Abstract
Operating techniques for zonal rotors have been developed, and computer programs written to simplify handling of the data obtained. In particular, the size distribution of fragments of the plasma membranes of liver and hepatoma cells was investigated. In liver, two distinct sizes of fragment were found, large sheets, deriving from several, adjacent cells, and small vesicles. The former were practically absent from the hepatoma studied. The A-XII zonal rotor was used to isolate the large sheets of membrane found in liver. Removal of erythrocytes by perfusion or homogenisation in 0. 08 M sucrose, was essential. Using a suitable gradient, plasma membrane fragments were separated as a band, clear of all other organelles, but contaminated by trapped material. This was removed by a further homogenisation, followed by flotation from sucrose of density 1.19. The specific activity of the plasma membrane marker, 5'-nucleotidase, was increase in this 'pure' plasma membrane fraction to 15 x that of the homogenate if unperfused liver was used as the starting material, 30 x if perfused liver was used. The enzymic composition of the isolated plasma membranes was studied, and computing programs developed which will be capable of quantitating the distribution of material between the plasma membrane and other cell organelles. In addition, the free cytoplasmic subribosomal particles of liver and hepatoma cells were studied by zonal centrifugation followed by concentration and either resedimentation on sucrose gradients, equilibrium banding on CsCl gradients, or extraction of the RNA followed by examination of its size distribution, labelling kinetics and base composition. In liver, the '40 S' subribosomal particles were heterogenous. At least two forms of small ribosome subunits, sedimenting at 35 S and 45 S respectively, were identified and it appeared probable that informosomes co-sedimented with the 35 S form. In hepatoma, only one form of the small ribosome subunit was found. Hepatoma contained equal numbers of large and small subunits. In liver, large subunits were much scarcer than small subunits.