Abstract
AbstractThe in vitro response of renal microbodies and lysosomes to a variety of altered physical and chemical conditions was examined. The influence of these conditions upon the integrity of the organelles was determined as that proportion of the total activity of various “marker” enzymes (α‐hydroxy acid oxidase, catalase and D‐amino acid oxidase for microbodies, and acid phosphatase for lysosomes) which was released from the organelles and recovered in the soluble phase following treatment.Microbodies and lysosomes were separated by differential centrifugation from 15% homogenates of male, rat kidney prepared in 0.25 M sucrose. After exposure to experimental treatments, the preparation was further separated into sediment and supernatant fractions by centrifugation at 100,000 × g for 30 minutes. The sediment and supernatant fractions were assayed to determine the distribution of the “marker” enzymes.Eighty to 100% of the activities of α‐hydroxy acid oxidase and catalase were found in the supernatant after exposure of the microbody‐lysosome fraction to low concentrations of Triton‐X 100 or digitonin, or after treatment by sonic vibrations or high speed laminar shearing. Lesser but significant proportions of the total activities of these enzymes were recovered in the soluble phase after exposure of the organelles to distilled water, 0.1 M Sorensen's phosphate buffer or 0.1 M TRIS‐HCl buffer prepared in distilled water. Acid phosphatase showed lower levels of release into the supernatant under most of these conditions.When microbody‐lysosome preparations were exposed to sucrose in concentrations of 0.1 M to 0.8 M to phosphate buffer in 0.25 M sucrose or to TRIS‐HCl buffer prepared in 0.25 M sucrose, the release of α‐hydroxy acid oxidase, catalase and acid phosphatase was insignificant. However, under these conditions D‐amino acid oxidase was released into the soluble phase to a significant extent.These results suggest that α‐hydroxy acid oxidase and catalase are soluble constituents of renal microbodies while D‐amino acid oxidase is associated firmly with a sedimentable component of the organelle, and that renal microbodies are more fragile than renal lysosomes.
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