Comparison of Surface Membranes Isolated from Cultured Murine Neuroblastoma Cells in the Differentiated or Undifferentiated State

Cloned cell lines derived from the murine C1300 neuroblastoma may be grown in suspension (Spinner) culture. Upon transfer to monolayer culture, morphological differentiation (appearance of long processes) may be induced under a variety of conditions. A method for the isolation of surface membranes from both undifferentiated and differentiated cultures has been developed. The yield (60 to 70 %) is very high and few contaminants were observed. The polypeptide composition of the surface membrane fraction from both types of cultures was very similar as determined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Following incorporation of radioactive leucine into the cultures for 60 hours, the distribution of radioactivity among surface membrane proteins was similar in preparations from differentiated and undifferentiated cells. When radioactive glucosamine was used in a similar experiment there was a preferential incorporation of radioactivity into a protein from differentiated cells with an apparent molecular weight of 105,000. Glucosamine was also preferentially incorporated into a soluble protein of the same molecular weight in differentiated cells. The appearance of this protein on the surface membrane was not due to contamination by the soluble protein, since in differentiated cells radioactive fucose was preferentially incorporated into this membrane protein, but not into the corresponding soluble protein. Synthesis of the glycoprotein of molecular weight 105,000 could also be induced by N6,O2'-dibutyryl cyclic 3' : 5'-adenosine monophosphate (Bt2cAMP) under conditions where morphological differentiation was prevented (Spinner culture). Iodination of proteins exposed on the cell surface was catalyzed by lactoperoxidase in the presence of hydrogen peroxide. A protein of molecular weight 78,000 was preferentially exposed on the surface of differentiated cells. This effect always accompanied morphological differentiation and could not be duplicated by Bt2cAMP treatment of suspension culture cells.