Lyt-2 and Lyt-3 alloantigens from NP-40 extracts of 125I-labeled mouse thymocytes were analyzed on 2-dimensional polyacrylamide gels by nonequilibrium pH gradient electrophoresis (NEPHGE) and by electrophoresis in 1-dimensional gels in sodium dodecyl sulfate (SDS-PAGE). Fractions of extracts not bound by DEAE-Sephadex A-50 were found to be rich in Lyt-2 and Lyt-3 antigens, and these were precipitated with monoclonal and conventional mouse anti-Lyt-2 and anti-Lyt-3 reagents. Results of NEPHGE of both anti-Lyt-2 and anti-Lyt-3 precipitates under reducing conditions were identical and revealed major labeled components that migrated as very basic molecular species of apparent m.w. 35,000 (35K) and 30K. A poorly labeled basic component of apparent m.w. less than 30K was also observed. The 30K species and, to a lesser extent, the 35K species exhibited charge heterogeneity, which could be eliminated by digestion with neuraminidase. Thus, the charge heterogeneity does not reflect extensive amino acid sequence variability, but variation in the number of sialic acid moieties attached to potentially homogeneous glycoprotein species. Under nonreducing conditions, 1- and 2-dimensional gels contained major labeled species of apparent m.w. 65K that could be partially resolved into 2 components. Upon reduction and re-electrophoresis, the higher and lower m.w. 65K components gave rise to primarily the 35K and 30K species, respectively and in each case, some material of m.w. less than 30K was also seen. Thus, the 35K and 30K species may exist in thymocyte extracts and on intact thymocytes as (35K)2 and (30K)2 disulfide-dependent homo-dimers. Alternatively, if the component of m.w. less than 30K is present in quantities greater than suggested by its weak labeling with 125I, the 2 65K species may consist of 1 molecule of m.w. less than 30K disulfide-bonded to 1 30K or 1 35K molecule. Evidence from this and previous studies indicate that these molecules are associated with each other to form higher aggregates. Although the precise relationships of 35K, 30K and less than 30K species to each other and to the Lyt-2 and Lyt-3 alloantigenic determinants are not known, the present results bear upon the arrangement and possible function of these components on the cell surface. Also, they demonstrate the means by which these species can be isolated for detailed structural analysis.
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Reilly et al. (1980) studied this question.