Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol

A novel method is described for the separation on a single gradient of the major intracellular organelles of the secretory pathway, the Golgi, the smo oth endoplasmic reticulum, and the rough endoplasmic reticulum. Total micro somes were prepared from rat; liver by differential centrifugation and resu spended in 20% iodixanol. The microsomal suspension was then layered. betwe en a 30% iodixanol cushion and a layer of 15% iodixanol and centrifuged in a vertical rotor for 2 h, The microsomes distributed in four visible bands, The gradients were collected by upward displacement and were characterized (i) by determination of UDP galactose-galactosyltransferase (Golgi marker) NADPH-cytochrome c reductase (endoplasmic reticulum marker) and RNA (rough endoplasmic reticulum marker); (ii) by immunoblotting for TGN38 (trans-Gol gi marker) and GS28 (cis-Golgi marker) and for protein disulfide isomerase (endoplasmic reticulum lumenal marker); (iii) by determination of the lipid composition; and (iv) by electron microscopy. The results suggest that the top band (density 1.045-1.090 g/ml), which contains 68% of the galactosylt ransferase activity, consists of vesicles derived from the Golgi. The secon d broad band in the middle of the tube (density 1.130-1.160 g/ml), which co ntains 54% of the NADPH-cytochrome c reductase activity, consists mainly of vesicles derived from the smooth endoplasmic reticulum, overlapped at the top by a small band of Golgi-derived lamellae. The two bands at the bottom of the tube (density 1.130-1.160 and density 1.180-1.220 g/ml) appear to co ntain two subfractions of vesicles derived from the rough endoplasmic retic ulum. (C) 1999 Academic Press.