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Biomedical subjects

A F Williams

Publications and source records attributed to A F Williams.

At least 253 records · Page 14Linked to original sources
PubMed1975

Analysis in deoxycholate of three antigenic specificities associated with the rat Thy-1 molecule.

Three antigens similar in tissue distribution can be identified on rat thymocytes; the Thy-1.1 antigen, a rat specific xenoantigen, and a rat-mouse cross-reacting xenoantigen. To determine if these three antigens were on the same molecule their behavior in detergent-solubilized extracts from thymocytes was studied. Membrane fragments containing Thy-1.1 activity were prepared by a rapid method involving the use of Tween-40 detergent, and were solubilized in deoxycholate. The 150 000 x g supernatant from this extract contained approximately 50% of the original Thy-1.1 and xenoantigen activity. The supernatant was chromatographed on Sephadex G-200, and subjected to zone sedimentation on sucrose gradients in H2O and 2H2O to determine the hydrodynamic properties of the antigens. The three antigens migrated in identical fashion in all cases, and behaved as a molecule of 28 000 daltons molecular weight. When the antigenically active fraction, recovered after chromatography on Sephadex G-200, was passed through an immunoabsorbent consisting of rabbit antibody to one of the xenoantigens, all three antigens were equally depleted compared with passage through a control column. The results of these experiments suggested that Thy-1.1 antigen and the two xenoantigens were closely associated and most probably all on the Thy-1 molecule.

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PubMed1975

Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

Animals↗
PubMed1975

Purification of the Thy-1 molecule from rat brain.

The present paper describes the characterization of the Thy-1 molecule from rat brain. The molecule was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate. In the solubilized form the Thy-1 antigens were associated with a homogenous component with the following hydrodynamic properties: s20,w=2.2s,v=0.72ml/g and Stokes radius=3.0nm. The mol.wt. of the deoxycholateantigen complex was estimated to be 27000; these values are not significantly different from those obtained thymocyte Thy-1. Brain Thy-1 was further purified by affinity chromatography with lentil lectin coupled to Sepharose 4B, and more than 80% of the antigen was bound. The material eluted with methyl alpha-D-glucopyranoside was then filtered on a column of Sephadex G-200, and only one glycoprotein was found in the antigenically active fraction. On sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the glycoprotein was very similar to the Thy-1 from thymocytes that binds to lentil lectin. Its apparent mol.wt. on 12.5% acrylamide gels was 24000, and it electrophoresed as a symmetrical band. Brain Thy-1 was antigenically indistinguishable from thymocyte Thy-1 when analysed with rabbit antisera raised against brain or thymocyte Thy-1.

Animals↗
PubMed1974

The origin of cell surface immunoglobulin of marrow-derived and thymus-derived lymphocytes of the rat.

The origin of immunoglobulin on the surface of TDL in the rat has been studied by comparing the binding of purified alloantibodies recognizing the Ig-1a allotype of rat light chain, with that of rabbit antirat Fab antibodies. Both reagents labeled all TDL from rats of the DA strain (Ig-1a) with two categories of cells being detected; one binding 100-2,000 molecules of antibody, the other 10,000-100,000 molecules. These categories were likely to be synonomous with T and B cells, respectively. The [(125)I]antiallotype antibodies did not bind to TDL from rats of the PVG/c strain (Ig-1b). When the binding to TDL from (PVG/c x DA)F(1) animals was studied it was found that allelic exclusion occurred in the heavily labeled cells, but not in the lightly labeled ones. Furthermore, when lymphocytes of one allotype were transferred to irradiated recipients of the opposite allotype and recovered from the TDL or spleen of the recipient 20-30 h later, the immunoglobulin on heavily labeled cells was of the donor type, while that of lightly labeled ones bore the recipient marker. Thus heavily labeled cells (B lymphocytes) had synthesized their own immunoglobulin while lightly labeled cells (T lymphocytes) had acquired theirs passively by adsorption. The class of immunoglobulin on lightly labeled cells was also studied and it was found that [(125)I]anti-IgM antibodies bound to an extent approaching the [(125)I]anti-Fab binding, while [(125)I]anti-IgG(2a+2b) antibodies gave much less binding.

Animals↗
PubMed1974

Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents.

1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s(20,w) values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen-detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).

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