SUMMARY
Serological and genetic analyses of H‐2 antigens indicate that each K or D region allele controls two highly polymorphic antigenic sites, the alpha site corresponding to the private specificity and the gamma site corresponding to the long public specificities. Studies of cell surface distribution and biochemical characteristics of the alpha type specificity H‐2.4 and gamma type specificity H‐2.28 in the product of a D region allele, Dd, demonstrate that these specificities are carried on two different polypeptide chains. Accordingly, two distinct and polymorphic genes are postulated to code for the product controlled by the D region of H‐2.
A purification procedure applied to H-2 antigens (controlled by the H-2 locus) was used for the characterization of HL-A substances (HL-A2 and HL-A7) (controlled by the HL-A locus) solubilized from lymphoid cell membranes by delipidation. Upon Sephadex chromatography the serologically active material had an apparent M , of 32000, and displayed, in relation to H-2 antigens, a faster electrophoretic mobility and higher p l values. One sulfhydryl group and one (intrachain) disulfide bond were present in partially purified HL-A preparations. Reduction and aminoethylation in 4 M urea did not influence the electrophoretic migration of HL-A active substances, suggesting that, as already demonstrated for H-2 alloantigens, in HL-A molecules the genetic determinants are present on single polypeptide chains. Highly purified HL-A alloantigens, like H-2 substances, revealed a high degree of microheterogeneity. These findings further support the concept of the genetic and structural homology of the main histocompatibility systems in mouse and man.
Mouse histocompatibility antigens were solubilized from lymphocyte membranes by limited papain degradation. Spleens from Balb/c mice (H-2&), enlarged by administration of lymphoma cells, were used for the membrane preparations. The solubilized protein components were purified by ion-exchange chromatography, gel filtration and discontinuous polyacrylamide-gel electrophoresis. A further characterization was achieved in a two-dimensional protein mapping system using disc-electrophoresis and isotachophoresis in the first and second dimension, respectively. The purification was monitored for alloantigens H-2.4(D) and H-3.21(K) ( H -2 D and H -2 K represent two regions of the H -2 system). On Sephadex (3-100 partially purified H-2-alloantigen chromatographed in a region of Mr = 35500. The p I of these substances showed a maximum a t pH 5.5 and 5.3 for H-2.4 and a t 5.2 for H-2.31. Free SH-and dithio-groups were determined with 5,5'-dithiobis(Z-nitrobenzoic acid). 1.56 pmol SH/pmol alloantigenic material was found, but no SS-bond could be detected after alkaline cleavage. This suggests the absence of covalently linked protein subunits in papain-solubilized molecules bearing single private specificites. This was confirmed after reduction and alkylation in 4 M urea: 30-50°/, of the alloantigenic activity was retained and shown, in polyacrylamide-gel electrophoresis, to migrate in the original region of activity (RBPB 0.29 and 0.33 for H-2.4; about 0.33 for H-2.31, BPB = bromophenol blue). After reductive alkylation gel filtration resulted in a further purification of the alloantigenic material. Highly purified substances showed a diffuse staining pattern and a broad serological profile after electrophoresis. We assume this heterogenity to be a genuine property of H-2 antigens. Structural and genetic implications OF these findings arc discussed.
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