The isolation of endosome-derived vesicles from rat hepatocytes.
Intracellular 5'-nucleotidase involved in membrane circulation in rat hepatocytes is latent, and is protected from inhibition when whole cells are incubated with inhibiting antiserum at 2 degrees C [Stanley, Edwards & Luzio (1980) Biochem. J. 186, 59-69]. These two criteria were used to identify intracellular membrane vesicles containing 5'-nucleotidase on Ficoll density gradients. A sharply defined turbid band containing intracellular 5'-nucleotidase isolated on density gradients was further fractionated by immunoadsorption of plasma-membrane fragments derived from the cell surface of surface-inhibited cells on to an anti-(immunoglobulin G) immunoadsorbent. The resulting non-adsorbed membrane fraction consisted of vesicles of uniform size (approx. 65 nm diam.), but was not identifiable as any known organelle. This fraction could account for approx. 5% of the total cell 5'-nucleotidase activity, and the enzyme activity measured was 55% latent. The fraction had a restricted polypeptide composition but similar phospholipid composition compared with plasma membrane. We suggest that the vesicles observed in this fraction were derived from the endocytic pathway.
D015720:5'-Nucleotidase; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D007163:Immunosorbent Techniques; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009708:Nucleotidases / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D051381:Rats
D010743:Phospholipids; D009708:Nucleotidases; D015720:5'-Nucleotidase