The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B 1 but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B 1 .

Rat liver peroxisomes have been separated according to size by zonal sedimentation. A method is described for calculating the size of the particles from their final position in the gradient. Peroxisomes seem biochemically homogeneous throughout their size distribution. 3 hr after injection of tritiated leucine, the specific radioactivity of catalase is the same in peroxisomes of different sizes, and it remains so for up to 1 wk after administration of the precursor. This observation rules out the possibility that peroxisomes have an extended period of independent growth. If individual particles maintain an independent existence, they must be formed very rapidly. The other possible explanation is that peroxisomes exchange material within the liver cell.

After preliminary experiments had established that the injection of Triton WR-1339 necessary for the separation of lysosomes and peroxisomes did not affect the turnover rate of catalase, the decay of 3 H-leucine incorporated into peroxisomes was studied in whole particles and in protein subfractions. It was shown that peroxisomes are destroyed in a completely random way, probably as wholes since the apparent half-life was the same for all subfractions, about 3½ days. In agreement with the results of Price et al. (11), the half-life of catalase derived from the rate of recovery from aminotriazole inhibition was about 11½ days, as was the apparent half-life of the heme prosthetic groups measured with 14 C-α-aminolevulinic acid. Guanidino-labeled arginine gave an apparent half-life of 2½ days with large statistical uncertainty. Either the leucine label was reutilized very extensively in our animals and the true half-life of peroxisomes is 1½ days, or the prosthetic groups of catalase turn over more rapidly than the protein part of the molecule.

Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D -amino acid oxidase, L -α-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D -amino acid oxidase, and L -α-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.

Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L -α-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.