Abstract
The main aims have been to separate membrane fragments as found in liver homogenates, and to develop and apply methods for cytochemical examination of the fractions obtained. Fixation of tissue fractions in glutaraldehyde and incubation in lead-containing media resulted in a marked inhibition of enzyme activities in biochemical and cytochemical assays. Acceptable cytochemical results were obtained with unfixed fractions and also with sections prepared by a novel method. Incubation studies indicated no increase with time in the proportion of microsomal membrane fragments exhibiting 5'-nucleotidase or glucose-6-phosphatase activities, but a lag period was noted for glucose-6-phosphatase. Zonal centrifugation of 'post-lysosomal' fractions in a 'B-IV' rotor with a sucrose gradient gave three main peaks corresponding to soluble material, smooth-surfaced membrane fragments and rough-surfaced fragments. 5'-Nucleotidase (a plasma membrane marker) and glucose-6-phosphatase (a cytomembrane marker) were demonstrated biochemically and cytochemically in the smooth-surfaced fraction. Separation of 5'-nucleotidase and glucose-6-phosphatase was obtained in the presence of magnesium but not with caesium. No marked change in the position of the enzyme peaks resulted from the use of 0.12 M sucrose or 0.14 M NaCl in place of 0.25 M sucrose, or from different centrifugation times. Effective separation of 5'-nucleotidase and glucose-6-phosphatase was obtained by using sigmoid gradients. The resolution was not improved by the presence of deoxycholate. The distribution of ADPase and magnesium-activated ATPase closely paralleled 5'-nucleotidase, while IDPase and UDPase showed a distribution intermediate between that of glucose-6-phosphatase and 5'-nucleotidase. Fractionation of a crude nuclear preparation led to the demonstration of 5'-nucleotidase on sheets of plasma membrane and bile canaliculi, while glucose-6-phosphatase activity was detected in small vesicles adhering to the plasma membrane. Cytochemical observations on hepatoma sections gave no evidence that the localizations of 5'-nucleotidase, glucose-6-phosphatase and ATPase within the 'parenchymal' cells differed from those found in normal liver. Zonal centrifugation of a 'post-lysosomal' fraction from a transplanted hepatoma showed a similar pattern to that of normal liver, as did a cytochemical study of hepatoma fractions.