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J Lamy

Publications and source records attributed to J Lamy.

At least 37 records · Page 2Linked to original sources
PubMed1985

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy.

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1983

Cell-free synthesis of hemocyanin from the scorpion Androctonus australis. Characterization of the translation products by monospecific antisera.

Translation of Androctonus australis poly(A)-RNA in vitro led to a number of polypeptides products (8-10) of 70-73 kDa analyzed by two-dimensional gel electrophoresis and identified by immunoprecipitation with an anti-(dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico-chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross-reacting subunits. Each polypeptide chain is coded by a different messenger without significant post-translational events. Hemocyanin could be detected among the translation products of the poly(A)-RNA isolated from the cuticle under the carapace.

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PubMed1983

Immunological correlates between multiple isolated subunits of Androctonus australis and Limulus polyphemus hemocyanins: an evolutionary approach.

Immunological cross-reactivities between isolated subunits of the scorpion Androctonus australis (Aa) and of the horseshoe crab Limulus polyphemus (Lp) hemocyanins were studied using subunit-specific antibodies prepared through immunoadsorption to pure immobilized subunits. Rocket immunoelectrophoreses of the various subunits of both hemocyanins were carried out at constant antigen concentration against the various subunit-specific antibody preparations. Then the data were analyzed through factorial correspondence analysis and compared to the respective intramolecular locations of the subunits in both hemocyanins. The results show that the dimeric subunits located in the central part of each (4 X 6)meric structure (Aa whole molecule and Lp half molecule) were strongly preserved. In addition, the (8 X 6)mer-forming subunit of Lp hemocyanin (LpIV) and the subunit occupying the same intramolecular position in Aa hemocyanin (Aa5A) were also strongly preserved. Besides the strong antigenic relatedness, less pronounced crossed immunoprecipitations or no precipitation at all were observed between subunits with homologous positions suggesting a minor structural and/or functional roles for these subunits. All the antigen-antibody combinations leading to an absence of immunoprecipitation were screened for the presence of soluble immunocomplexes by radioimmunological tests. In all cases, soluble immunocomplexes were observed. These results suggest the following evolution scenario. First, the central dimeric subunits, responsible of the dodecamer aggregation (Aa3C and 5B and LpV and VI) were already differentiated when Merostomata diverged from Arachnida. Second, the differentiation of the (8 X 6)mer-forming subunit occurred in the Merostomata ramification in a preserved subunit already possessing a functional advantage. Third, the differentiation of subunits Aa3A and Aa3B recently occurred in the scorpion ramification.

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PubMed1982

Architecture of Limulus polyphemus hemocyanin.

The architecture of the 48-meric hemocyanin of the horseshoe crab Limulus polyphemus has been determined from electron micrographs of whole (48-mer) molecules and half- (24-mer) molecules. The assembly of hexamers of kidney-shaped subunits can produce two dodecameric enantiomorphs, designated as right and left. The assembly of 24-mers can again result in two enantiomorphs. By taking into account the rocking effect described by Van Heel and Frank [Van Heel, M., & Frank, J. (1981) Ultramicroscopy 6, 187-194], we deduced that the 24-meric half-molecule is made up of two copies of the left dodecameric enantiomorph. In addition, the two constituent dodecamers of the half-molecule are shifted with respect to a symmetric head-to-tail arrangement, which makes it possible to distinguish two different faces of the 24-mer, termed flip and flop. A model of the whole molecule was built from two copies of the 24-meric half-molecule. This model presents the four distinct views observed in the electron microscope (pentagon, ring, cross, and bowtie). In addition, the model shows the pentagonal view to exist in two varieties: symmetric and asymmetric. An analysis of electron micrographs presenting the pentagonal view by image processing using the statistical technique of correspondence analysis confirmed the existence of two types of pentagonal view, representing projections of a molecule built from two copies of the left 24-meric enantiomorph. In addition, the best fit between the averaged molecule images and the possible models was observed with a flop-flop inter 24-mer contact. The final model is shown in a series of stereo views produced by computer graphical techniques.

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PubMed1982

A refined quaternary structure of Androctonus australis Hemocyanin.

The quaternary structure of the (4 X 6)-mer hemocyanin from the Scorpion Androctonus australis previously published [Lamy, J., Bijlholt, M. M. C., Sizaret, P.-Y., Lamy, J., and van Bruggen, E. F. J. (1981) Biochemistry, 20, 1849-1856] has been refined. The relative positions in the half molecule of subunits Aa 3A and Aa 3B compared to those of Aa 3C and Aa 5B have been established by double labeling of the (2 X 6)-mer with binary mixtures of subunit-specific Fab fragments. The results show that subunits Aa 3B and Aa 5B are located in the same hexamer while Aa 3A and Aa 3C are in the other half of the (2 X 6)-mer. The choice of the enantiomer was deduced from a careful examination of electron micrographs of the native molecule. Finally a position was assigned to each of the 24 subunits on the flip and flop faces as defined by Van Heel and Frank [Ultramicroscopy, 6, 187-194 (1981)].

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PubMed1979

Hemocyanins in spiders, VII. Immunological comparison of the subunits of Eurypelma californicum hemocyanin.

The isolated subunites of Eurypelma californicum hemocyanin were studied by aid of antibodies raised against whole, dissociated hemocyanin. The proportion of impurities was found to be low in almost all subunits. There was no cross reaction between the individual chains, and the total number of antigenically different subunits was found to be seven, confirming results obtained by different methods. If an artificial mixture prepared from purified subunits is compared to whole, dissociated hemocyanin, an overall very similar pattern is obtained but differences appear which are due to specific interaction.--The dimeric subunit 4D was shown to be a heterodimer (asymmetric dimer) composed of chains b and c4.

Animals↗
PubMed1977

[Structure of scorpion hemocyanin: heterogeneity of its sub-units].

Six dissociation products isolated from the Androctonus australis scorpion haemocyanin are studied through analytical ultracentrifugation and acrylamide-SDS electrophoresis: the results show that one of these fractions exists as a dimer and that the five others are monomers. A genetical study shows that two subunits at least can undergo mutations.

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