Mapping of lymphocyte surface polypeptide antigens by chemical cross-linking with BSOCOES.

A base-reversible, 13 Å homo-bifunctional chemical cross-linking reagent, selected for its reactivity at 0°C but not previously used in membrane studies, was used to measure topographic relationships between Rauscher murine leukemia virus envelope glycoprotein (R-MLV gp70, 70,000 daltons) or mouse major histocompatibility (H-2,46,000 and 12,000 daltons) antigens and their neighboring polypeptide antigens on the surface of R-MLV-transformed and virus-producing C57BL/6 (H-2b) mouse leukemia cells (RBL-5A). RBL-5A cell surface polypeptides were radioiodinated and then cross-linked with bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSO-COES) at 0°C. An NP-40 extract was prepared, polypeptides were precipitated with specific antiserum and analyzed by two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In a first dimension SDS-polyacryl-amide gel, the polypeptides were separated with BSO-COES-links intact, and then their cleavage products were separated by using SDS-PAGE in a second dimension. Anti-R-MLV gp70 serum precipitated 140,000-dalton, 210,000-dalton, and high molecular weight (HMW) polypeptides from the surface of BSOCOES-linked RBL-5A cells. These homo-dimers (140,000 daltons) and homotrimers (210,000 daltons) of a 70,000 dalton polypeptide may be the R-MLV gp70-containing knob structures visualized by electron microscopy on the surfaces of R-MLV-infected cells. Anti-H-2b serum precipitated 58,000-dalton dimers comprised of one heavy polypeptide chain (46,000 daltons) and one light chain (12,000 daltons), intrachain-linked heavy chain monomers, and several other species of higher M.W. polypeptides. BSOCOES cross-linking of cells at 0°C in conjunction with surface radioiodination, immunoprecipitation with specific antiserum and separation of cross-linked polypeptides and their cleavage products by two-dimensional reducing SDS-PAGE can be used in the mapping of nearest polypeptide neighbors on the cell surface.