DISSECTION OF HEPATIC RECEPTOR-MEDIATED ENDOCYTIC PATHWAYS USING SELF-GENERATED GRADIENTS OF IODIXANOL (OPTIPREP)

Iodixanol is a new, nonionic, iodinated density gradient medium which has an advantage over other similar media in that it rapidly forms sel f-generated gradients in vertical or near-vertical rotors. Endocytosis of Tc-99m-labeled neogalactosyl albumin (Tc-99m-NGA), a synthetic lig and for the asialoglycoprotein receptor, was studied by administering the ligand as a short pulse to perfused rat livers operating under sin gle-pass conditions. Intracellular processing was arrested at various times after the pulse and the resultant homogenate cleared of nuclei a nd heavy mitochondria by centrifugation at 3000g for 10 min. After adj ustment to 12.5% (w/v) iodixanol, the 3000g supernatants were centrifu ged at 350,000g for 60 min to form the gradients in which early, clath rin-containing vesicles, low-density endosomes, and lysosomes were wel l-resolved. Tc-99m-NGA bound to the sinusoidal membrane could be parti ally resolved from clathrin-containing vesicles by inclusion of 1 mM C aCl2 in the homogenization and gradient buffers. Two populations of ea rly clathrin-containing vesicles could be resolved by rate-zonal centr ifugation in preformed iodixanol gradients. Thus, iodixanol is an exce llent density gradient medium for the rapid and efficient resolution o f endo some compartments. (C) 1998 Academic Press.