Reduction Of Disulphide Bonds Research Articles

Disulphide bonds in wheat gluten: cystine peptides derived from gluten proteins following peptic and thermolytic digestion.

Gluten from the wheat variety Rektor was extracted with 70% aqueous ethanol. The insoluble portion (whole glutenin) was partially hydrolysed with trypsin at pH 6.5 and separated on a Sephadex G25 column. The high molecular weight fraction 1 was further hydrolysed with pepsin at pH 2.0. To remove low molecular weight proteins, a portion of whole glutenin was extracted with dilute acetic acid. The residue (enriched glutenin), which contained mostly LMW and HMW subunits of glutenin, was hydrolysed with thermolysin at pH 6.5. The peptic and tryptic hydrolysates were separated on a Sephadex G25 column and the peptide fractions with the highest cystine content were separated further by reversed-phase high-performance liquid chromatography (RP-HPLC). Cystine peptides were detected by differential chromatography (RP-HPLC prior to and after reduction of disulphide bonds) and then isolated by preparative RP-HPLC. After reduction, cysteine peptides were alkylated and analysed for their amino acid sequence. Altogether, 19 cystine peptides were characterized and assigned to known sequences of gluten proteins; 16 peptides confirmed the positions of disulphide bonds present in LMW subunits and gamma-gliadins, as described previously. For the first time, a cystine peptide has been isolated, representing an intermolecular disulphide bond between the y-type of HMW and LMW subunits. Furthermore, a cystine peptide was assigned to gamma-gliadins; thus, all cysteine residues of gamma-gliadins are documented by at least one cystine peptide. One peptide analysed came from the alpha-amylase inhibitor CM 16. Altogether the results indicate that the intramolecular linkages of gluten proteins are not formed at random, but are strongly directed.(ABSTRACT TRUNCATED AT 250 WORDS)

Organization of the major and minor capsid proteins in human papillomavirus type 33 virus-like particles

The organization of the major (L1) and minor (L2) proteins in the human papillomavirus capsid is still largely unknown. In this study we analysed the disulphide bonding between L1 proteins and the association of L2 proteins with capsomers using virus-like particles obtained in insect cells by co-expression of the L1 and L2 genes of human papillomavirus type 33. About 50% of the L1 protein molecules in these particles (1.29 g/cm3) formed disulphide-bonded trimers. Reduction of the intermolecular disulphide bonds by dithiotreitol (DTT) treatment caused disassembly of virus-like particles into capsomers. This indicates that disulphide bonds between capsomers at the threefold symmetry positions of the capsid are essential for the assembly of the papillomavirus capsid. In contrast, the L2 protein was not engaged in intermolecular disulphide bonding. The L2 protein remained associated with capsomers on disassembly by treatment with DTT. When the disassembly was carried out in 0.65 M-NaCl, complete L2 protein molecules bound preferentially to capsomer oligomers, whereas truncated L2 protein molecules bound only to monomers. In 0.15 M-NaCl only complete L2 protein molecules remained bound to capsomers. This indicates that different regions of the L2 protein molecule are differentially involved in the association of the papillomavirus capsid.

The location of disulphide bonds in α-type gliadins

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two α-type gliadins (α3- and α8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from α3-gliadin and three cystine peptides from α8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both α-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from α8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of α-type gliadins described in the literature, the cystine peptides from α3- and α8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively 11. The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of α-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined for low M r subunits of glutenin and glutenin-bound γ-type gliadins 6. Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed.

Biochemical and topological analysis of adult T-cell leukaemia-derived factor, homologous to thioredoxin, in the pregnant human uterus.

Adult T-cell leukaemia-derived factor (ADF), homologous to thioredoxin, displays various biological activities, such as radical scavenging action and the reduction of protein disulphide bonds. We examined the biochemical and immunohistochemical localization of ADF in the pregnant human uterus, using two heteroantibodies to ADF, antibody C and W. Immunohistochemically, decidua and trophoblast cells were intensely stained by antibody C. The concentration of ADF-like substance in the decidua was 95.9 ng/mg protein, determined by enzyme-linked immunosorbent assay. The molecular weight of ADF-like substance in these tissues was determined by gel electrophoresis to be 13 kDa, the same as that of recombinant ADF. These findings indicate that abundant ADF is present in decidua and trophoblast cells; the localization of such a potent dithiol reducing substance may be beneficial in protecting the fertilized egg and placental trophoblasts from the cytotoxic effects of oxygen radicals.

The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi.

Dithiothreitol (DTT) blocks the endoplasmic reticulum (ER)-Golgi transport of newly synthesized immunoglobulin (Ig) molecules, whereas 2-mercaptoethanol (2ME) allows secretion of unpolymerized Igs otherwise retained intracellularly by disulphide interchange reactions. To understand this dichotomy, we have compared the effects of DTT and 2ME on the assembly, intracellular transport, and secretion of a panel of chimeric Igs that are either constitutively secreted or retained intracellularly. Our results demonstrate that DTT, but not 2ME, reduces some of the inter- and intrachain disulphide bonds and causes partial disassembly of H2L2 complexes and unfolding of individual chains in the ER. Upon DTT removal, heavy (H) and light (L) chains reform hapten-binding H2L2 molecules, which are later secreted. Reduction of the H2L2 interchain disulphide bonds can occur along the entire secretory pathway; however, in or beyond the Golgi this does not result in efficient H-L disassembly or unfolding. As a consequence, DTT does not block the exit from the Golgi. Moreover, unpolymerized Igs--normally retained in a pre-Golgi compartment--no longer require reducing agents to be secreted once they have reached the Golgi. Thus, little if any thiol-mediated retention seems to take place in or beyond the Golgi complex.

Cysteine-524 is not the only residue involved in the formation of disulphide-bonded dimers of the insulin receptor.

The human insulin receptor (hIR) is a member of the transmembrane tyrosine kinase receptor family. It is a disulphide-linked homodimer which can be reduced to two insulin-binding monomers by mild reduction of class-I disulphide bonds. The number of disulphide bonds between the alpha- and beta-chains within the monomer or between the monomers in the dimer is not known, although one dimer bond involving hIR Cys-524 has recently been identified [Schaffer and Ljungqvist (1992) Biochem. Biophys. Res. Commun. 189, 650-653]. In the present report hIR Cys-524 was converted into alanine by site-directed mutagenesis and expressed at high levels in Chinese hamster ovary (CHO) cells. The mutant receptor was processed normally and shown to bind insulin normally, with ED50 and KD values not different from those of the wild-type hIR. It was still a disulphide-linked dimer as judged by SDS/PAGE, indicating that there are alpha-alpha-chain disulphide bonds additional to the Cys-524 linkage in the insulin receptor dimer. Insulin-stimulated receptor autophosphorylation and kinase activity of the mutated receptor were both impaired compared with that of the wild-type receptor by 49% and 53% respectively. CHO cells overexpressing the mutant receptor, however, did not show a reduced capacity to stimulate glucose utilization, indicative that the level of receptor expression was sufficient to saturate downstream insulin action. These findings indicate that alpha-alpha disulphides additional to that provided by Cys-524 hold the receptor dimer together and that mutagenesis of Cys-524 reduces the ability of the receptor to signal insulin action subsequent to hormone binding.

Surface Properties and Emulsification Behavior of Denatured Soy Proteins

ABSTRACTSoy protein isolate was physically (heat pretreatment) and chemically (urea, guanidine·HCl, and cleavage of SS bonds) modified in order to dissociate subunits, unfold the protein and improve surface properties: hydrophobicity, emulsification capability, and stability. Heat pretreatment as well as chemical treatment with urea or guanidine·HCl or reduction of disulphide bonds, will improve emulsification performance in comparison to native soy protein isolate. Significant differences in reduction of surface tension of water in the presence of native and modified proteins were observed (45 and 35 dynes/cm respectively). Measurements of fluorescence indicated that the relative hydrophobicity of the soy protein was also improved (from 600 to 1360) after heat pretreatment or contacting the soy protein with 8M urea solution.

Collective isolation of two major, close molecular weight coagulum proteins occurring in human semen before liquefaction.

Two major, close molecular weight basic coagulum proteins of freshly ejaculated human semen identifiable on reducing SDS-PAGE, have been isolated together from washed seminal coagulum. The latter was solubilized in urea, followed by reduction and carboxymethylation of disulphide bonds. By high-resolution chromatography on carboxymethy-Sephadex and Sephacryl S-300 using 8 M urea in 0.1M Tris-HCl (pH 7.4) as eluate these proteins were isolated together (purity 99%) with a yield of 10.5%. The molecular weights of the isolated proteins on SDS-PAGE were 75 and 79 kD.

Isolation of the predominant coagulum protein of human semen before liquefaction

The predominant protein of freshly ejaculated human semen and seminal coagulum in reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis was purified for the first time prior to cleavage of coagulum proteins during liquefaction. The method involves three steps using semen immediately after ejaculation. Washed seminal coagulum was first collected from fresh semen samples in acetate buffer at pH 4.5 at 0 degrees C, solubilized immediately in deionized urea followed by reduction and carboxymethylation of disulphide bonds among the aggregating coagulum proteins. Then using cation exchange (CM-Sephadex) chromatography, eluted with stepwise ionic strength gradient and finally Sephacryl S-300 high resolution chromatography in the presence of urea, the predominant coagulum protein was highly purified (99%) with a yield of 6.3%. The mol. wt of the purified protein was 57,000 Da. At physiological pH, the protein is rich in positive charge.

Biochemical characterization of air-filled albumin microspheres.

Heating and sonication of a solution of human serum albumin (HSA) yields air-filled microspheres that can be used as a contrast agent in ultrasound examinations. The microspheres are stabilized by a thin-layer of protein surrounding the air bubbles. As long as the microspheres were intact, the protein was insoluble in aqueous solutions. After disintegration of the microspheres, the protein could be solubilized in several solutions. The intermolecular interactions in the microsphere protein have been elucidated from its solubility properties. The microspheres were disintegrated by several detergents which also solubilized the protein. After pressure disintegration of the microspheres, the protein was solubilized immediately in urea and guanidinium chloride, and also in phosphate-buffered saline after incubation overnight. These results indicate that the protein was mainly stabilized by non-covalent forces. The solubilization in buffer was inhibited by a high salt concentration, suggesting that hydrophobic interactions were involved in stabilizing the microsphere structure. Analysis of the solubilized protein by gel filtration showed that the protein contained substantial amounts of soluble aggregates of HSA. Reduction of the disulphide bonds dissolved these aggregates into monomeric HSA, showing that intermolecular disulphide bonds were also involved in stabilization of the microspheres. The solubilized protein also contained less fatty acids than the soluble HSA used for the production of microspheres. These results show that the microsphere protein has the same characteristics as heat-denatured HSA.

Protein-protein interactions controlling acrosin release and solubilization during the boar sperm acrosome reaction.

In this study we used previously characterized monoclonal antibodies to acrosin (ACR.2) and to an acrosomal matrix antigen (ACR.4) to analyze the acrosin-binding activity of a 28-kDa putative acrosin-binding protein from the acrosomal matrix. The 28-kDa protein bound proacrosin and the 49-kDa form of acrosin (alpha-acrosin) but it did not bind the 36-kDa acrosin form (beta-acrosin). The acrosin-binding activity of the 28-kDa protein was stimulated by Ca2+, inhibited by Mg2+, and removed by disulphide bond reduction. Induction of the acrosome reaction by a calcium ionophore resulted in proteolytic cleavage of the 28-kDa protein, giving rise to a 12-kDa degradation product that was the only form of ACR.4 antigen released to incubation media; the release of the ACR.4 antigen was closely correlated with that of acrosin. The release of alpha-acrosin to incubation media was accelerated in the presence of ACR.4 antibody. In a cell-free system, a limited cleavage of the purified 28-kDa protein into immunoreactive degradation products was catalyzed by acrosin but not by trypsin or chymotrypsin. The data suggest that the 28-kDa acrosomal protein helps to maintain acrosomal matrix integrity and controls the acrosin release from acrosome-reacted cells.

Biochemical characterization of a rat oncofetal colonic antigen defined by a monoclonal antibody raised against gastric surface epithelium

The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.

Metachromatic staining of human sperm nuclei after reduction of disulphide bonds

Intra- and interprotamine cross-linking by disulphide bonds are the main factors responsible for the highly compact and stable structure of chromatin in mammalian spermatozoa. Unfixed or methanol fixed smears of human sperm and sperm suspensions from fertile donors and oligospermic patients were subjected to a reductive cleavage of disulphide bonds by using 2-mercaptoethanol (ME) or dithiothreitol (DTT). Untreated (control) and ME or DTT treated samples were stained with toluidine blue (TB) and examined in light microscopy; spectral characteristics of TB stained sperm nuclei were also analyzed. Untreated smears from fertile donors showed an orthochromatic (pale blue) staining of most sperm heads, while a variable proportion of metachromatic nuclei was found in spermatozoa from patients with oligospermia. After treatment with DTT followed by TB staining, fixed and unfixed smears showed metachromatic sperm heads. ME treatment only induced a scarce colour shift, whereas a striking metachromatic reaction and variable nuclear swelling were observed in DTT treated sperm suspensions. These results indicate that after cleavage of disulphide bonds, phosphate groups from chromatin DNA are unmasked and available for TB binding and metachromatic staining.

Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity.

Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.

Pseudomonas exotoxin: recombinant conjugates as therapeutic agents.

Pseudomonas exotoxin: structure and function Pseudornonas exotoxin (PE) is a bacterial protein produced by €3. aeruginosa that is toxic for most mammalian cells [for reviews see 1, 21. The mature protein is a proenzyme and, after activation, exhibits both NAD hydrolase activity and ADP-ribosyltransferase activity. When added to mammalian cells the toxin binds to the alpha 2-macroglobulin receptor which mediates internalization to the endosoma1 compartment [ 31. In the endosome the toxin is cleaved (‘nicked’) by a cellular protease. Disulphide bond reduction then results in the release of an enzymatically active toxin fragment which translocates to the cytosol where it inhibits protein synthesis. In the cytosol; PE hydrolyses NAD and transfers the ADP-ribose moiety of NAD to elongation factor 2 (EF-2). When EF-2 is modified in this way it can no longer participate in the synthesis of new protein. The inactivation of EF-2 is lethal for the cell. Structural analysis of PE crystals by McKay and colleagues [4] revealed three prominent domains. These were termed domains I, 11, and 111: domain I was further divided into domains Ia and Ib. This study was followed first by a deletion analysis of the structural gene [5] and then by many individual studies focused on determining the location of important sequences within each domain. The results from all these studies has revealed that domain Ia (amino acids 1-252) is responsible for the toxin binding to its surface receptor; domain I1 (amino acids 253-364) is the substrate for the cellular protease and, after cleavage, has translocating activity; domain Ib (amino acids 365-399) has no known function and most of it can be deleted without loss of toxin activity; and domain I11 (amino acids 400-61 3) has the ADP-ribosylating activity and contains an endoplasmic retention sequence. A closer look at the interactions of PE with cells has revealed that a lysine at position 57 in domain I is needed for cell binding [6]. Changing this basic residue to an acidic residue such as glutamic acid or inserting a dipeptide reduces binding by 100-fold. After binding the toxin is

Anomalies in reduction-mediated technetium-99m labelling of monoclonal antibodies

A reduction-mediated technetium-99m labelling method has been evaluated with a range of tumour-specific monoclonal antibodies. Antibodies reduced with 2-mercaptoethanol (2-ME) had free sulphydryl groups, but their number was much higher than could be accounted for by only limited intra-chain reduction of disulphide bonds. Reduced antibody could be labelled efficiently with 99mTc using an methylene diphosphonate (MDP) bone-scanning kit, although this seemed to depend on the presence of residual 2-ME in the preparations. With a carcinoembryonic antigen (CEA)-specific antibody, immunoreactivity of labelled antibody was confirmed, and after injection into nude with CEA-producing xenografts there was localisation into the tumours. Sephacryl S300 gel filtration showed the radiolabel eluting at a single discrete peak at the expected 150 kDa. However, examination of all of the labelled antibodies by polyacrylamide gel electrophoresis (PAGE) and autoradiography showed the presence of a large number of radiolabelled low molecular weight degradation products of the antibodies. These degradation products seemed to be formed in previously reduced antibodies during processing for PAGE, indicating some fragility of the reduced antibody.

Biochemical and Immunological Analysis of Discontinuous Epitopes in the Family of Human Cytomegalovirus Glycoprotein Complexes Designated gC-I

The envelope of human cytomegalovirus contains a family of disulphide-linked glycoprotein complexes designated gC-I which contain two glycoproteins of 52,000 Mr (gp52) and 93,000 to 130,000 Mr (gp93-130). Epitopes recognized by several of our gC-I gp52-specific monoclonal antibodies (MAbs) were previously assigned to three domains based on reactivity with gC-I in a competitive binding assay. In this report, we have used additional gC-I MAbs to characterize three distinct discontinuous epitopes in the gC-I complexes. Two of these epitopes were in Domain I and one in Domain III. These epitopes were resistant to proteolysis, heat denaturation and SDS treatment. However, the discontinuous epitopes were lost after reduction of disulphide bonds. After digestion of gC-I complexes with chymotrypsin, two fragments of 43,000 (43K) Mr and 34,000 (34K) Mr were obtained which contained all discontinuous and continuous epitopes recognized by our gp52 MAbs. The Mr of these fragments could not be reduced further by longer digestion or by use of other proteases such as trypsin or pronase. The 43K fragment contained N-linked oligosaccharides not detected in the 34K fragment. These oligosaccharides may have prevented a complete proteolytic digestion so that the 34K fragment was not always obtained. It was established that 80 to 90% of the mass of these fragments was contributed by gp52. Thus the discontinuous epitopes were composed primarily of gp52 and not gp93-130.

Role of disulphide bonds in burst‐like activity of nicotinic acetylcholine receptor channels in rat sympathetic neurones.

1. The effects of reduction of disulphide bonds in nicotinic acetylcholine receptors (nicotinic AChRs) with dithiothreitol (DTT) were studied in rat superior cervical ganglion neurones using the patch-clamp method in whole-cell and cell-attached recording modes. 2. Dithiothreitol (1 mM) markedly reduced the ACh-induced membrane current, while the action of ACh remained reversible. Conversely, bromoacetylcholine (BrACh), if applied after the treatment with DTT, caused irreversible activation of nicotinic AChRs manifested in the appearance of a non-declined steady-state component in BrACh-induced currents accompanied by increased membrane current fluctuations. The successive reoxidation of sulphydryl groups by potassium ferricyanide (1 mM-ferricyanide) restored the response to ACh. Ferricyanide itself had a weaker inhibitory effect on the ACh-induced current, compared to the effect of DTT. 3. As a result of the action of DTT (1 mM), the spectrum of BrACh-induced current noise shifted to a higher frequency range. 4. The distributions of durations of the gaps (closed states) and the bursts (the states identified as open states after the shortest gaps were ignored) in single-channel activity of native (non-treated with DTT) nicotinic AChRs caused by ACh (30 microM) and BrACh (30 microM) were similar and both revealed four to five and two to three components for gap intervals and burst durations respectively. 5. Single-channel behaviour of reduced nicotinic AChRs was similar for both ACh and BrACh as agonists, but significantly differed from that in the native one. The first difference was the marked increase in the frequency of the appearance of long closed states of the channel that was presumably due to enhanced receptor desensitization. The second difference was an almost complete disappearance of long bursts associated with disappearance of the fastest component in gap interval distribution. 6. Mean conductance of single nicotinic AChR channels decreased by approximately 20% in the reduced receptor compared with that in the native one, for both agonists. 7. The results suggest a critical role of disulphide bonds for the functioning of native neuronal nicotinic AChRs: the disruption of disulphide bonds leads to the loss of burst-like kinetics of the nicotinic AChR ionic channel.

Isolation and characterization of a monoclonal rheumatoid factor specific for the hinge region of rat IgG2b

The isolation and characterization of an isotype-specific autoantibody-secreting hybridoma NET/2/3 from rats bearing the syngeneic tumour HSN is described. This rheumatoid factor of the IgM class recognizes an epitope within the hinge region of rat immunoglobulins of the IgG2b subclass which is destroyed by reduction of disulphide bonds. The specificity of NET/2/3, although not allotype-restricted, is highly isotype-restricted, as it does not bind to rat Ig other than IgG2b, nor does it react with the majority of mouse IgG, although some reactivity occurs with mouse IgG3. One remarkable feature of NET/2/3 is that it binds more strongly to F(ab') 2 and Fab' fragments of rat IgG2b, obtained by digestion with pepsin, than to the whole molecule. This anti-isotype response is not peculiar to the HSN tumour model as NET/2/3-like antibodies have been found in the sera of rats immunized with various protein and cellular antigens. The possible biological role of this anti-isotype antibody is discussed.

Macromolecular properties and polymeric structure of canine tracheal mucins

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.