Separation of visual pigments on columns of controlled-pore glass beads

Plasma membranes have been isolated from 3T3 and SV-3T3 cells grown in culture. Cells were harvested mechanically and disrupted in simple isotonic buffered salt solutions without resorting to hypotonic swelling or chemical membrane “hardeners.” The method of storing collected cells, the cell concentration during disruption, and the method of mechanical disruption were found to be significant variables affecting the yield of plasma membranes. The plasma membranes were separated from mitochondria and other cellular organelles by a single centrifugation through a step sucrose gradient containing a viscosity barrier of Dextran T-500 (modified fromA. S. Sun and B. Poole (1975)Anal. Biochem.68, 260). The isolated plasma membranes were located by assay for the “marker” enzyme, alkaline phosphatase (EC 3.1.3.1). The isolated plasma membrane fraction was free of mitochondrial and essentially free of lysozymal and endoplasmic reticulum contamination, which were assayed by measuring cytochrome c reductase, arylsulfatase, and hydrolysis of α-naphthol acetate, respectively. Of the enzymes tested, the phosphodiesterase activity was found to be the most specific assay for the plasma membrane from culture mouse fibroblast cells. The 5′-nucleotidase (EC 3.1.3.5) activity, the other plasma membrane marker, was extremely low in activity and gave an additional peak of activity when 5′-adenilic acid was used as substrate as compared to the expected single peak obtained with 5′-cytidilic acid as substrate. Overall recovery of isolated plasma membranes was greater than 75% as measured by the final recovery of phosphodiesterase activity.