THE ISOLATION OF LYSOSOMES FROM EHRLICH ASCITES TUMOR CELLS FOLLOWING PRETREATMENT OF MICE WITH TRITON WR-1339

A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80–90-fold purification over the homogenate, and a 15–18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-β-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction

Non-glycolytic pathways of metabolism of glucose

ix+276hlm.;24c

Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II

alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed