1. We have measured specific [ 3 H]ouabain binding and ouabain sensitive 86 rubidium influx in intact human lymphocytes incubated for up to 7 days in media containing different concentrations of fetal calf serum and human serum. 2. Incubation for periods of up to 7 days with fetal calf serum and human serum produced increases in both specific [ 3 H]ouabain binding and ouabain sensitive 86 rubidium influx that were dependent on concentration and time. 3. Neither specific [ 3 H]ouabain binding nor ouabain sensitive 86 rubidium influx was altered when dialysed serum was used, suggesting that both fetal calf serum and human serum contain a dialysable factor or factors which stimulate specific [ 3 H]ouabain binding and ouabain sensitive 86 rubidium influx in intact human lymphocytes. 4. To further elucidate the mechanisms underlying these changes we also measured the activity of two other enzymes of the lymphocyte plasma membrane, 5′-nucleotidase and γ-glutamyltransferase, the uptake of [ 3 H]thymidine by the intact cells, and the effects of cycloheximide, puromycin, and anisomycin, inhibitors of protein synthesis. 5. The activity of 5′-nucleotidase was increased after incubation of the lymphocytes in fetal calf serum for 72 h, but the activity of γ-glutamyltransferase was not changed, suggesting some selectivity of the stimulatory effect. 6. Measurements of [ 3 H]thymidine uptake by the lymphocytes showed that the major part of the observed changes in specific [ 3 H]ouabain binding and ouabain sensitive 86 rubidium influx was not attributable to transformation of the lymphocytes to lymphoblasts. 7. All three inhibitors of protein synthesis prevented the increase in specific [ 3 H]ouabain binding due to fetal calf serum.

1. Parenchymal, Kupffer and biliary tract cells were isolated from normal rat liver by perfusion with collagenase solution. 2. The specific activities (munits of enzyme activity/mg of protein) of marker enzymes for the principal subcellular organelles were determined in the isolated cell homogenates and compared with whole liver homogenates. 3. The cells were disrupted and the extracts subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. Lysosomal integrity was determined by assaying latent β- N -acetylglucosaminidase in the extracts. 4. Similar subcellular distributions were found for lysosomal, endoplasmic reticulum and plasma membrane marker enzymes in the whole liver and in parenchymal and biliary tract cells. In Kupffer cells, the proportion of these enzymes in the cytosol was significantly increased compared with the other fractions. In addition the equilibrium densities of the various organelles in these cells were lower than those from parenchymal cells.

1. The distribution of three membrane-bound enzymes, alkaline phosphatase, 5′-nucleotidase and γ-glutamyl transferase, has been examined in rat liver fractionated into parenchymal cells, a Kupffer cell fraction and a biliary tract fraction by perfusion with collagenase solution. 2. In control rat livers the highest enzyme concentrations were found in Kupffer cell preparations. The fraction enriched with biliary tract cells showed concentrations two- to fourfold those in isolated parenchymal cells. 3. At 24 h after ligation of the bile duct there was a fourfold increase in the alkaline phosphatase activity in parenchymal cells and a 2·5-fold increase of the same enzyme in the biliary tract fraction. The concentration of alkaline phosphatase in Kupffer cells remained unchanged. 4. 5′-Nucleotidase and γ-glutamyl transferase concentrations tended to decrease after bile-duct ligation for 24 h, the most marked changes being found in Kupffer cells. 5. After bile-duct ligation for 7 days the overall activities of hepatic alkaline phosphatase and γ-glutamyl transferase increased fourfold and there was a small rise in 5′-nucleotidase. For alkaline phosphatase and γ-glutamyl transferase the changes were found primarily in parenchymal cells and in the biliary tract fraction. There was a small rise in the activity of 5′-nucleotidase in parenchymal cells and a fall in activity in Kupffer cells. 6. These data show marked differences in the distribution and behaviour of three enzymes which are often used as alternatives in the assessment of patients with liver disease.