Solute traffic across mammalian peroxisomal membrane – single channel conductance monitoring reveals pore-forming activities in peroxisomes

Abstract.

Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient followed by an Optiprep density gradient centrifugation. Peroxisomes contributed 90–96% of the total protein content in the fraction, as confirmed by marker enzyme assays, protein pattern in SDS-PAGE, immunoblotting, and electron microscopy. Solubilized peroxisomal membrane proteins were reconstituted into a planar lipid bilayer. A single-channel conductance monitoring of the reconstituted lipid bilayer revealed the presence of two pore-forming components with a conductance in 1 M KCl of 1.3 nS and 2.5 nS. Control experiments with fractions enriched in mitochondria, lysosomes, and fragments of endoplasmic reticulum showed that the peroxisomal channel-forming activities were not due to admixture of isolated peroxisomes with other cellular organelles. The peroxisomal channels were well preserved in membrane preparations but became unstable after solubilization from the membranes by detergent.