binding to D domains of other fibrin(ogen) will lead to the formation of the trimer and bring additional molecules to fibrin N-terminal region, and the combined steric congestion may lead to a complete alphaC repulsion from the overcrowded E domain. (4) Repulsion of the large Aalpha 220-609 fragments may unmask multiple polymerization sites beyond the fibrin N-terminal region.

Amino Acid Sequence↗
PubMed1992

Synthesis of alkoxy-substituted diaryl compounds and correlation of ring separation with inhibition of tubulin polymerization: differential enhancement of inhibitory effects under suboptimal polymerization reaction conditions.

A number of cytostatic compounds (2-4, 7, and 8), which can be described as "diaryl", inhibit tubulin polymerization, cause cells to accumulate in mitotic arrest, and competitively inhibit the binding of colchicine to tubulin. They differ, however, in the separation of the two aryl moieties. To attempt to understand this variability we prepared a series of analogues modeled on 3 and 4 ("benzodioxole series") and on 7 and 8 ("combretastatin series") which differed only in the number of methylene units (ranging from none to four) separating the aryl moieties. These compounds were evaluated for their effects on tubulin polymerization, colchicine binding, and the growth of L1210 murine leukemia cells. In terms of inhibitory effects on tubulin polymerization, for the combretastatin series there was an optimal separation of the two phenyl rings by a two-carbon bridge (compound 24), with progressively decreasing inhibitory activity when the separation was by one carbon (20), three carbons (25), or four carbons (28) (the biphenyl analogue 16 was inactive). The benzodioxole series, however, did not permit us to generalize this finding, because the least active agents prepared (39 and 40) had a two-carbon bridge, while those with one- (5 and 6) and three-carbon (46 and 47) bridges were nearly equivalent in potency. Submicromolar IC50 values for inhibition of L1210 cell growth were only obtained for compounds 20 (IC50, 0.2 microM), 24 (0.07 microM), and 25 (0.4 microM). While evaluating the effects of these agents on tubulin polymerization, we noted with the combretastatin series and with several standard agents that apparent potency (in terms of IC50 values) was always lower if the reaction was performed at 30 degrees C, with 0.25 mM MgCl2, than at 37 degrees C, with 1.0 mM MgCl2. This enhancement of IC50 values in the former system as compared with the latter was particularly dramatic for the less active agents (e.g., 28) as compared with the more active (e.g. 24).

Animals↗
PubMed1998

Anionic Polymerization of 1,3-Cyclohexadiene with Alkyllithium/Amine Systems. Characteristics of n-Butyllithium/N,N,N',N'-Tetramethylethylenediamine System for Living Anionic Polymerization.

The n-butyllithium (n-BuLi)/N,N,N',N'-tetramethylethylenediamine (TMEDA) system (with the molar ratio of TMEDA to n-BuLi higher than 4/4) has been found to polymerize 1,3-cyclohexadiene to produce "living" polymer having a narrow molecular weight distribution with well-controlled polymer chain length. The rate of polymerization and polymer yield increased with increasing of the ratio of TMEDA to n-BuLi. The molecular weight distribution of obtained polymers became narrower with the ratio of TMEDA to n-BuLi. The formation of benzene generated by termination reaction was found to decrease with the ratio of TMEDA to n-BuLi. In contrast to 1,3-cyclohexadiene, low yield and broad molecular weight distribution were observed in the polymerization of 1,3-hexadiene and 2,4-hexadiene initiated by the n-BuLi/TMEDA (4/5) system. The microstructure of poly(1,3-cyclohexadiene) was determined by 2D-NMR. A high content of 1,2-units was found in the polymerization initiated by the alkyllithium/TMEDA system, while the high content of 1,4-units was observed in the cases of alkyllithium or alkyllithium/1,4-diazabicyclo[2,2,2]octane system. The 7Li NMR signals of the n-BuLi/TMEDA systems and poly(1,3-cyclohexadienyl)lithium /TMEDA were in higher magnetic field than the signals of n-BuLi and poly(1,3-cyclohexadienyl)lithium, respectively, indicating the disaggregation of the lithium species.

Journal Article↗
PubMed1997

Effect of composite type, light intensity, configuration factor and laser polymerization on polymerization contraction forces.

PURPOSE: To investigate the effect of composite type, light intensity, configuration factor and laser polymerization on polymerization contraction force. MATERIALS AND METHODS: Glass rods (10 pairs/group) were etched with HF acid, silanated, unfilled resin applied and light cured for 20 s. Rods were held vertically in chucks on a Zwick machine. A cylindrical matrix was filled with Silar chemical cure, Silux Plus microfill or Z-100 hybrid composite and the crosshead of the UTM positioned at an inter-rod distance corresponding to a specific ratio of bound to unbound composite surface area (configuration factor or C). Exposure time with the Demetron 401 conventional visible light curing unit (D401) was 40 s/side (80 s total). Exposure times for the ILT Model D5500 air cooled laser (LAC) and Model 5500ABL water cooled laser (LWC) was 20 s/side (40 s total). Experimental groups, n = 10 with constant factors in parentheses, included: (1) Silar chemical-cured (C = 3); (2) Z-100 hybrid (C = 3, D401, 100% intensity); (3) Silux Plus microfill (C = 3, D401, 100% intensity); (4) D401 100% light intensity = 476 mW (Z-100, C = 3, D401); (5) D401 50% intensity = 238 mW (Z-100, C = 3, D401); (6) D401 25% intensity = 119 mW (Z-100, C = 3, D401); (7-9) C = 5, 3 & 1 respectively (Z-100, D401, 100% intensity); (10) D401 with 13 mm tip = 391 mW/cm2 (Z-100, C = 3; D401); (11) D401 with Turbo Tip = 811 mW/cm2 (Z-100, C = 3; D401); (12) LAC = 265 mW, 689 mW/cm2 (Z-100, C = 3); (13) LWC = 365 mW, 1100 mW/cm2 (Z-100, C = 3). One Way ANOVA and Duncan's Multiple Range Test (alpha = 0.05) were performed separately for each variable. RESULTS: Homogeneous subsets by variable were: composite type Group 1 (25N) < Group 3 (65.8N) < Group 2 (90.4N); intensity Group 6 (73.9N) = Group 5 (77.7N) < Group 4 (90.4N); C-Factor Group 7 (81.8N) < Group 8 (90.4N) < Group 9 (103.4N); light source Group 12 (77.4N) = Group 13 (79.1N) < Group 10 (90.4N) = Group 11.(89.4N). The chemical-cured composite had the lowest maximum polymerization contraction force, the microfill was intermediate and the hybrid composite had the highest recorded force. Increases in light intensity increased the maximum force on the force/time curve. Maximum forces were inversely related to C-factor (C5 < C3 < C1) and directly related to composite volume in a non-rigid system which allowed compliance. Maximum force was not significantly different with the two tips tested on the conventional curing light. Forces obtained with laser polymerization were similar for the two laser groups, which were both statistically lower than the conventional light tested.

Analysis of Variance↗
PubMed1998

Antibody against the human J chain inhibits polymeric Ig receptor-mediated biliary and epithelial transport of human polymeric IgA.

To emphasize the requirement for a J chain in native polymeric immunoglobulins for their selective transport into exocrine secretions, IgG, purified from two different antisera specific for the human J chain, was shown to: (i) bind in vitro to human polymeric IgA (pIgA) by density gradient ultracentrifugation; (ii) inhibit binding in vitro of rat secretory component to human pIgA; (iii) inhibit hepatic transport of human pIgA into rat bile in vivo; and (iv) inhibit apical transcytosis of pIgA in vitro by polarized human polymeric immunoglobulin receptor (pIgR)-expressing Madin-Darby canine kidney cells. Inhibition of biliary transport increased with the molar ratio of anti-J chain antibodies against pIgA and their incubation time. Anti-J chain F(ab')2 and Fab fragments also inhibited biliary transport, excluding a role for phagocytic clearance or excessive size of the immune complexes. Anti-human-Fc alpha Fab, bound to human pIgA in complexes of larger size than those with anti-J chain Fab, did not inhibit biliary transport of human pIgA. Propionic acid-denatured human pIgA, although containing J chains, was very poorly transported into rat bile. Altogether, our data strongly support, now also by in vivo experiments, the crucial role of the J chain of native pIgA in its selective pIgR-mediated transport into secretions, as suggested long ago by in vitro data only. Recent data on J chain-knockout mice, with low IgA levels in bile and feces, cannot explain the role of the J chain in contributing to the secretory component/pIgR-binding site of normal pIgA, but otherwise agree with our study.

Animals↗
PubMed2000

A new assay system for detection of polymeric immunoglobulin A-polymeric immunoglobulin receptor binding.

A new system for detection of polymeric immunoglobulin A (pIgA)-polymeric immunoglobulin receptor (pIgR) binding was established. Cell lysates of a mouse pIgR cDNA transfectant, 2S9.1, were incubated with mouse polymeric immunoglobulin A (pIgA) or immunoglobulin G (IgG). The resulting immunocomplexes were precipitated with protein G-Sepharose and blotted with polyclonal anti-mouse pIgR antibody. The mouse pIgR molecule was specifically precipitated with pIgA, indicating successful detection of the pIgA-pIgR complex. Using this system, the role of N-glycosylation in pIgA-pIgR binding was examined. The pIgR molecule (molecular mass 100 kDa) after complete deglycosylation by tunicamycin treatment was still able to bind to pIgA, indicating that N-glycosylation of pIgR is not necessary for pIgA-pIgR binding. This novel system will be useful for detecting pIgA-pIgR complexes containing intact pIgR molecules.

Animals↗