Antibody against rabbit immunoglobulin VH and L chain allotype determinants in heterologous anti-Ig antisera.

Heterologous anti-rabbit IgG antisera have been examined by radioimmunoassay for the occurrence of anti-VH antibody. Most of the sera contained antibody specific for VH allotype determinants (Aal). The antisera contained as much or more anti-al antibody as estimated in conventional alloantisera. It has further been shown that the anti-L chain antibody in all these sera was specific for L chain allotype determinants. Antibody against H chain-dependent L chain conformational determinants or cross-reacting determinants in CHlgamma and CHlalpha could not be demonstrated.

The subunit and polypeptide-chain structure of rabbit secretory immunoglobulin A. Isolation of a proteolytic fragment suitable for sequence studies on the variable region of alpha-chain.

A method was developed for the preparation of a proteolytic fragment of rabbit secretory immunoglobulin A (sIgA) which contains the variable region of the alpha-chain; this fragment is suitable for primary-sequence studies. The serologically defined subclasses of sIgA are shown to correlate partially with the nature of the binding of a constituent chain of sIgA, called secretory piece. Data are also presented on the relative resistance of sIgA to enzymic and reductive cleavage, compared with immunoglobulin G.

Sequence studies on the heavy chain of rabbit immunoglobulin A of different alpha-locus allotypes.

The amino acid sequence was determined of part of the variable region of heavy chain from rabbit immunoglobulin A of allotypes a1 and a3. Two corrections of the primary sequence of Aa1 gamma-chains are reported; most of the structural correlates of the alpha-locus allotypes are confirmed. The amino acid sequence of the N-terminal 20 residues of alpha-negative molecules was also determined and found to be homologous to the human VhIII subgroup. These molecules are present in a much higher proportion in the alpha-chain pool than in the gamma-chain.

Estimation by radioimmunoassay of VH determinants (Aa1) associated with rabbit T lymphocytes.

A radioimmunoassay with specificity for the a1 allotype determinant of rabbit immunoglobulin (Ig) heavy chains was used for the quantitation of VH in detergent extracts of lymph node cells from a1a1/b4b4 rabbits. B cell-derived Ig in the same extracts was estimated with a radioimmunoassay specific for L-chain. About 2 X 10(5) molecules/cell was found by both assays when assuming a molecular weight of 1.5 X 10(5) dalton. Extracts prepared from lymphocytes depleted of B cells (surface Ig+ cells) were also found to contain corresponding amounts of a1 and L-chain, with values varying from about 10(3) to 10(4) molecules/cellmthe estimates were not influenced by the presence of inhibitors of proteolytic enzymes. Supernatants, obtained after keeping the lymphocytes in culture for 16 h, were analyzed by the same methods. Again, matching amounts of a1 and L-chain were found in supernatants from nonfractionated as well as from B cell-depleted populations. The results obtained indicate that T cells do not carry or produce an excess of a1 compared to L-chain, as would have been expected if the T cell antigen receptor carried the same variable region as the Ig heavy chain while being otherwise composed of nonimmunoglobulin-like structures.

Localization of the papain cleavage site of H-2 glycoproteins.

The antigenic products of the murine H-2K and H-2D genes are glycoproteins of about 45,000 molecular weight which are tightly integrated within the cell surface membrane. A glycoprotein fragment (FAg, antigenic fragment) of 37,000 daltons carrying the carbohydrate, antigenic sites, and the associated putative beta2-microglobulin of 12,000 daltons can be generated by papain cleavage either of the native molecules in the cell membrane or of immune precipitates made from the antigen solubilized by nonionic detergent. Partial NH2-terminal sequence analyses of the native H-2 glycoprotein and of the papain-cleaved glycoprotein fragment establish that the fragment is, in fact, the NH2-terminal portion of the native molecule. Thus, the cleavage by papain proteolysis is near the COOH-terminus, and removal of the COOH-terminal portion (Fm, membrane fragment) converts the glycoprotein to a water-soluble form. This observation suggests that the NH2-terminus of the native glycoprotein extends out of the hydrophobic bilayer of the cell membrane, and that the COOH-terminus contains the membrane binding region and is buried within the bilayer.

A gamma Bence-Jones protein in guinea pigs.

1. The L2C lymphocytic leukaemia in strain-2 guinea pigs is accompanied by a protein in the urine resembling a homogenous immunoglobulin light chain. 2. The amino acid sequence over the first 20 residues demonstrates a close analogy with a human gamma chain of V region subgroup IV. 3. The protein is apparently synthesized by the leukaemic cells and thus represents a monoclonal light chain, i.e. a Bence-Jones protein.

Sequence studies on the constant region of the Fd sections of rabbit immunoglobulin G of different allotype.

The amino acid sequence has been completed for the constant region of the Fd fragments of heavy chains from rabbit IgG (immunoglobulin G) of allotype Aa1 and Aa3. The amino acid sequence given by Fruchter et al. [(1970) Biochem. J. 116, 249-259] for the constant region of the Fd fragment from Aa1 IgG was extended and in in part corrected to give a continuous sequence of 140 residues. No allotype-related sequence variation was found in the constant section of the Fd fragment. This evidence confirms the view that the differences in sequence between the variable regions of Aa1 and Aa3 IgG [Mole et al., (1971) Biochem. J. 124, 301-318] are responsible for the allotypic specificities.

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.

The isolation and characterization of the V-H domain from rabbit heavy chains of different a locus allotype.

The Fd fragment of rabbit gamma-chains was split by papain to yield a smaller fragment with a molecular weight of approximately 14,000 and dialyzable small peptides and amino acids. The domain size fragment was identified as intact variable region from its amino acid content, its blocked amino-terminus, and two characteristic cysteine-containing peptides, while the small peptides and amino acids were accounted for by the degradation of the C-H1 region. The variable regions isolated from Aa1 and Aa3 Fd fragments not only reacted quantitatively with immunoadsorbents conjugated with the homologous anti-a allotype antibody, but also completely inhibited the binding of the parent Fd fragment to the homologous antibody as measured by radioimmune assay. These data provide direct evidence that the group a allotypic determinants are contained entirely in the variable portion and are independent of the constant portion of rabbit heavy chains.

The amino acid sequence of ragweed pollen allergen Ra5.

The complete amino acid sequence of Ra5, a ragweed pollen allergen, has been determined. Allergen Ra5 is a low molecular weight protein of 45 residues derived from Ambrosia elatior, the short ragweed. It contains no detectable carbohydrate or lipid and has four disulfide bridges. The total structure was determined on 1.4 mumol of material and indicates that structural analysis is increasingly possible on relatively small amounts of highly purified material when a combination of automated and manual sequencing techniques and highly sensitive detection systems is employed. This represents the first complete amino acid sequence of a ragweed allergen and it should provide a basis for many structure-function correlative experiments in the field of immediate hypersensitivity.

The specificity of chain interactions among immunoglobulins. Combinations of gamma chains with kappa chains of the same subgroup as in the parent immunoglobulin G.

1. The specificity of combination of heavy and light chains from selected human immunoglobulins was examined in the light of greater structural information than in previous studies. Heavy (gamma) chains from immunoglobulin G (kappa) myeloma proteins were allowed to combine with their homologous light (kappa) chains or with other kappa chains of the same variable-region subgroup. The affinity of each such pairing was assessed by having the test kappa chain compete with a standard population of normal light chains. 2. There was a spread of affinities among the heavy-light pairings with the homologous pairings having an average affinity significantly higher than the heterologous pairings. 3. It follows that (a) the preference shown for homologous heavy-light pairings is not explicable simply in terms of the known subdivisions of the variable and constant regions of the chains, and (b) it is unlikely that those residues specifying the subgroups of kappa-chain variable regions have a predominant role in the formation of interchain bonds with the gamma-chain variable regions.

Allotypically related sequences in the Fd fragment of rabbit immunoglobulin heavy chains.

The sequence has been completed of the N-terminal 94 residues of the variable section of the Fd fragment of heavy chains from rabbit immunoglobulin G (IgG) of allotype As1. Most of the sequence of the same section from IgG of allotype Aa3 is also reported. These results, in conjunction with a substantial sequence of the variable region of allotype Aa2 reported elsewhere (Fleischman, 1971), show the presence of 16 positions (including six consecutive positions) in which the residue present correlates with the allotype. No allotype-related sequence variation has been found in the constant section of the Fd fragment. This evidence supports the view that two genes code for the heavy chain and it can be used as evidence in favour of somatic mutation as the origin of the variability in the sequence of the N-terminal section. The evolutionary origin of the ;a' locus allotypes of rabbit immunoglobulins remains obscure.

Sequence studies of the Fd section of the heavy chain of rabbit immunoglobulin G.

A partial amino acid sequence was given by Cebra, Steiner & Porter (1968b) of the N-terminal half of the heavy chain of rabbit immunoglobulin G. This was extended and in part corrected to give a continuous sequence of 136 residues, which together with other work accounts for three-quarters of the total sequence. Evidence is given suggesting that there is a limited region of 10-15 residues that are exceptionally variable in the heavy chains from pooled rabbit immunoglobulin G.