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 self-generated gradients in vertical or near-vertical rotors. Endocytosis of 99mTc-labeled neogalactosyl albumin (99mTc-NGA), a synthetic ligand for the asialoglycoprotein receptor, was studied by administering the ligand as a short pulse to perfused rat livers operating under single-pass conditions. Intracellular processing was arrested at various times after the pulse and the resultant homogenate cleared of nuclei and heavy mitochondria by centrifugation at 3000 g for 10 min. After adjustment to 12.5% (w/v) iodixanol, the 3000 g supernatants were centrifuged at 350,000 g for 60 min to form the gradients in which early, clathrin-containing vesicles, low-density endosomes, and lysosomes were well-resolved. 99mTc-NGA bound to the sinusoidal membrane could be partially resolved from clathrin-containing vesicles by inclusion of 1 mM CaCl2 in the homogenization and gradient buffers. Two populations of early clathrin-containing vesicles could be resolved by rate-zonal centrifugation in preformed iodixanol gradients. Thus, iodixanol is an excellent density gradient medium for the rapid and efficient resolution of endosome compartments.