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D W Sears

Publications and source records attributed to D W Sears.

33 records · Page 2Linked to original sources
PubMed1982

Unexpected complexity of the dm1 mutation revealed in the structure of three H-2D/L-related antigens.

The H-2L-dm1 and H-2Ddm1 MHC antigens of the B10.D2 (H-2dm1) mutant mouse strain (formerly known as M504 or H-2da) have been compared to the H-2Ld and H-2Dd antigens of the B10.D2 (H-2d)mouse strain. Ldml and Ld are 45 000 Mr antigens and both are reactive with anti-H-2."28" (k/r anti-h2) serum and unreactive with anti-H-2.4 (k/b anti-a) serum which detects private determinants of the Ddm1 and Dd antigens. However, the tryptic peptide compositions of these two antigens are different and, based on the number of major tryptic peptides which coelute during ion-exchange chromatography, the estimated peptide homology between Ldm1 and Ld is 80 percent. A newly defined antigen (mr = 39 000), designated gp39dm1, was found in glycoprotein extracts of the dm1 strain but not of the d strain. This antigen coprecipitates with Ldm1 but does not coprecipitate with Ddm1 indicating that it lacks the H-2.4 determinant. In comparison with Ldm1, gp39dm1 appears to contain far fewer Arg and Lys residues and is most likely not a simple proteolytic fragment of Ldm1. Finally, peptide maps of the Ddm1 antigen show that the majority of its Arg peptides are identical to Dd Arg peptides, whereas at least five of its Lys peptides and three of its Arg peptides correspond not to Dd peptides but to Ld and Ldm1 peptides. These data raise the possibility that the Ddm1 antigen is a hybrid molecule and they have also revealed an unexpected level of complexity in the dm1 mutant phenotype.

Animals↗
PubMed1981

Biochemical evidence for structurally distinct H-2Dd antigens differing in serological properties.

H-2Dd antigens, as defined by the private H-2.4 determinant, exist as two immunochemically distinct populations in H-2a and H-2dm2 splenocytes and in the transformed cell line, RADA1 (H-2a). The two populations are distinguishable by the anti-H-2.28 serum, k/r anti-h2, which is directed, in part, against the H-2.28 family of public determinants encoded by the D end of the b haplotype. Sequential precipitates of lentil-lectin-purified glycoprotein extracts metabolically labeled with radioactive amino acids reveal that approximately one-quarter to one-third of the H-2Dd antigens, designated H-2Dd (b28+), react with this antiserum, whereas two-thirds to three-quarters, designated H-2Dd (b28-), do not. Paired-label tryptic peptide maps in this and a previous study indicate that H-2Dd (b28+) and H-2Dd (b28-) are closely related structurally and are more likely to represent modified forms of the same gene product rather than products of different genes, although the existence of closely related genetic loci is not rigorously excluded. Together, H-2Dd (b28+) and H-2Dd (b28-) have a radioactivity level seven times higher than H-2Ld, which also reacts with the anti-H-2.28 serum but which lacks the H-2.4 determinant. As yet unresolved, however, is the question of whether the observed quantitative differences between these three antigens reflect actual molar differences at the cellular level, or whether the variation is the result of metabolic or compositional factors. In any case, a complex serological and structural relationship is found to exist between antigens encoded by the D/L end of the MHC.

Amino Acids↗
PubMed1980

Biochemical evidence for a separate, MHC-linked locus encoding H-2.28 antigens.

In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2 alpha) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linked H-2L region encodes H-2L antigens separately from the H-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k X r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also suprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.

Animals↗
PubMed1977

Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Reoxidative assembly in vitro.

We recently reported results of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol (Sears, D.W., et al. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 353). In that work, we described the reoxidative behavior of the molecule under nondissociating conditions. In the present paper, results are presented of the reoxidation of heavy (H) and light (L) chains of this protein alone, or mixed in varying proportions after separation, or mixed with the L chains modified prior to recombination and reoxidation. The overall reoxidative asembly patterns in experiments with H and L separated prior to recombination are similar to those observed when the chains remain noncovalently associated throughout. With equimolar mixtures of H and L, the reoxidation rates also are similar to those of unseparated chains. However, when L chains are present in excess, the overall in vitro rates of covalent assembly are generally diminished, probably indicating transient nonproductive interactions. At the highest molar excesses of L (3:1), the assembly pathways may also be modified. In all experiments with excess L chains, covalent L2 dimers form at rates which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the first time. In the first, the free half-cystine of L is irreversibly blocked by reaction with iodoacetamide, and the alkylated L chains are recombined with reduced H chains. This experiment isolates the reactions in which H2 disulfides are formed without the accompanying formation of HL bonds. Although the alkylated L chains do not play a direct role in the reoxidation, their presence is required to inhibit aggregation and precipitation of high-molecular-weight products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the second kind of experiment, covalent L2 dimers are mixed with reduced H chains. L2 rapidly disappears with the concurrent appearance of HL, H2L, and fully assembled H2L. H2 dimers are also reactive in this process. Special procedures were developed for analyzing the data from these experiments. A complete format is given for the quantitative determination of the concentration of each of the molecular components directly from spectroscopic scans of the gels. The computational methods solve the general analytical problem posed when staining is not proportional to mass and are applicable to a wide variety of systems utilizing gel electrophoresis to study subunit interactions. A theoretical analysis of pathway and kinetic cooperatively in this system is presented in the following paper (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (following paper in this issue)).

Bence Jones Protein↗
PubMed1977

Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Theoretical reoxidation models.

A theoretical format suitable for analyzing diverse complex kinetic systems where reaction pathways may exhibit cooperativity, is developed to account for the in vitro kinetics of air reoxidation of a human IgGlkappa immunoglobulin, in which the four interchain disulfide bonds have been reduced (Sears, D.W., et al. (1977), Biochemistry 16 (preceding paper in this issue)). The equations relate experimentally determined concentrations of the reactants, product, and macroscopic intermediates to the probability of occurrence of any of 12 distinct microscopic states. The concentrations of the macroscopic species--light chains (L), heavy chains (H), covalently associated intermediates HL, H2, H2L, and product H2L2--are also explicitly related to an observable function of those concentrations, the sulfhydryl titer, r. Since values of r can be calculated for any microscopic state of the system, the theory is in principle capable of a complete description of the experimental reaction course. In practice, limited experimental information precludes a unique solution of the equations at present, and it was not judged worthwhile to attempt curve fitting or approximation of probability terms with adjustable parameters of unknown physical significance. Certain special cases of the theory are, however, readily and exactly solved. These include models in which reoxidation is a random process and others in which the probabilities for inter-HL and inter-HH bond formation are different but independent of one another. The experimental results in the case of the IgGlkappa studied here clearly depart form the predicted behavior in either of these models. The initial probability for formation of a bond between a heavy and light chain is 1.5 to 2 times greater than for a bond between heavy chains. From the fact that this ratio changes as the reaction proceeds, and from the pattern of variation in concentration of intermediates during the reaction, it is concluded that the reoxidation process is not random, and that the bonds do not form independently, exhibiting instead kinetic cooperativity. The results are discussed in terms of assembly pathways in this and related systems. A novel feature of the theory is that it eliminates time as an explicit variable in the treatment of the kinetic process. This makes it especially useful for discerning whether the formation of one bond influences the reaction probability of another in a system where several similar or intrinsically identical reactions occur, and where kinetic order is difficult to establish. While the theory here is formulated in terms of the reoxidation reactions, it is, for example, equally applicable to the reduction process described (Sears, D.W., et al. (1977b), Biochemistry 16 (following paper in this issue)).

Disulfides↗
PubMed1977

Relative susceptibilities of the interchain disulfides of an immunoglobulin G molecule to reduction by dithiothreitol.

The reduction by dithiothreitol (DTT) of the four interchain disulfides of a human IgGlkappa immunoglobulin has been studied by two methods: variation of the concentration of DTT relative to the protein concentration (incremental reduction); and variation of the time of reduction at fixed levels of DTT and protein (kinetic reduction). In both cases, the results depend on whether the reduction is carried out aerobically or anaerobically. Under aerobic conditions, the relative levels of intermediates (HL, H2, and H2L) which are generated as native molecules (H2L2) are converted to reduced heavy (H) and light (L) chains depend on the concentrations of protein and DTT as well as on the exposure time to DTT; no stable equilibrium is reached between reduced and oxidized states and conditions gradually revert from those favoring reduction to those favoring reoxidation. By contrast, anaerobic reduction is independent of protein concentration or time of exposure to DTT, beyond about 30 min, indicating that an equilibrium between partially reduced and oxidized states is achieved. The distribution of intermediates observed under anaerobic conditions has been analyzed according to theoretical models (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in a series of three articles in this issue)). Within experimental error, both kinds of anaerobic experiments resemble a random reduction process wherein the four disulfides are equivalent and independent of each other with respect to rate and extent of reduction by D. It is concluded that there are no readily detected pathways in the process, as would occur if the intrinsic reactivities of the bonds were distinct, and no marked cooperatively between the four reaction sites, as would be observed if reduction of one bond materially facilitated or hampered reactivity at another site. Both of these characteristics of the reduction are in direct contrast to those of the reoxidative process, which is marked by the initial preference for formation of a bond between heavy and light chains, and by kinetic cooperativity in bond formation during the course of the reaction (Sears, D.W., et al. (1977), Biochemistry 16 (first in a series of three articles in this issue); Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in this series)).

Disulfides↗
PubMed1975

A kinetic study in vitro of the reoxidation of interchain disulfide bonds in a human immunoglobulin IgGLk. Correlation between sulfhydryl disappearance and intermediates in covalent assembly of H2L2.

After reduction by dithiothreitol and removal of the reductant by molecular sieve chromatography, the four interchain disulfide bonds of the human IgGlk protein Fro reoxidize in the presence of oxygen and trace metal ions. The six molecular components of the reoxidation--L (light chain), H (heavy chain), HL, H2, H2L, H2L2--are quantitatively determined from polyacrylamide gels containing sodium dodecyl sulfate and the time-dependent sulfhydryl titer is measured with 5,5'-dithiobis-(2-nitrobenzoic acid). The rates of H2L2 covalent assembly depend on pH in an unexpected way: If the reduced protein is chromatographed at pH 3.2 and then adjusted to pH 7.5 (25 degrees, ionic strength equals 0.14), H2L2 formation proceeds rapidly, with half-times ranging between 20 and 40 min. In contrast, if chromatography is carried out at pH 5.5 before adjusting to the same final conditions, the half-times for H2L2 formation are considerably longer (120-180 min). The half-times in the former case approach the somewhat faster rates of H2L2 assembly observed in pulse-chase experiments with various types of mouse, IgG-producing cells [Baumal, et al. (1971) J. Exp. Med. 134, 1316-1334]. To facilitate comparison of experiments and models, we plot the concentrations of the six components against the corresponding number of sulfhydryl equivalents per mole of Fro. The respective plots for the pH 3.2 leads to 7.5 and 5.5 leads to 7.5 experiments are very similar despite the rate differences. Moreover, these plots differ significantly from the calculated plot for a hypothetical random reoxidation in which the intrinsic probability for formation of each correct HL and H2 disulfide bond is assumed equal and independent. It is concluded that the in vitro reoxidation of Fro (i) is other than random; (ii) involved a pathway of pathways with HL, H2, and H2L precursors; and (iii) involves at least some kinetic cooperativity in bond formation, since no model bases solely on independent bond formation adequately accounts for the results. The models were used also to examine the cellular assembly pathways of mouse IgG proteins.

Disulfides↗