Time-dependent alteration in endoplasmic reticulum membrane profiles during in vitro incubation of Krebs II ascites cells.

Krebs II ascites cells were harvested from the mouse peritoneum 6-8 days after inoculation and incubated in vitro in roller suspension culture for up to 22 hr. Within 2 hr of incubation a large portion of the cells entered S phase as judged by the incorporation of 3H-thymidine into DNA. The incorporation of radioactive precursors into phospholipid, protein and RNA increased rapidly during in vitro incubation indicating a high degree of macromolecular synthesis. The rates of incorporation were maximal within 4 hr of incubation. When cells labeled with 3H-choline were disrupted by nitrogen cavitation and endoplasmic reticulum (ER) membranes analyzed for subfractions on discontinuous sucrose gradients, it was observed that only small amounts of radioactivity could be detected in the HR region after 1/2 hr incubation while 63.2% of total radioactivity in ER membranes appeared in the LR fraction. Between 8-18 hr there was a considerable increase in the amount of HR membranes. Of the total radioactivity in ER membranes that in the HR fraction increased from 16.9% to 53.9% during this period. There were only small changes in the amounts of radioactivity in LR and S membranes between 8 and 18 hr. The results suggest a time-dependent appearance of ER membrane subfractions during a 22 hr period of in vitro incubation of Krebs II ascites cells.