Immunochemical characterization of submicrosomal rat liver membranes.

Rat liver microsomes were separated into three subfractions by means of ultracentrifugation through sucrose media of different densities and in the presence of different cations. Detergent extracts of these preparations were analysed by double diffusion in agar gel and immunoelectrophoresis with rabbit antisera against each of the subfractions. One subfraction was derived from the rough' and another from the smooth' part of the endoplasmic reticulum of the liver parenchymal cells. Their extracts contained at least thirteen soluble antigens in common. However, fraction-specific antigens were also present. Thus, the extracts of the rough (R) membranes contained at least one typical antigen, not found in any of the other fractions. An antiserum against this component also precipitated a non-migrating antigen present only in the extracts of smooth (Sa) membranes. These two antigens may represent different molecular forms of a substance involved in binding of the ribosomes or in the assembly of membrane subunits. At least eleven of the antigens common for the two fractions exhibited enzymatic activities when assayed after precipitation with antibody in the immunoelectrophoretic plates. Six immunologically and electrophoretically distinct antigens had esterase activity with α-naphthyl propionate as substrate. Two of these esterases also split indoxyl acetate. Three other antigens with acid phosphatase activity split both α-naphthyl acid phosphate and β-glycerophosphate. Three antigens had nucleoside diphosphatase activity when tested with uridine- or inosine-diphosphate. Preliminary experiments also suggested that two additional antigens possessed NADH-diaphorase activity and thus could belong to the microsomal electron transport systems. The third subfraction consisted of electron-microscopically smooth membranes (Sb), enzymatically different from those of the endoplasmic reticulum. The antigens typical for the latter were either absent or present only in minor and variable concentrations. Its extracts contained at least two typical antigens. One of these was identified as contaminating ferritin. The nature and origin of the other antigen has not yet been established. All antigens described in this paper were immunologically different from the common rat serum proteins.