Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation.
We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.
Ripoche
V
V
International Institute of Cellular and Molecular Pathology, Université de Louvain, Belgium.
Beauwens
R
R
Bouisset
M
M
Amar-Costesec
A
A
Beaufay
H
H
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002024:Bufo marinus; D002374:Catalase / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000201:enzymology / Q000648:ultrastructure; D002498:Centrifugation / Q000379:methods; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000648:ultrastructure; D004848:Epithelium / Q000201:enzymology / Q000648:ultrastructure; D006867:Hydrolases / Q000032:analysis; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D010088:Oxidoreductases / Q000032:analysis; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure; D001743:Urinary Bladder / Q000201:enzymology / Q000648:ultrastructure*
D015415:Biomarkers; D010088:Oxidoreductases; D002374:Catalase; D006867:Hydrolases