Shuichi Karasaki

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Abstract Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μ moles·(mg protein) −1 ·h −1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.

Ultrastructural analysis of malignant conversion in vitro of epithelial cells derived from normal adult rat liver

Abstract A tumorigenic cell line of epithelial-type was established from a long-term culture of isolated liver cells from a normal adult Wistar rat. Electron microscopic examinations of foci of transformed epithelial cells in the primary culture revealed production of virus particles in association with membranes of the nuclear envelope, endoplasmic reticulum and cell surface. The epithelial cells were found in quiescent state and had intranuclear canalicular membranes. By trypsinization, the cells were initiated into a rapid replication and showed subcellular differentiation. Annulate lamellae and paired cisternae emerged in passage 1 . Viral production ceased at this stage. Close apposition of adjacent cells became prominent in passage 9 , when the cell line first revealed tumorigenicity upon transplantation into neonatal Wistar rats. Concomitantly, cytochemical tests gave a positive reaction of ATPase activity on the plasma membranes. Surface ATPase activity continued to show in the epithelial cells following passages in vitro as well as in tumor cells in vivo . The possible origin of epithelial cells in vitro from hepatocytes was suggested by the demonstration of peroxisomes and bile canalicular-like structures in the carcinoma cells.