Peroxisome subpopulations of the rat liver: Isolation by immune free flow electrophoresis

Peroxisomes (POs) are a heterogenous population of cell organelles which, i n mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and th eir sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-ca lled "heavy" POs with a buoyant density of 1.22-1.24 g/cm(3) have been high ly purified from rat liver, whereas the other subpopulations also present i n that tissue have escaped adequate characterization. The purification of t hese subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal s ubpopulations in the biogenesis of this organelle. Here we used an alternat ive novel approach to density gradient centrifugation, called immune free f low electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use o f the fact that the electrophoretic mobility of a subcellular particle comp lexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electr ophoresis buffer is adjusted to pH similar to 8.0, the pl of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles ca n be separated in an electric field from those that are noncoupled and henc e more mobile. The IFFE technique has been recently applied for isolation o f regular POs (rho = 1.22-1.24 g/cm3) from a light mitochondrial fraction o f rat liver. We succeeded in isolating different subpopulations of POs by a pplying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent disti nct subpopulations of rat hepatic POs.