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Peroxisomes were purified from liver homogenates from rats, treated with the peroxisome proliferator clofibrate, by a combination of differential centrifugation and isopycnic centrifugation in iso-osmotic self-generating Percoll gradients. Structural integrity of the peroxisomes appeared to be preserved as evidenced by a high degree of catalase latency, the absence of catalase release during purification and the exclusion of inulin (mol.wt. +/- 5000). Spaces for water and solutes were measured after incubation of the peroxisomes in iso-osmotic sucrose with radioactive water or solutes and separation of the organelles from their media by centrifugation through an organic layer. Extraperoxisomal water was corrected for by the use of radioactive dextran or inulin. The sucrose, glucose, urea, methanol and acetate-accessible spaces were identical, suggesting that these spaces represent the volume in which molecules that can cross the membrane distribute. This volume equalled 50-65% of the water space. Urate and NAD+, a cofactor of peroxisomal beta-oxidation of fatty acids, also distributed in this volume, but were also partly bound. Urate and NAD+ binding was not abolished by sonication, which released the bulk of matrix catalase activity, but NAD+ binding was seriously diminished. The peroxisomal water and sucrose spaces were estimated to be 107 microliters and 55 microliters per g of liver tissue from a clofibrate-treated rat. From quantitative morphometric data [Anthony, Schmucker, Mooney &amp; Jones (1978) J. Lipid Res. 19, 154-165] and our marker enzyme analyses, as well as from our experimentally determined water spaces of mitochondrial and microsomal fractions, it could be calculated that the volume contamination by lysosomes, mitochondria and microsomes did not exceed 1, 8 and 6% respectively. Our data indicate that apparently intact peroxisomes are permeable to a number of small molecules, including NAD+. Whether the NAD+-binding sites in sonicated peroxisomes mirror the likely existence of a membrane carrier requires further investigation.

Water- and solute-accessible spaces of purified peroxisomes. Evidence that peroxisomes are permeable to NAD+