Abstract
Rat liver plasma membranes (p.m.) were isolated from the crude nuclear fraction by the method of Hinton et al. (1970). Minor modifications, notably the use of younger rats, were made to overcome aggregation due to nuclear lysis which vitiated some preparations. The ribonuclease and phosphodiesterase activities of purified liver p.m. were examined and compared. The p.m. was found to be responsible for between 65 and 90% of the alkaline phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH 8.7 in the presence of 7.5mM MgCl2. Both enzymes were most active between pH 8.5 and 8.9, and both were at least 12 times more active in the presence of Mg than of EDTA. Density-gradient centrifugation indicated that after membrane solubilisation by detergent treatment most of the ribonuclease was released into a small fragment of the same size as that containing the phosphodiesterase. The HS zonal rotor was used to isolate plasma-membrane fragments from hepatoma. Initially, there were two major problems: aggregation of the p.m. with intact erythrocytes, and the appearance of a nucleoprotein gel from lysed nuclei. A method was developed for the preparation of p.m. but as the tumour progressed there was a fall in yield of membranes and an increase in contamination, probably by debris from necrotic cells. These difficulties were overcome by flotation of a partially purified p.m. fraction from homogenates adjusted to density 1.18. The resulting fraction was further purified by rate-zonal centrifugation in the HS rotor followed by a second flotation step to remove trapped material. The specific activity of the marker enzyme, 5'-nucleotidase, in this 'pure' p.m. preparation was over 20 times that of the homogenate. The specific activity of several phosphatases was found to be much higher than in liver p.m. The alkaline phosphodiesterase and ribonuclease activities of purified hepatoma p.m. were investigated. Phosphodiesterase specific activity was very similar to that in liver p.m. Alkaline ribonuclease specific activity in the presence of Mg was also very similar to liver, but there was more activity in the presence of EDTA in the hepatoma p.m. than in liver p.m.