Recycling of surface sialoglycoconjugates in HTC and HeLa cells

Abstract

Surface sialoglycoconjugates of HeLa and HTC cells were labeled with NaB[3H]4 after oxidation of NaIO4. The labeling procedure cleaves the sialic acids to a neuraminidase-sensitive 7-carbon derivative, 5-acetamido-3,5-dideoxy-L-arabino-heptulosonic acid, termed AcNeu7 (Van Lenten, L., and Ashwell, G. (1971) J. Biol. Chem. 246, 1889-1894). After labeling, the radioactivity is lost from both cell types with biphasic kinetics. The half-time for the fast phase is about 4-5 h; the slow phase has a half-time of 100-200 h. About 30 h after labeling and at later times, approximately 30% of the cell-associated radioactivity is susceptible to removal by external neuraminidase, suggesting an exchange with an internal pool that is twice the size of the surface pool. An internal pool of relatively high specific activity compared to the surface was generated by labeling as above, followed by a period of time to allow internalization and enzymatic removal of external neuraminidase-sensitive radioactivity. During subsequent reincubation in growth medium, the surface became relabeled from the internal pool, again reaching a 30% neuraminidase-sensitive plateau. The relabeling of the surface was confirmed by radioactivity measurements on isolated plasma membranes. [3H]AcNeu7 cannot be reutilized by these cells in the de novo membrane biosynthetic pathway. The argument is made that the labeled sialoglycoconjugates are recycling intact through the internal pool.