Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats.
A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.
D015073:2-Acetylaminofluorene / Q000633:toxicity; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D004032:Diet; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000201:enzymology / Q000648:ultrastructure; D006965:Hyperplasia / Q000139:chemically induced; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000187:drug effects / Q000473:pathology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D008055:Lipids; D011506:Proteins; D012313:RNA; D015073:2-Acetylaminofluorene