The importance of salt concentration during nitrogen cavitation of MPC-11 cells for the isolation of heavy rough endoplasmic reticulum membranes.
When MPC-11 cells are disrupted by nitrogen cavitation in the presence of buffer containing 25-40 mM KCl then endoplasmic reticulum membranes can be separated into three subfractions by sucrose density gradient centrifugation: heavy rough (HR), light rough (LR) and smooth (S) membranes. An increase in the salt concentration of the buffer to 50 mM or above results in the occurrence of only the LR and S membranes in gradients. However, when cells equilibrated at high pressure in the bomb in 100 mM KCl buffer were expelled into a diluting buffer such that the final buffer concentration was reduced to 25 mM KCl upon cell disruption, then appreciable amounts of HR membranes are observed in sucrose gradients. The results would suggest that salt concentrations above 25-40 mM KCl stabilize the interaction between HR membranes and the cytoskeleton to such a degree that these membranes are pelleted at low speed together with the nuclei. The yields of LR and S membranes are apparently not affected to any significant degree by altered salt concentration.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D004721:Endoplasmic Reticulum / Q000648:ultrastructure*; D051379:Mice; D009994:Osmolar Concentration; D013395:Sucrose
D013395:Sucrose