Structural difference between the two forms of guinea-pig beta 2-microglobulin and their occurrence in inbred guinea-pig strains

The structural difference between two forms (basic and acidic) of guinea-pig beta 2-microglobulin (beta 2m) has been established. Both forms are present in urine from inbred guinea-pig strains. The beta 2m forms were each digested with carboxypeptidase Y and carboxypeptidase A contaminated with carboxypeptidase B. Released amino acids were separated from remaining protein, dansylated and analysed by 2-dimensional TLC on polyamide layer sheets. From the results it was concluded that the basic beta 2m form has lysine and the acidic beta 2m form has asparagine as their respective C-terminal amino acids. The acidic form is also 1 amino acid (lysine) shorter than the basic form, which is supported by electrophoretic studies on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the 2 forms of beta 2m in urine from inbred guinea-pig strains 2 and 13, shown by gel filtration and ion exchange chromatography, makes it unlikely that the 2 forms are a result of genetic polymorphism.     

Distribution of the two forms of guinea-pig β2-microglobulin originally detected in urine

Two distinct forms of β₂-microglubin (β₂m) have been detected in pooled guinea-pig serum from outbred animals using ultrafiltration, gel filtration and ion exchange chromatography. These two forms probably correspond to the two β₂m forms, with different electrophoretic mobility, that have been previously identified in guinea-pig urine. In solubilized membranes from perfused guinea-pig livers only the basic form of β₂m could be found. These results may indicate that the two β₂m forms should not be ascribed to genetic polymorphism but rather are a result of post-translational modification.     

The molecules recognized by anti-MT2 alloantisera and anti-MT2-like (DRw52) monoclonal antibodies are different

The human class II alloantigens include the HLA-DR, DQ, and MT determinants. Previous reports in the literature suggest that while the DQ determinants appear to be on a molecule separate from DR, the MT determinants are variably present on DR or DQ molecules. We have previously reported, using the homozygous DR5 cell line Swei, that the MT4 determinant defined by the allosera, MGH88B, was only on the DQw3 molecule, while MT2, defined by the functionally monospecific anti-MT2 alloantiserum MGH87B, was present on both the DR5 and DQw3 molecules. We now report using the monoclonal antibody ILR2 directed against an MT2-like determinant DRw52, that DRw52 is present on the DR molecules only. The MT2 determinant(s) recognized by the functionally monospecific alloantisera MGH87B appear to include the DRw52 determinant(s) recognized by the monoclonal antibody ILR2.     

Proteolysis of red cell membrane proteins by immunoglobulin G preparations

Red cell integral membrane proteins obtained by mild alkaline extraction were degraded by isolated IgG preparations. Erythrocyte or ghost membranes were not significantly affected under comparable conditions. The IgG appeared pure following ¹²⁵I-labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis by both densitometric scanning and radioelectrophoretic profiling. In addition, no proteolytic activity was detectable with azocoll under standard conditions. Despite this, the integral protein fraction, which consisted predominantly of band 3 and the major sialoglycoproteins, showed extensive degradation following incubation at 37°C for 1.5 hr with the IgG. The process was partially inhibitable with phenylmethylsulfonyl fluoride and totally inhibited by Trasylol. Proteolysis of band 3 led to the formation of fragments retained in the membranes of apparent mol. wt of 65,000 and lower fragments appearing in the 4.5 region. Band 7 present in the starting membrane fraction appeared resistant to degradation.The problems when working with purified membrane fractions are illustrated by data showing the IgG-binding membrane components that are obtained with IgG preparations having different levels of protease activity.Proteolytic susceptibility appears to be a general characteristic of isolated membrane proteins or receptors. UnIike soluble proteins which are resistant to proteolysis in the native state, enhanced protease degradation may be inherent in isolated membrane proteins even when obtained under mild non-denaturing conditions. Protease-sensitive sites on membrane proteins or receptors which in native membranes are protected by protein-protein or protein-lipid interactions probably become protease-vulnerable following isolation and purification.     

Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization

The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.     

Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. alpha-Bungarotoxin purified from this venom failed to block transmission at 50 micrograms/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1-3 micrograms/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from alpha-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by alpha-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [125I]toxin F bound to the shared site and had no effect on [125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.     

Interaction of C3 and C3b with immunoglobulin G

Human C3b as well as native C3 were found to bind to solid phase human and rabbit IgG. Haemolytically active C3 had significantly higher binding capacity to IgG than the C3b fragment. Inhibition experiments proved that C3 and C3b have common binding sites on the Fab and Fc part of the IgG molecule but the character of these binding sites was different. As a functional consequence of C3-IgG interaction, C3 binding was found to inhibit the specific precipitation of an IgG antibody preparation.     

Insolubilized anithyroglobulin antibodies: properties and interest in the study of thyroglobulin

Antithyroglobulin antibodies have been insolubilized, either through polymerization of the immune serum by ethyl chloroformate or through covalent binding to cyanogen bromide activated Sepharose 2B. These immunosorbents are able to bind thyroglobulin specifically, however, they have lost a part of the immune serum antibody activity. Thyroglobulin immunosorption is reversible. The degree of reversibility increases respectively with a pH 2 buffer, 3 M NaSCN and 0.1% sodium dodecylsulfate. After desorption by the first two procedures, thyroglobulin remains entirely precipitable by homologous antibodies, however, it undergoes conformational changes, some of them being partially reversible (unfolding of the 19S species, dissociation into 12S subunits). These immunosorbents may be used, under the conditions described, in the quantitative estimation and purification of thyroglobulin.     

Multiple non-repeated epitopes on the circumsporozoite protein of Plasmodium knowlesi

The Plasmodium knowlesi circumsporozoite (CS) protein contains a repetitive immunodominant epitope. Here we show that the serum of rabbits repeatedly immunized with P. knowlesi sporozoites contains antibodies which bind to immobilized synthetic peptides ('C2', 'N2', and 'charged') representing two different polar regions of the CS polypeptide. These reactions are specific since the binding is inhibited only by the homologous peptides. Antisporozoite antibodies were isolated from the rabbit serum by affinity chromatography on Sepharose beads coupled to two synthetic peptides, 'C2' and 'charged'. Both purified antibodies recognized the CS protein and the intracellular precursors as shown by Western blotting analysis using sporozoite extracts. These results demonstrate that the corresponding areas of the native CS molecule are immunogenic, accessible to interaction with antibody, and therefore constitute potential targets for vaccine development. In addition, the present findings confirm the published amino acid sequence of a large portion of the CS protein which has been deduced from the nucleotide sequence of the corresponding gene.     

Distribution of substance P-like immunoreactivity in the central nervous system of the rat--I. Cell bodies and nerve terminals.

The distribution of substance P-immunoreactive structures in the central nervous system of young (1–3 weeks old), adult and colchicine-treated adult male rats has been studied, using the indirect immunofluorescence technique of Coons and collaborators. Substance P-positive cell bodies were observed in more than thirty areas including the spinal cord and many parts of the brain stem. Extensive networks of substance P-positive nerve terminals of varying densities were found in most areas of the central nervous system. The cerebral and cerebellar cortices contained only few substance P-positive structures. It was difficult to identify substance-P immunoreactive axons in the rats studied. Some pathways could, however, be described but further experimental studies are necessary to elucidate the projections of the substance P-immunoreactive neurons in the rat central nervous system.     

One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-gel blue chromatography

Monoclonal antibodies can be purified directly from ascitic fluids by chromatography on a DEAE Affi-gel blue column. Optimal conditions were determined for the recovery of immunoglobulins free of contaminating protease and nuclease activities.     

Isolation of a polypeptide from the venom of Bungarus multicinctus that binds to ganglia and blocks the ganglionic transmission in mammals

A 15,000 dalton polypeptide purified from Bungarus multicinctus venom (which normally copurifies with alpha-bungarotoxin) was characterized biochemically and its biological effects were studied. This polypeptide, P15, had an aminoacid composition and molecular weight different from those of both alpha- and beta-bungarotoxin. It inhibited the ganglionic transmission in the guinea-pig hypogastric nerve-vas deferens preparation and did not block, even at very high concentrations, the neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. In the same preparations alpha-bungarotoxin was unable to block the response at the ganglionic synapse while it was fully active in blocking the neuromuscular transmission. However, a pretreatment of the vas deferens preparation with alpha-bungarotoxin prevented the inhibitory effect of P15. 125I-Labeled P15 showed a specific and saturable binding to rat superior cervical ganglia homogenate and to a Torpedo postsynaptic membrane fraction. The binding of P15 to ganglia was inhibited by curare. The binding was Ca2+ dependent. The density of binding sites was of 300 fmol/mg of protein in the ganglion and 500 fmol/mg of protein in Torpedo membranes. The amount of P15-binding sites in ganglia was not modified by denervation, indicating that P15 binds to postsynaptic receptors. The binding of 125I-labeled P15, both in ganglia and Torpedo membranes, was inhibited by alpha-bungarotoxin. P15 had a Ca2+-dependent phospholipase A2 activity. Lowering Ca2+ concentration in incubation media affected the phospholipase A2 activity more than binding properties and inhibition of phospholipase activity with p-bromophenacyl bromide did not affect the activity of P15 on vas deferens preparation, suggesting that the phospholipase activity is not necessary for the activity of P15 on nicotinic receptors. Our results suggest that P15 toxin may be a specific and valuable probe for studying the ganglionic nicotinic receptor.     

Factors influencing the quantitation of antibodies to influenza virus by indirect solid-phase radioimmunoassay

An indirect solid-phase radioimmunoassay involving purified influenza haemagglutinin bound to polyvinyl microtitre plates has been used to quantitate the humoral response to influenza infection in ferrets. Sialic acid residues, present on normal ferret immunoglobulin, caused non-specific binding of immunoglobulin to the haemagglutinin, resulting in a non-linear relationship between serum dilution and primary antibody bound to antigen. Direct proportionality was achieved by removal of sialic acid residues from serum immunoglobulin or by inclusion of free sialic acid in the incubation buffer. Absolute quantitation of IgG and IgM antibodies was achieved by determining the relationship between primary antibody bound to antigen and secondary anti-immunoglobulin antibodies using ¹³¹I-IgG or -IgM and the homologous ²⁵I-labelled monospecific antiserum. Examples of antibody quantitations in sequential post-infection sera are presented.     

Glycosyl transferase activity in homogenates of adult Dirofilaria immitis

Homogenates of adult Dirofilaria immitis possess a microsomal enzyme system able to transfer mannose from GDP-mannose to endogenous lipid intermediate(s) and exogenous dolichol monophosphate. A divalent metal was required with Mn²⁺ being the most effective; other requirements for optimal activity included Triton X-100, EDTA and either ATP or NaF. The maximal rate of mannose transfer to the lipid acceptor by the filarial system, 1.6 pmol · min^?1 · mg^?1 protein, occurred at 37°C and pH 7.0, and this was inhibited 50% by 8 μM diumycin and not at all by 100 μM tunicamycin. D. immitis microsomes also were shown to promote the transfer of mannose to derivatives of α-lactalbumin, resulting in the synthesis of a mannose-labeled glycoprotein.     

Structural characterization of the 'melanoma-specific' antigen detected by monoclonal antibody 691I5Nu-4-B

A cell surface antigen complex detected by a monoclonal antibody specific for human melanoma cells has been isolated and its molecular structure determined. The antigen consists of four polypeptide chains with mol. wts of 116,000; 95,000; 29,000; and 26,000 dallons. In the native state the two smaller polypeptide chains are linked to the 116,000 dalton species by disulphide bonds but the 95,000 unit is attached only by noncovalent interactions. 2-Nitro-5-thiocyanobenzoic acid (NTCB) cleavage of the two major polypeptides reveals distinct patterns of cleavage indicating that the two proteins have different primary amino acid structures.     

Further characterization of two “DR supertypic” specificities additional evidence that they reside on molecules different from those carrying HLA-DR locus specificities

Peripheral lymphocytes from a panel of individuals who had been assayed for DR specificities by the conventional cytotoxicity assay were typed for DR “supertypic” specificities, DC1 and BR4 × 7, by the radioimmunoassay. A positive and a negative population were clearly distinguished for both specificities and the strong association of the DC1 specificity with DR1, 2, and w6 was confirmed as well as the BR4 × 7 specificity with DR4 and 7. Family study also supported this strong association. Appropriate papain digestion separated molecules carrying DC1 determinant from those carrying DR2 as well as from those carrying DRw6, and separated molecules carrying BR4 × 7 from those carrying DR4. Specificity analysis of the 8th Workshop antisera by use of these separated antigen preparations showed that some anti-DC1 antisera do not possess appreciable anti-DR1, 2, or w6 activity and vice versa. The same was found for BR4 × 7 in its relationship with DR4 and 7. The existing evidence could be explained most economically by assuming a genetic model of two loci in linkage disequilibrium each coding for analogous but distinct forms of the small (beta) subunits of Ia molecules.     

Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes

We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins.     

Monoclonal antibodies (IgM) against a murine carcinoma, Lewis lung carcinoma (3LL): production, purification and in vitro selectivity

C57BL/6 mice were immunized against a syngeneic murine carcinoma, Lewis lung carcinoma (3LL). Monoclonal antibodies were prepared by fusing spleen cells of immunized mice with SP2-O-Ag mouse myeloma cells. Hybridoma clones producing anti-3LL antibodies were selected on the basis of a solid-phase radioimmunoassay. Two clones were selected and subcloned to give the 5B5 and the 6B6 hybrid lines which were found to secrete IgM monoclonal antibodies. Large quantities of monoclonal antibodies were purified from ascitic fluids by gel filtration chromatography. Specificities of these antibodies were tested on 3LL tumor cells, 3LL metastases, lymphoid cells and leukemic L1210 cells. The 5B5 IgM monoclonal antibody was found to be selectively directed against primary tumor cells and metastatic cells.     

Pronase and proteinase K digestion of human immunoglobulin M

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.