FIGURE 3.
Tail-to-tail ChEL homodimerization. Upper gradient: 293 cells transiently expressing secretory ChEL-CD were pulse-labeled with 35S-amino acids and chased, and the secretion was resolved on 5–10% linear sucrose velocity gradients as described under “Experimental Procedures.” Fractions collected from the bottom (left side) were immunoprecipitated with anti-Tg antiserum and analyzed by non-reducing SDS-PAGE and fluorography. Monomers sedimented between fractions 20 and 25; dimers sedimented between fractions 15 and 20. Monomeric ChEL-CD migrated by SDS-PAGE with an apparent molecular mass of ∼70 kDa; covalent ChEL-CD dimer migrated with an apparent molecular mass of ∼140 kDa. Lower gradient: 293 cells transiently expressing the secretory ChELG-CD mutant were analyzed as above. None of the ChELG-CD contained an intermolecular covalent bond; all molecules migrated to the monomer peak in the gradient.