Purification and characterization of two biosynthetic intermediates of ovalbumin

Abstract

Ovalbumin was extracted with a buffered Triton X-100 solution from oviduct slices incubated with [35S]methionine or [2-3H]mannose and was purified by CM-cellulose and Sephacryl S-200 column chromatography. The labeled ovalbumin was fractionated on a concanavalin A (Con A)/Sepharose column at room temperature. Six fractions were separated: Two unadsorbed fractions, OA and OB; and four adsorbed fractions, OC, OD, OE, and OF. Fractions OA, OB, OC, and OD corresponded to the four fractions prepared from unlabeled ovalbumin as previously reported (Iwase, H. et al. (1981) J. Biol. Chem. 256, 5638-5642). Fractions OE and OF were novel constituents of the labeled ovalbumin, although a small amount of OE was also present in unlabeled ovalbumin. Pulse-chase experiments indicated that both OE and OF behaved as biosynthetic intermediates of the other ovalbumin fractions, namely OA, OB, OC, and OD. Partial structural analyses involving endo-beta-N-acetylglucosaminidase treatment and subsequent Bio-Gel P-4 chromatography showed that both OE and OF were composed of components bearing high mannose-type sugar chains which consisted of Man9GlcNAc2, Man8GlcNAc2, or Man7GlcNAc2. We failed to distinguish OF from OE on the basis of their carbohydrate chains. However, on SDS slab gel electrophoresis, the OF band migrated slower than the OE band.