IMMUNOISOLATION OF HIGHLY PURIFIED PEROXISOMES USING MAGNETIC BEADS AND CONTINUOUS IMMUNOMAGNETIC SORTING

Immune-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic pr operties. We have established a method for immune-isolation of peroxis omes (PO) from both rat liver and the human hepatoblastoma cell line H epG2 using magnetic beads as solid support. A polyclonal antibody rais ed against the cytoplasmic C-terminal 10 amino acids of the rat 70 E:D a peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitoch ondrial fraction and the organelle-bead complexes formed were separate d by magnetic sorting in a free-flow system without pelleting the comp lexes during the isolation procedure. Scanning electron microscopy rev ealed decoration of beads with particles measuring 150-400 nm in diame ter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polya crylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existen ce of inherited diseases such as the Zellweger syndrome in which intac t PO are lacking, but peroxisomal remnants called ''ghosts'' are obser ved instead. Peroxisomal disorders are usually studied using skin fibr oblast cell lines derived from afflicted patients and immune-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fat al peroxisomal disorders.