scholarly journals The gross architecture of an antibody-combining site as determined by spin-label mapping

1977 ◽  
Vol 165 (2) ◽  
pp. 177-197 ◽  
Author(s):  
Brian J. Sutton ◽  
Peter Gettins ◽  
David Givol ◽  
Derek Marsh ◽  
Simon Wain-Hobson ◽  
...  
Keyword(s):  
Light Chain ◽  
Metal Binding ◽  
Spin Label ◽  
Immunoglobulin A ◽  
Variable Region ◽  
Spin Labels ◽  
Myeloma Protein ◽  
Polarity Profile ◽  
Resonance Relaxation ◽  
Hyperfine Splittings

1. A series of Dnp (dinitrophenyl) nitroxide spin labels was used to map the dimensions of the combining site of the Dnp-binding immunoglobulin A myeloma protein MOPC 315. The method compares the observed e.s.r. (electron-spin-resonance) hyperfine splittings with those calculated on the basis of different postulated motions for the spin label. The analysis is complicated by the sensitivity of the e.s.r. hyperfine splitting to the overall ‘tumbling’ time of the antibody–hapten complex and the polarity of the spin-label's environment. When these effects are considered quantitatively, it is then possible to determine the degree of mobility of each hapten which is allowed by the shape of the combining site. 2. The dinitrophenyl ring is rigidly held, and the depth of the site is 1.1–1.2nm and has lateral dimensions at the entrance to the site ≥0.6nm×0.9nm. The analysis of the results for spin-labelled haptens with chiral centres allows these lateral dimensions to be refined to 0.8nm and 1.1nm, and it is shown that the site is asymmetric with respect to the plane of the dinitrophenyl ring. 3. A polarity profile of the combining site was also obtained and a positively charged amino acid residue, possibly arginine-95L (light chain), was located at the entrance to the site. 4. The binding of Gd(III) to the antibody–hapten complexes results in quenching of the e.s.r. signal of the nitroxide. By using La(III) as a control, the paramagnetic contribution to the quenching is measured. 5. Analysis of the differential quenchings of the enantiomers of two five-membered nitroxide ring spin labels gives two possible locations of the metal-binding site. One of these is equidistant (0.7nm) from each of the three dinitrophenyl aromatic protons, and nuclear-magnetic-resonance relaxation studies, at 270MHz, on solutions of dinitrobenzene, Gd(III) and the Fv fragment (variable region of heavy and light chain) from protein MOPC 315 support this location for the metal site. 6. The e.s.r. and metal-binding data were then compared with the results of a model of the combining site constructed on the basis of framework invariance in immunoglobulins [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol.41, in the press]. The overall agreement is very good. Assignments of possible chelating groups for the metal can be made.

An Immunoglobulin Light Chain of Subgroup III1 of λ Type with a Free Sulfhydryl Group

1971 ◽  
Vol 49 (8) ◽  
pp. 900-902 ◽  
Author(s):  
Barbara M. Buchwald
Keyword(s):  
Light Chain ◽  
Sulfhydryl Group ◽  
Variable Region ◽  
Cysteine Residue ◽  
Immunoglobulin Light Chain ◽  
Myeloma Protein ◽  
Free Sulfhydryl Group ◽  
Bence Jones Proteins

A myeloma protein, Du, (γ1)2 (λ)2, is shown to have an extra cysteine residue in the light chain. This light chain is from the same λ variable region subgroup as are two Bence-Jones proteins, X and Bau, which also have an extra cysteine residue. The position of this residue is the same in all three chains.

scholarly journals The natural abundance of lambda2-light chains in inbred mice.

1978 ◽  
Vol 148 (5) ◽  
pp. 1388-1399 ◽  
Author(s):  
T Cotner ◽  
H N Eisen
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Variable Region ◽  
Fold Increase ◽  
Light Chains ◽  
Mouse Strains ◽  
Mouse Serum ◽  
Myeloma Protein

The amino acid sequence of the constant (C) domain of the light chain of the mouse myeloma protein M315 has not been identified so far in any other myeloma protein. In this study, serological analysis with antiserum to the C-domain of this light chain (L315) showed that approximately equal to 1% of Igs in normal mouse serum have L chains of the L315 type (called lambda2). Corroborative evidence was obtained by analysis of the carboxyterminal amino acid removed from normal light chains by carboxypeptidase A. A survey of 35 inbred mouse strains showed that all had lambda2; the serum level of Igs with lambda2-chains ranged from approximately equal to 140 microgram/ml in AL/N mice to approximately equal to 25 microgram/ml in SJL, BSVS, and eight other strains. In accord with the anti-Dnp activity of M315, sera from mice immunized with Dnp-KLH had three- to fivefold more lambda2 than sera from control mice immunized with KLH. It was also possible to measure serum immunoglobulin molecules bearing the lambda2 variable region of M315 (VL315). In BALB/c sera, the concentration of VL315 was about sixfold lower than that measured for lambda2. Thus, lambda2-chains are divided into at least two subsets: those whose V domain is indistinguishable from VL315 and those whose VL differs from VL315. A 10-fold increase in VL315 was obtained by immunizing BALB/c mice with Dnp-KLH. The relationship of the VL domains of normal immunoglobulin lambda2-chains to the embryonic Vlambda gene recently sequenced by Tonegawa et al., is discussed.

scholarly journals Expression of an idiotype (Id-460) during in vivo anti-dinitrophenyl antibody responses. I. Mapping of genes for Id-460 expression to the variable region of immunoglobulin heavy-chain locus and to the variable region of immunoglobulin kappa-light-chain locus.

1980 ◽  
Vol 152 (3) ◽  
pp. 720-729 ◽  
Author(s):  
E A Dzierzak ◽  
C A Janeway ◽  
R W Rosenstein ◽  
P D Gottlieb
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Regulatory Gene ◽  
Variable Region ◽  
Immunoglobulin Heavy Chain ◽  
Kappa Light Chain ◽  
Myeloma Protein ◽  
Variable Regions ◽  
Chain Locus

The genetic contro of the expression of an idiotype (Id-460) associated with the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC 460 was studied using congenic strains of mice. It was shown that the expression of high levels of Id-460 during secondary in vivo anti-DNP-ovalbumin responses was determined by genes governing immunoglobulin heavy-chain variable and kappa-light chain variable regions (V kappa). Appropriate alleles at both loci were required for the expression of Id-460. Genes in the major histocompatability complex and the X-linked immune deficiency gene found in strain CBA/N did not greatly affect Id-460 expression. The V kappa gene controlling Id-460 expression can be differentiated from Lyt-3, and it is the first instance in which expression of an idiotype subdivides the V kappa genes associated with the Lyt-3a allele. Although it is likely that the V kappa gene(s) involved are structural, the involvememt of a regulatory gene linked to the structural gene can not be excluded.

scholarly journals T helper cells recognize an idiotope located on peptide 88-114/117 of the light chain variable domain of an isologous myeloma protein (315).

1983 ◽  
Vol 158 (6) ◽  
pp. 2183-2188 ◽  
Author(s):  
T Jørgensen ◽  
B Bogen ◽  
K Hannestad
Keyword(s):  
Light Chain ◽  
T Helper Cells ◽  
Cyanogen Bromide ◽  
Hypervariable Region ◽  
Variable Region ◽  
T Helper ◽  
Myeloma Protein ◽  
The Third ◽  
Helper Cells ◽  
Terminal Fragment

Isolated variable region light chain 315 (VL-315), the VL domain of a myeloma protein of BALB/c origin, induces T cells of BALB/c (H-2d) mice that help the adoptive secondary anti-4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP) antibody response to NIP-Fab315. The location of the epitope recognized by helper cells was examined with two fragments of VL-315, obtained by cleavage with cyanogen bromide at Met 87. Both N-terminal fragment 1-86 and C-terminal fragment 88-114/117 elicited BALB/c antibodies that bound to the respective fragments and to VL-315. By contrast, only fragment 88-114/117, which consists of the third hypervariable region, J region, and 5-7 amino acids of the C region, induced helper cells that augmented the anti-NIP response to NIP-Fab 315.

scholarly journals Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315

1976 ◽  
Vol 155 (1) ◽  
pp. 37-53 ◽  
Author(s):  
R A Dwek ◽  
D Givol ◽  
R Jones ◽  
A C McLaughlin ◽  
S Wain-Hobson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Spin Label ◽  
Binding Sites ◽  
Binding Constants ◽  
Kinetic Scheme ◽  
Difference Spectrum ◽  
Protein Fluorescence ◽  
Myeloma Protein ◽  
Fluorescence Studies

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a ‘slow’ conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment.

Molecular analysis of the murine lupus-associated anti-self response: involvement of a large number of heavy and light chain variable region genes

1987 ◽  
Vol 17 (1) ◽  
pp. 91-95 ◽  
Author(s):  
Reinhard Kofler ◽  
Daniel J. Noonan ◽  
Robert Strohal ◽  
Robert S. Balderas ◽  
Niels P. H. Moller ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Light Chain ◽  
Variable Region ◽  
Murine Lupus ◽  
Variable Region Genes

scholarly journals 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 α-chain

1977 ◽  
Vol 167 (1) ◽  
pp. 245-253 ◽  
Author(s):  
A P Johnstone ◽  
L E Mole
Keyword(s):  
Immunoglobulin G ◽  
Polypeptide Chain ◽  
Immunoglobulin A ◽  
Variable Region ◽  
Chain Structure ◽  
Secretory Immunoglobulin A ◽  
Primary Sequence ◽  
Relative Resistance ◽  
Proteolytic Fragment ◽  
Secretory Immunoglobulin

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.

Structure of immunoglobulin A. Cysteine-containing peptides of the α chain of an immunoglobulin A1 myeloma protein

Biochemistry ◽  
1973 ◽  
Vol 12 (25) ◽  
pp. 5186-5194 ◽  
Author(s):  
Enrique Mendez ◽  
Blas Frangione ◽  
Edward C. Franklin
Keyword(s):  
Immunoglobulin A ◽  
Myeloma Protein ◽  
Α Chain
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