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F J Stevens

Publications and source records attributed to F J Stevens.

At least 73 records · Page 4Linked to original sources
PubMed1988

Dual conformations of an immunoglobulin light-chain dimer: heterogeneity of antigen specificity and idiotope profile may result from multiple variable-domain interaction mechanisms.

The structure of an immunoglobulin antigen-binding fragment (Fab) has been thought to be invariantly defined by well-conserved amino acid residues in the variable domains of the heavy and light chains. These conserved residues enable folding of the polypeptide segments into the characteristic immunoglobulin fold domains and are the major controllers of interactions between domains. However, crystallographic studies of some immunoglobulin light-chain dimers have suggested and the crystallographic structure of the Fab in an Fab-neuraminidase complex may have proven that antibodies are not restricted to a single, invariant relative positioning of the two variable domains. We propose that in some cases the detailed quaternary structural relationships between the variable domains of heavy and light chains are not restricted to those of the canonical Fab. It is unclear whether alterations of these relationships occur only after complex formation with antigen or, if in ligand-free solution, Fab conformers might coexist in relative concentrations determined by isomerization rates. In the latter case, antibody-presenting lymphocytes may be polyspecific, and the specificity of lymphocytes might be modulated by anti-idiotopic antibodies complexed to cell surface receptors. In either case, the idiotopic repertoire displayed by an antibody or lymphocyte surface receptor might be changed by the presence or absence of antigen.

Antibody Diversity↗
PubMed1988

Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties.

An antigen histochemically localized in the nuclei and cytoplasmic granules of normal and leukemic human myeloid cells has been identified as myeloperoxidase (MPO; EC 1.11.1.7). The localization and amount of the enzyme was determined by using a murine monoclonal antibody designated H-43-5 raised against nuclear proteins derived from human promyelocytic HL-60 leukemia cells. The highest amount of nuclear MPO (3.5 micrograms per 10(6) nuclei) was found in granulocytes; less than half of this amount was detected in nuclei from HL-60 cells. Still lower levels were found in nuclei from monocytes and a series of human monomyelocytic leukemia cells. MPO from HL-60 cells was purified by immunoaffinity chromatography and fractionated into three components (forms I, II, and III) by CM-cellulose chromatography. Chromatography of these MPO forms on DNA-Sepharose columns confirmed that all three forms of MPO were tightly bound to DNA with apparent relative affinities in the order of form III greater than form II greater than form I. The affinity of MPO form III for DNA was sufficient to enable the formation and elution of DNA-MPO complexes during size-exclusion chromatography at high ionic strength and neutral pH. This form of MPO was also able to shield DNA from strand scission induced by active oxygen species generated by xanthine oxidase acting aerobically on xanthine. These data suggest that intranuclear MPO may help to protect DNA against damage resulting from oxygen radicals produced during myeloid cell maturation and function.

Antibodies, Monoclonal↗
PubMed1987

Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody.

A recently described procedure for the evaluation of the affinity of monoclonal antibodies [Friguet et al., J. Immun. Meth. 77, 305-319 (1985)] uses an ELISA system to determine the quantity of free antibody present in a mixture of antigen and antibody. However, an intact IgG may bind antigen by either of two binding sites, and an IgG can bind to a solid-phase antigen whether one or two of its binding sites are free. Therefore, this procedure does not directly provide the concn of liganded binding sites, the quantity necessary for calculation of the thermodynamic association constant. A binomial probability distribution relates the fraction of liganded binding sites to the concn of unliganded, singly liganded, and doubly liganded IgG assuming that the binding of each Fab to antigen is independent. Simulated experiments were used to compare the apparent binding characteristics of bivalent IgG and monovalent Fab and to calculate apparent association constants in each case. It was found that the affinity of binding sites on intact IgG was underestimated by a factor of at least 2 and that the error was inversely related to the fraction of liganded binding sites. Binding site affinity of an antibody may be underestimated by several orders of magnitude. On the basis of binomial analysis, it is possible to convert apparent concns of bound IgG to actual concns of liganded binding site resulting in the calculation of valid association constants for intact IgG without alteration of the experimental protocol.

Antibody Affinity↗
PubMed1986

Relationships between liquid- and solid-phase antibody association characteristics: implications for the use of competitive ELISA techniques to map the spatial location of idiotopes.

A liquid-phase assay system based on small-zone size-exclusion chromatography was used to examine the binding of a monoclonal anti-idiotopic antibody, F6, to its idiotope on the murine plasmacytoma IgA, TEPC-15. Chromatographic behavior revealed a strong association between T-15 and F6, which was previously characterized by solid-phase immunoassay as recognizing a nonbinding site epitope of the T-15. This chromatographic pattern suggests that the inability of the hapten phosphorylcholine to inhibit the anti-idiotope:idiotope relationship in solid-phase immunoassay might be equally explained by the low affinity of the hapten relative to the high affinity of the anti-idiotope antibody. Bivalent interactions between solid-phase IgA and liquid-phase IgG should enhance the binding of the anti-idiotope to an extent that would prevent the hapten from dissociating the complex. Under these solid-phase assay conditions, observation of hapten inhibition may, in some cases, indicate site specificity, but absence of inhibition cannot be interpreted. Computer simulations of solid-phase hapten inhibition scenarios were used to evaluate the qualitative nature of binding inhibition profiles expected under various conditions of liquid- and solid-phase reactant affinities and concentrations. The apparently unusual inhibition curves previously observed in the T-15:anti-T-15 studies in which the degree of binding inhibition by hapten appeared to be independent of anti-idiotope concentration may be predictable in cases of excess solid-phase epitope; the plateau inhibition value is a function of relative affinity constants and concentrations of solid-phase and inhibitor components. The results additionally suggest that the affinity of a liquid-phase antibody may modulate the effective concentration of solid-phase epitope.

Antibody Affinity↗
PubMed1986

Analysis of protein-protein interaction by simulation of small-zone size-exclusion chromatography: application to an antibody-antigen association.

The association of two or more macromolecules results in the formation of a complex characterized by a larger Stokes radius than that of its components. Therefore, analytical procedures such as ultracentrifugation and size-exclusion gel chromatography that resolve molecules on the basis of size have been used to characterize the association. In this paper we describe an iterative computer simulation of small-zone size-exclusion gel filtration. The simulation describes univalent and bivalent interactions of proteins of equal and nonequal molecular weight and appears to have both qualitative and quantitative application to the evaluation of protein-protein interaction as revealed by alteration of chromatographic elution profiles. To test the validity of the simulation, the model was applied to an antibody-antigen interaction by determining the association constant (Ka) for the interaction between the binding fragment derived from a human immunoglobulin A rheumatoid factor and the antigenic fragment obtained from a human myeloma immunoglobulin G. The self-consistency of the estimated Ka values obtained with a valence value of 2 in contrast to the lack of self-consistency if an antigenic valence of 1 was assumed was taken to support the ability of the algorithm to reasonably emulate the chromatographic processes of interacting proteins. In conjunction with the computer simulation, a sensitive microcomputer-interfaced chromatography system was assembled, which is capable of analyzing 300 ng of protein in less than 1 h. This combination of rapid reagent-conservative chromatography and simulation analysis may contribute to the usefulness of small-zone gel filtration in studies of protein-protein interaction.

Antigen-Antibody Complex↗
PubMed1986

Evidence for growth heterogeneity among foci with different phenotypes in the population of altered hepatocyte foci induced by a single neonatal treatment with carcinogen.

Relationships between phenotypic and growth characteristics of carcinogen-induced altered hepatocyte foci were investigated. Male and female rats were given a single i.p. injection of carcinogen (diethylnitrosamine or benzo[a]pyrene) within 1 day after birth and were exposed to dietary promoter (phenobarbital) beginning at weaning. Groups of these rats were then killed at intervals, and their livers were examined for foci exhibiting various phenotypic markers through the use of serial frozen sectioning techniques, histochemical staining and computer-assisted image analysis. These procedures permitted the identification and sizing of foci with different specific phenotypes (identities of focus markers) within each phenotypic complexity level (number of markers per focus). The data suggest that foci growth rates differ with respect to specific focus phenotypes within complexity levels. This observation complements previous demonstrations of a direct relationship between foci growth rates and levels of phenotypic complexity and indicates that the observed diversity of focus phenotypes reflects true biological diversity within the focus population. Given the prior evidence for (i) the stability of focus phenotypes; (ii) the rapid emergence of phenotypically dissimilar foci following a single carcinogen treatment; and (iii) the production of foci by single initiation events, we suggest that each proliferatively and phenotypically distinct member of the focus population reflects the occurrence of a lesion at a unique genetic locus during initiation.

Animals↗
PubMed1985

Formation of an infinite beta-sheet arrangement dominates the crystallization behavior of lambda-type antibody light chains.

The packing interactions in crystals of human lambda-type antibody light chain dimers have been reviewed. These homologous proteins are composed of individually specific variable domains, but all have very similar constant domain sequences. The proteins do not emulate each other in their overall crystallization behavior: each attains an individually characteristic space group or unit cell dimensions. However, each of these protein crystals has one unit cell dimension in common, 72.4(+/- 0.2) A. Examination of the protein packing in these crystals reveals that the common cell dimension is a consequence of a packing arrangement of their constant domains, which is conserved in all three crystals. In this striking arrangement, beta-sheets of adjacent constant domains are placed in juxta-position to form an "infinite chain". Although this constant domain packing pattern is rigorously conserved, the variable domain packing arrangements in each of these crystals are different. The conservation of the "infinite" beta-sheet pattern suggests that the constant domain interactions dominate the thermodynamic energy of lattice formation, probably through a combination of specific hydrogen bond formations and by a decrease in the solvent-accessible surface. A single amino acid substitution prohibits this characteristic interneighbor hydrogen bond pattern in the homologous kappa-type light chains. This may explain the observation that very few kappa-type light chains have been crystallized.

Crystallography↗
PubMed1985

Novel arrangement of immunoglobulin variable domains: X-ray crystallographic analysis of the lambda-chain dimer Bence-Jones protein Loc.

We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)

Bence Jones Protein↗
PubMed1982

Small-angle neutron scattering study of Bence-Jones protein Mcg: comparison of structures in solution and in crystal.

Immunoglobulins fragments are composed of globular domains linked by extended polypeptide segments. The molecular flexibility inherent in this arrangement allows for significant potential differences between structures observed in the crystalline state and those attained in solution. Small-angle neutron scattering measurements in dilute solution were performed on the Mcg Bence-Jones protein dimer, for which performed on the Mcg Bence-Jones protein dimer, for which accurate atomic coordinates have been determined by crystallographic methods [Edmundson, A. B., Ely, K. R., Abola, E. E., Schiffer, M., & Panagiotopoulos, N. (1975) Biochemistry 14, 3953-3961; Schiffer, M. (1980) Biophys. J. 32, 230-232]. The measured radius of gyration (Rg) in H2O buffer is 24.0 +/- 0.4 A and in D2O buffer is 23.3 +/- 0.1 A; the calculated value of Rv (Rg in vacuo) is 24.0 A. The above values compare well with the calculated Rg value of 23.6 A when refined coordinates of the trigonal crystal form of the Mcg Bence-Jones protein are used. On the basis of a match point of 44.2% D2O concentration, the experimental partial specific volume is 0.74 cm3/g. The experimentally derived molecular weight of 47 000 is in very good agreement with that (45 500) calculated from the amino acid composition. For comparison with different Fab's (antigen binding fragments) exhibiting various "elbow bends" due to the flexibility of the switch peptide between variable and constant domains of the immunoglobulin chains, calculation of the Rg value of the Mcg dimer was performed as a function of the elbow bend. The Rg varied from 22.8 to 26.0 A as the elbow bend was opened from 100 degrees to 180 degrees; the maximum radius of gyration of the particle was 26.5 A with the switch peptide stretched by separating the variable and constant domains by an additional 1.5 A at an elbow bend of 180 degrees.

Bence Jones Protein↗
PubMed1982

Cytochemical localization and measurement of aerobic 3,3'-diaminobenzidine oxidation reactions in photosynthetically grown Rhodospirillum rubrum.

Photoheterotrophically grown Rhodospirillum rubrum mutant C oxidized 3,3'-diaminobenzidine (DAB) by two aerobic reactions. One reaction was light dependent. The other respiratory reaction occurred in the dark and could be inhibited by 0.1 M KCN. DAB was not oxidized under anaerobic conditions. Cytochemical results, obtained by reacting viable cells with DAB, indicated that the two reactions were associated with different regions of the cellular membrane system. Under dark conditions, oxidized DAB was deposited along the cytoplasmic membrane. In the light, the cytochemical reagent accumulated at both the cytoplasmic and the intracytoplasmic photosynthetic membranes. DAB oxidation activity was measured by sensitive spectrophotometric and polarographic techniques. Under 880 nm actinic illumination, the rate of DAB oxidation was about 5.5-fold faster than in the dark. Determination of O2 consumption associated with the two DAB oxidation reactions showed that white light stimulated the rate of O2 uptake by about the same extent.

3,3'-Diaminobenzidine↗
PubMed1981

Computer simulation of protein self-association during small-zone gel filtration. Estimation of equilibrium constants.

A simulation is developed that qualitatively describes the small-zone-gel-filtration behaviour of a reversibly associating protein. The results reflect the dependence of the apparent molecular weight of a reversibly associating protein on the equilibrium constant (KD) and initial concentration of the protein as well as the column length. The behaviour of a protein on an individual column is characterized and thus a means is provided for estimation of KD. The procedure is extended to describe the behaviour of a mixture of two proteins capable of heterologous as well as homologous association. This computer simulation has been applied in association studies of immunoglobulin light chains [Stevens, Westholm, Solomon & Schiffer (1980) Proc. Natl. Acad. Sci. 77, 1144--1148]. The KD value determined for the Bence--Jones protein Au (10(5) M-1) is close to the value (6.6 X 10(4) M-1) determined by other methods [Maeda, Steffen & Engel (1978) Biophys. Chem. 9, 57-64].

Bence Jones Protein↗